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UNIVERSIDADE FEDERAL DE GOIÁS
PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA TROPICAL
E SAÚDE PÚBLICA
SIMONE SCHNEIDER WEBER
Análise Proteômica de Superfície Celular e Secretoma
de Paracoccidioides
Goiânia
2012
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Autor(a): SIMONE SCHNEIDER WEBER
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Agência de fomento: CNPq Sigla:
País: Brasil UF: GO CNPJ:
Título: Análise Proteômica de Superfície Celular e Secretoma de Paracoccidioides
Palavras-chave: Paracoccidioides, secretoma, proteômica, virulência, macrófagos
Título em outra língua: Proteomic analysis of Paracoccidioides celular surface and secretome
Palavras-chave em outra língua: Paracoccidioides, secretome, proteomics, virulence,
macrophages
Área de concentração: MICROBIOLOGIA
Data defesa: (05/12/2012)
Programa de Pós-Graduação: Medicina Tropical e Saúde Pública
Orientador(a): Célia Maria de Almeida Soares
CPF: E-mail: [email protected]
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________________________________________ Data: 09 /02 /2013
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1 Em caso de restrição, esta poderá ser mantida por até um ano a partir da data de defesa. A extensão deste prazo
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SIMONE SCHNEIDER WEBER
Análise Proteômica de Superfície Celular e Secretoma
de Paracoccidioides
Tese de Doutorado apresentada ao Programa de Pós-
Graduação em Medicina Tropical e Saúde Pública da
Universidade Federal de Goiás para obtenção do
Título de Doutor em Medicina Tropical e Saúde
Pública.
Orientador: Professora Dra CÉLIA MARIA DE
ALMEIDA SOARES
Goiânia
2012
Dados Internacionais de Catalogação na Publicação (CIP)
GPT/BC/UFG
W373a
Weber, Simone Schneider.
Análise proteômica de superfície celular e secretoma
de Paracoccidioides [manuscrito] / Simone Schneider
Weber. - 2012.
Vi, 72 f. : figs, tabs.
Orientadora: Profª. Drª. Célia Maria de Almeida
Soares.
Tese (Doutorado) – Universidade Federal de Goiás,
Instituto de Patologia Tropical e Saúde Pública, 2012.
Bibliografia: f. 64-72.
Inclui listas de figuras, tabelas, símbolos, siglas e
abreviaturas.
1. Paracoccidioides brasiliensis. 2. Paracoccidioides
- Análise proteômica. I. Título.
CDU: 616.992:543.645.6
Este trabalho foi realizado no Laboratório de Biologia Molecular, Departamento de
Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal
de Goiás. Com apoio financeiro: CNPq, FINEP e FAPEG.
Programa de Pós-Graduação em Medicina Tropical e Saúde Pública
da Universidade Federal de Goiás
BANCA EXAMINADORA DA DEFESA DE DOUTORADO
Aluna: SIMONE SCHNEIDER WEBER
Orientadora: Professora Dra CÉLIA MARIA DE ALMEIDA SOARES
Membros:
1. Dra. Célia Maria de Almeida Soares - ICB/UFG
2. Dr. Márcio Lourenço Rodrigues - UFRJ
3. Dr. Sébastien Olivier Charneau - UNB
4. Dr. Alexandre Melo Bailão - ICB/UFG
5. Dr. Milton Adriano Pelli de Oliveira - IPTSP/UFG
6. Dra. Ana Flávia Aves Parente - ICB/UFG, Suplente
7. Dr. Clayton Luiz Borges - ICB/UFG, Suplente
8. Dra. Juliana Alves Parente - ICB/UFG, Suplente
9. Dra. Maristela Pereira - ICB/UFG, Suplente
Data: 05/12/2012
`` Sou um pouco de todos que conheci,
um pouco dos lugares que fui,
um pouco das saudades que deixei,
sou muito das coisas que gostei.
Entre umas e outras errei,
entre muitas e outras conquistei´
Ramon Hasman
AGRADECIMENTOS
Em especial, agradeço a Prof
a Dr
a Célia Maria de Almeida Soares, pela orientação. Obrigada
pela oportunidade e por confiar na minha capacidade. Seu exemplo de dedicação e
profissionalismo foi para mim um aprendizado. Muito obrigada!
Aos meus familiares, pelo apoio nos momentos de desânimo e compreensão nas horas de
ausência. Ao meu marido, agradeço pelo incentivo e por me amar. Aos meus filhos, agradeço
por suportar a minha falta.
Aos amigos, colegas, colaboradores do LBM pelo convívio e valiosas sugestões. Meu MUITO
OBRIGADO!!!
Ao CNPq pela bolsa de estudo, ao Programa de Pós-graduação em Medicina Tropical de
Saúde Pública (IPTSP/UFG) pela oportunidade de estudo.
SUMÁRIO
Pág.
FIGURAS E TABELAS ................................................................................................. I
SÍMBOLOS, SIGLAS E ABREVIATURAS ................................................................ II
RESUMO ......................................................................................................................... V
ABSTRACT ..................................................................................................................... VI
1. INTRODUÇÃO ........................................................................................................... 1
1.1. O gênero Paracoccidioides ....................................................................... 1
1.2. Parede celular de Paracoccidioides .......................................................... 2
1.3. Proteínas de parede celular de Paracoccidioides ...................................... 4
1.4. Vias de secreção em Eucariotos ................................................................ 8
1.5. Proteínas extracelulares em fungos patogênicos .......................................
1.6. Brefeldina A, um inibidor da via de secreção .........................................
1.7. Interação do patógeno com as células imunes do hospedeiro ...................
11
14
16
2. JUSTIFICATIVA .....................................................................................................
19
3. OBJETIVO .................................................................................................................. 20
4. MANUSCRITO ...........................................................................................................
21
5. RESULTADOS DE TRABALHOS EM DESENVOLVIMENTO ......................... 42
5.1. Proteoma da superfície celular de Paracoccidioides ................................ 42
5.1.1. Padronização da extração de proteínas da parede celular ........... 42
5.1.2. Análise proteômica da superfície celular de Paracoccidioides .. 45
6. DISCUSSÃO ................................................................................................................ 55
7. CONCLUSÃO ............................................................................................................. 63
8. REFERÊNCIAS .......................................................................................................... 64
I
FIGURAS E TABELAS
Pág. Figura 1 - Parede celular de Paracoccidioides ..................................................................
3
Figura 2 - Representação esquemática de proteínas covalentemente ligadas à PC ...........
7
Figura 3 - Representação esquemática da via clássica e não-clássica de secreção de
proteínas através da parede celular em leveduras ...............................................................
10
Figura 4. Estrutura química da Brefeldina A ..................................................................... 14
Figura 5. Mecanismo de ação da Brefeldina A ................................................................. 15
Figura 6. Modelo da participação dos exossomos secretados por Leishmania na
liberação de moléculas efetoras ........................................................................................... 18
Figura 7. Representação esquemática das etapas da extração de proteínas da parede
celular padronizada para Paracoccidioides ......................................................................... 44
Figura 8. Validação do método de extração das proteínas da parede celular de
Paracoccidioides .................................................................................................................
45
Figura 9. Mapa proteico da superfície celular de levedura de Paracoccidioides .............
46
Figura 10. Classificação funcional das proteínas identificadas na superfície celular de
leveduras de Paracoccidioides ............................................................................................
47
Tabela 1. Proteínas associadas à parede celular de Paracoccidioides, Pb01 ...................
48
Tabela 2. Proteínas de superfície celular de Paracoccidioides, Pb01 .............................. 52
II
SÍMBOLOS, SIGLAS E ABREVIATURAS
2D-PAGE: eletroforese bidimensional
ACN: acetronitrila
ASL-PPCs: proteínas de parede celular sensíveis a álcalis
(alkali-sensitive linkage-PPCs)
Bg12p: proteína β-1,3-glucanosiltransferase
BMDM: macrófagos derivados de medula óssea (bone marrow-derivated macrophages)
CFU: Unidade formadora de colônias (colony forming unit)
Cts1p: proteína quitinase
CW: parede celular (cell wall)
CWPs: Proteínas de parede celular (cell wall proteins)
DNA: ácido desoxirribonucleico (deoxyribonucleic acid)
DTT: ditiotreitol (dithiotreitol)
EDTA: ácido etilenodiamino tetra- acético (ethylenediaminetetraacetic acid)
Exg1p: proteína β-exoglucanase
FSD: Banco de dados de secretoma de fungos (Fungal secretome database)
GAPDH: enzima gliceraldeído 3-fosfato desidrogenase
gp43: glicoproteína secretada de 43 kDa
GPI: glicosilfosfatidilinositol (glycosylphosphatidylinositol)
GPI-CWP: proteína com âncora de GPI ligadas a parede celular
(glycosylphosphatidylinositol-bound cell-wall protein)
GPI-PPC: proteínas com âncora de GPI ligadas a parede celular
HCl: ácido clorídrico (hydrogen chloride)
HF-piridina: fluoreto hidrogenado de piridina (hydrofluoride pyridine)
H2O2: peróxido de hidrogênio
H2O: água
IEF: focalização isoelétrica (Isoelectric focusing)
IPGphor: equipamneto usado para IEF
MALDI: método de ionização e dessorção a laser assistida por matriz
(matrix-assisted laser desorption/ionization)
MEC: matriz extracelular
MP: membrana plasmática (plasmatic membrane)
III
MS: espectrometria de massas (mass spectrometry)
MSMS: espectrometria de massas em tandem
NaCl: cloreto de sódio (sodium chloride)
P: fosfato (phosphate)
PI: índice de fagocitose (Phagocytic índex)
Pb01: isolado 01 de Paracoccidioides
PbCAT: proteína catalase de Paracoccidioides
PbENO: proteína enolase de Paracoccidioides
PbFMD: proteína formamidase de Paracoccidioides
PbGAPDH: proteína gliceraldeído 3-fosfato desidrogenase de Paracoccidioides
PbGST: proteína glutationa S transferase de Paracoccidioides
PbSOD: proteína superóxido dismutase Paracoccidioides
PC: parede celular
PCR: Reação em cadeia da polimerase (polymerase chain reaction)
PCM: Paracoccidioidomicose (Paracoccidioidomycosis)
pH: potencial hidrogeniônico
pI: ponto isoelétrico (isoelectric point)
PIR: proteínas com repetições internas
PIR-PPCs: proteínas da parede celular com repetições internas
PMF: método de identificação das proteínas pelo padrão de digestão enzimático
(peptide mass fingerprint)
PMSF: fenil metil sulfonil fluoridro (phenylmethylsulfonyl fluoride)
PPCs: proteínas de parede celular
RE: reticulo endoplasmático (endoplasmatic reticulum)
RNA: ácido ribonucléico (ribonucleic acid)
RGD: motivo Arg-Gly-Asp em uma sequência protéica
rpm: rotações por minutos (rotations per minute)
SDS: dodecil sulfato de sódio (sodium dodecyl sulfate)
SDS-PAGE: eletroforese unidimensional
SI: Sistema imune
SOD: superóxido dismutase
TNF-: fator de necrose tumoral alfa
TOF: tempo de vôo (time-of-flight mass spectrometer)
IV
TPI: proteína triose fosfato isomerase
-1,3: ligação alfa 1,3 das glucanas da PC
-1,6: ligação alfa 1,6 das glucanas da PC
β-1,3: ligação beta 1,3 das glucanas da PC
β-1,6: ligação beta 1,6 das glucanas da PC
V
RESUMO
Paracoccidioides é um complexo de espécies filogenéticas que causam
paracoccidioidomicose (PCM). As proteínas de superfície celular constituem uma importante
classe de biomoléculas por se localizarem na interface da célula com o meio extracelular. Assim
como as proteínas extracelulares podem participar da interação do parasita com o hospedeiro,
desempenhando diversos papeis biológicos com intuito de garantir a sobrevivência e
multiplicação do micro-organismo. Proteínas secretadas por fungos patogênicos são conhecidas
por desempenhar funções essenciais, tais como: captação de nutrientes, comunicação célula-
célula, detoxificação e mais especificamente desempenham funções importantes na virulência e
patogênese. Com objetivo de descrever o perfil de proteínas secretadas em ambas as fases,
micélio e levedura, de Paracoccidioides, Pb01, nós usamos uma metodologia proteômica
combinando eletroforese bidimensional (2-DE) e espectrometria de massas (MS). Foram obtidas
três replicadas biológicas de três amostras biológicas independentes. A análise proteômica
revelou 356 e 388 spots no secretoma de micélio e levedura, respectivamente. Esse estudo
permitiu a identificação de 160 proteínas não redundantes, as quais correspondem a 86
diferentes proteínas. Nós identificamos 30 e 24 proteínas preferencialmente secretadas em
micélio e levedura, respectivamente. As análises in silico mostraram que 65% das proteínas
extracelulares identificadas foram preditas de ser secretada, a maioria usando vias não clássicas
de secreção. Os dados mostraram que 12,5% das proteínas identificadas apresentaram peptídeo
sinal enquanto que 52,5% foram preditas de ser secretada por mecanismos não convencionais.
As 160 proteínas identificadas foram agrupadas em 8 categorias funcionais de acordo com o
catálogo funcional do MIPS. Investigamos a influência do bloqueio da via de secreção sobre a
fagocitose de células leveduriformes de Paracoccidioides pelos macrófagos. Observamos que a
adição de brefeldina A ao meio de cultura diminuiu significativamente o número de proteínas
secretadas pelo fungo, bem como o número de leveduras internalizadas pelos macrófagos. Em
contraste, a adição de sobrenadante de cultura concentrado, ao co-cultivo, aumentou
significativamente o número de células de levedura internalizadas pelos macrófagos. É
importante notar que proteínas detectadas no secretoma também foram identificados dentro de
macrófagos. Esses dados indicam que proteínas extracelulares de Paracoccidioides são
importantes para a interação do fungo com o hospedeiro. Foram identificadas ainda na forma
leveduriforme de Paracoccidioides, Pb01 um total de 40 proteínas associadas à parede celular e
22 proteínas de parede celular sensível ao tratamento com álcali (ASL-PPCs). Este estudo
representa uma análise global das proteínas que participam da interação do fungo com células
do hospedeiro, através da descrição de proteínas secretadas ao meio extracelular, bem como de
proteínas que constituem a superfície celular de Paracoccidioides, Pb01.
VI
ABSTRACT
Paracoccidioides is a complex of phylogenetic species that cause paracoccidioidomycosis
(PCM). The cell surface proteins constitute an important molecules class because they were
situated on interface between the cell and extracellular medium. As well, extracellular proteins
are involved in host-parasite interactions, playing several roles in biological order to ensure the
survival and multiplication of fungus in the host environment. Secreted proteins by pathogenic
fungi are known to perform essential functions such as nutrient uptake, cell-cell communication,
detoxification and more specifically play important roles in virulence and pathogenesis. In
order to describe the profile of secreted proteins in the both phases, mycelia and yeast cells, of
Paracoccidiodes, Pb01, we performed a proteomic methodology combining two-dimensional
electrophoresis (2-DE) with mass spectrometry (MS). Three analytical replicates were produced
for three independent biological samples. The proteomic analysis revealed 356 and 388 spots in
the mycelium phase and in the yeast cells secretomes, respectively. This work allowed the
identification of 160 non-redundant proteins, which corresponded to 86 different proteins. We
identified 50 and 42 proteins preferentially secreted in mycelia and yeast cells, respectively. In
silico analysis showed that 65% of extracellular identified proteins were predicted to be
secreted, mostly using non-conventional secretory pathways. Data showed that 12.5% of
extracellular identified proteins presented a classical secretion signal whereas 52.5% were
predicted to be secreted following nonclassical secretion mechanisms. The 160 identified
proteins were classified into 8 functional categories according to the MIPS Functional
Catalogue Database. We investigated the influence of secretion pathway blocking in the
phagocytosis of Paracoccidioides yeast cells by macrophages. Addition of brefeldin A in the
culture medium decreased significantly the amount of secreted proteins by fungal, as well the
number of internalized yeast cells by macrophages. In contrast, the addition of concentrated
culture supernatant to the co-cultivation significantly increased the number of internalized yeast
cells by macrophages. Importantly, the proteins detected in the fungal secretome were also
identified within macrophages. These results indicate that Paracoccidioides extracellular
proteins are important to fungal interaction with the host. Were also identified in the
Paracoccidiodes, Pb01 yeast cells, a total of 40 cell wall associated proteins, and 22 cell wall
proteins alkali-sensitive treatment (ASL-PPCs). This study represents a comprehensive analysis
of proteins involved in the host-fungus interactions through the description of proteins secreted
to extracellular medium, as well proteins that constitute the Paracoccidiodes, Pb01 surface.
1
1. Introdução
1.1. O gênero Paracoccidioides
Paracoccidioides é o agente etiológico da paracoccidioidomicose (PCM), uma das
micoses sistêmicas mais frequentes que acomete a população rural da América Latina (Restrepo &
Tobon 2005). O gênero compreende quatro linhagens filogenéticas (S1, PS2, PS3 e Pb01-like)
(Matute et al. 2006; Carrero et al. 2008). Análises filogenéticas dos isolados de Paracoccidioides
tem resultado na diferenciação do gênero em duas espécies: P. brasiliensis que agrupa um
complexo de três espécies filogenéticas e P. lutzzi que representa o isolado Pb01-like (Teixeira et
al. 2009; Desjardins et al. 2011).
Tendo sido descrito pela primeira vez em 1908 por Adolpho Lutz, Paracoccidioides é um
fungo termo-dimórfico, que cresce na forma de micélio em temperaturas inferiores a 28ᵒC, e na
forma de levedura em tecidos do hospedeiro ou quando cultivadas in vitro a 36ᵒC (San-Blas et al.
2002; Restrepo et al. 2008). A forma miceliana ou infectiva é caracterizada por filamentos de hifas
septadas e multinucleadas com conídios terminais ou intercalares. Enquanto que a forma
parasitária ou leveduriforme é constituída por células unicelulares com múltiplos brotamentos,
onde uma célula mãe grande e central é circundada por células periféricas menores apresentando
um aspecto de roda de leme de navio, estrutura determinante para o diagnóstico da presença de
Paracoccidioides em amostras biológicas (Restrepo-Moreno 2003). A infecção se dá através da
inalação de propágulos de micélio, como conídios pela via respiratória. Nos alvéolos pulmonares,
essas estruturas transitam para a forma leveduriforme, de onde podem disseminar-se via
hematogênica e/ou linfática para diferentes órgãos e tecidos (San-Blas 1993).
A transição dimórfica se dá pela mudança na temperatura e constitui-se uma etapa
essencial para o estabelecimento da infecção e para a fase inicial da interação do fungo com o
hospedeiro. Acredita-se que essa etapa seja importante como mecanismo de virulência, uma vez
2
que isolados que não possuem a capacidade de se diferenciar em levedura não são virulentos (De
Moraes Borba & Schäffer 2002).
1.2. Parede Celular de Paracoccidioides
A superfície celular, em especial a parede celular (Assumpção et al.), é o ponto de contato
entre o micro-organismo e o hospedeiro, e desempenha um papel importante de proteção ativa do
fungo contra mecanismos de defesa do hospedeiro (San-Blas & San-Blas 1977). A PC é uma
estrutura dinâmica e complexa que confere proteção e rigidez à célula, está continuamente
mudando como resultado de alterações na condição de cultivo e estresse ambiental (Latgé 2007).
A composição da PC varia entre as diferentes espécies de fungos, mas basicamente é
composta por carboidratos, proteínas, glicoproteínas e lipídeos (De Groot et al. 2005). Em
Paracoccidioides, não existem diferenças significativas na quantidade de lipídeos (5-10%) e
glicanas (36-47%) entre ambas as fases do fungo. O teor protéico é maior em micélio (24-41%)
que em levedura (7-14%), enquanto que a fase patogênica possui uma quantidade maior (37-48%)
de quitina comparada com a miceliana (7-18%). Em Paracoccidioides, a forma leveduriforme
contém -1,3-glucana como principal polímero de glicose na parede celular, enquanto que o
micélio possui β-1,3-glucana (Kanetsuna et al. 1969).
A quitina, responsável pela integridade da parede celular, é constituída por resíduos de N-
acetilglicosamina unidos por ligações β-1,4 (Munro & Gow 2001). Uma alteração na síntese de
quitina resulta em malformação e instabilidade osmótica da célula fúngica (Bago et al. 1996).
Estruturalmente, a quitina da forma de levedura encontra-se associada a pequenas quantidades de
-1,6-glucana e β-1,3-glucana na camada interna. Já a quitina de micélio encontra-se associada a
β-glucanas e proteínas em uma única camada de parede celular. As β-glucanas de micélio
possuem principalmente ligações glicosídicas do tipo β-1,3-glucana e, em menor quantidade, β-
1,6-glucana (Carbonell 1969; Kanetsuna et al. 1969; San-Blas et al. 1987).
3
Estruturalmente, os componentes da parede celular de Paracoccidioides são dispostos em
camadas (Figura 1). A parede celular de levedura é constituída por três camadas, sendo a interna
formada por quitina e β-glucanas, e a externa por -1,3-glucana. Enquanto que a parede celular do
micélio é constituída por uma única camada, onde quitina, proteínas, β-1,3-glucana, e em menor
quantidade β-1,6-glucana, se interconectam (San-Blas & San-Blas 1977).
Figura 1. Parede celular de Paracoccidioides (A) Parede celular de levedura x
40.000 mostrando três camadas distintas, duas eletrodensas intercaladas por uma
camada translúcida. A camada externa é formada por -1,3-glucana, enquanto a
interna por uma rede de filamentos de quitina e β-glucanas. (B) Parede celular da
forma miceliana x 38.000 mostrando uma única camada constituída por uma mistura
de quitina e glucanas. Adaptado de San-Blas & San-Blas (1977).
Os principais polímeros de carboidratos (quitina, -glucana e β-glucana) são propostos
como os responsáveis pela integridade estrutural e forma da parede celular (Kanetsuna et al. 1969)
e contribuem para a transição dimórfica de Paracoccidioides (San-Blas & San-Blas 1994), sendo a
β-1,3-glucana vista como um imunomodulador enquanto que a -1,3-glucana relacionada com a
virulência. Assim, a mudança na composição da parede celular pode funcionar como um
mecanismo de escape do sistema imune, onde -1,3-glucana estaria localizada mais externamente
na parede celular de leveduras (Kanetsuna et al. 1972) protegendo a β-1,3-glucana, que possui
propriedades antigênicas, dos mecanismos de defesa do hospedeiro (San-Blas & San-Blas 1982),
uma vez que β-1,3-glucana é capaz de induzir a liberação de fator de necrose tumoral alfa (TNF-
4
) por células fagocíticas resultando na morte do fungo (Anjos et al. 2002). Os polímeros de -
1,3-glucana estão presentes externamente apenas na parede celular da forma patogênica de
Paracoccidioides, sendo substituídos por β-1,3-glucana quando este fungo encontra-se na forma
miceliana (Kanetsuna et al. 1972).
1.3 Proteínas de parede celular
As proteínas de parede celular (PPC) constituem uma importante classe de biomoléculas
por se localizarem na interface da célula com o meio extracelular. Estudos têm atribuído inúmeras
funções a proteínas de superfície, como enzimas, moléculas de adesão, antígenos e receptores de
superfície (Hoyer et al. 2001; Sundstrom 2002; Chatterjee et al. 2006; Pereira et al. 2007;
Nogueira et al. 2010). São reportadas ainda, como imunogênicas e referidas como importantes
fatores de virulência (Hung et al. 2002; McGwire et al. 2002). Alem disso, algumas proteínas de
parede celular possuem propriedades enzimáticas que podem atuar na biossíntese e
remodelamento da parede celular (Hartland et al. 1996; Mouyna et al. 2000).
Apesar da estrutura e organização da PC ter sido investigada mais extensivamente em
Saccharomyces cerevisiae, um modelo molecular semelhante é também aplicável para outros
ascomicetos, incluindo Paracoccidioides. A parede celular varia entre as diferentes espécies,
porém é denominada uma estrutura complexa formada basicamente por quitina, glucanas e
manoproteínas (N e O-glicosiladas). As β-glucanas e quitina formam um esqueleto de
polissacarídeo em torno da membrana plasmática, sobre os quais são ligadas as manoproteínas
através de ligações covalentes e não covalentes, levando a uma alta complexidade estrutural (de
Groot et al. 2004). As PPCs por sua vez podem ser agrupadas dependendo da estrutura do C-
terminal e do tipo de ligação à glicana, incluindo proteínas que se ligam de forma direta a β-1,3-
glucana ou indiretamente via β-1,6-glucana. O modelo molecular proposto para a parede celular
5
de S. cerevisiae e C. albicans (Figura 2) descreve inúmeros tipos diferentes de ligações
covalentes entre proteínas e componentes da parede celular (Pitarch et al. 2008).
As PPCs estariam localizadas principalmente no lado externo da rede de quitina e β-1,3-
glucana e, em menor quantidade ao longo da parede celular determinando sua porosidade e
poderiam ser agrupadas em três classes:
1. Classe 1: Proteínas solúveis da parede celular, um grupo importante de proteínas
ligadas de modo não covalente e/ou por pontes dissulfeto à parede celular. Essas proteínas podem
ser extraídas utilizando detergentes como SDS ou agentes redutores como DTT. Essa classe de
PPCs compreende proteínas relacionadas à biossíntese e modulação de constituintes da parede,
como β-1,3-glucanosiltransferase (Bg12p), β-exoglicanase (Exg1p) e quitinase (Cts1p), e também
proteínas que se localizam na superfície celular por mecanismos de secreção (Pitarch et al. 2008).
2. Classe 2: Proteínas covalentemente ligadas à β-1,3-glicana
2.1. Diretamente via uma ligação sensível a álcali, como as PIR-PPCs (proteínas da
parede celular com repetição interna) e outras PPCs que não apresentam homologia com as PIR-
PPCs, mas são da mesma forma, ligadas covalentemente à β-1,3-glucana da parede celular através
de uma ligação sensível ao pH alcalino (Figura 2). Por esse motivo, essa classe foi renomeada
para ASL-PPCs (proteínas de parede celular sensíveis a álcali), (De Groot et al. 2005). Esse grupo
de proteínas de parede celular, o segundo mais abundante, pode ser solubilizadas em condições
alcalinas ou com tratamento enzimático com β-1,3-glucanase, mas não com β-1,6-glucanase.
2.2. Indiretamente através da β-1,6-glucana via âncora glicosilfosfatidilinosiltol (GPI)
remanescente, como as GPI-PPCs. As GPI-PPCs são proteínas altamente O-glicosiladas com um
peptídeo sinal N-terminal e um sinal para adição da âncora GPI na região C-terminal e regiões
ricas em serina e treonina, fornecendo assim sítios para O-glicosilação (Hamada et al. 1999).
6
Essas proteínas são predominantemente localizadas na camada externa da parede celular. Esse
grupo de proteínas pode ser liberado pelo tratamento enzimático com β-1,3 ou β-1,6-glucanase
bem como, pelo tratamento com ácido HF-piridina, o qual cliva a ligação fosfodiéster no
glicosilfosfatidilinosiltol remanescente. Essa classe denominada de GPI-PPC1, é o complexo
proteico mais abundante da parede celular (Figura 2).
3. Classe 3: Proteínas covalentemente ancoradas a quitina pela β-1,6-glicana via
âncora GPI, como algumas GPI- PPCs. Essa classe de PPCs pode ser extraída pelo tratamento
enzimático com quitinase e β-1,6-glucanase bem como pelo uso do ácido HF-piridina. Este
complexo denominado GPI-PPC4 é comumente observado em resposta a estresse da parede
celular (Pitarch et al. 2008).
O modelo molecular descrito acima para S. cerevisiae e C. albicans inclui ainda dois
grupos adicionais de complexo GPI-PPCs definidos como GPI-PPC2 e GPI-PPC3 (Figura 2). O
primeiro inclui GPI-PPCs ligadas à β-1,3-glucana via uma ligação sensível a álcali (GPI-PPC2) e
o segundo, GPI-PPCs simultaneamente ligadas à β-1,3-glucana através da β-1,6-glucana via
âncora GPI remanescente e ao mesmo tempo via uma ligação sensível a álcali (GPI- PPC3).
Inúmeras proteínas têm sido descritas na parede celular de Paracoccidioides
desempenhando papel importante na virulência e biogênese da parede celular, algumas são ainda
caracterizadas como antígenos de superfície e moléculas de adesão (Barbosa et al. 2006; Pereira et
al. 2007; Castro 2008; Castro et al. 2008; Nogueira et al. 2010). A aderência de micro-organismos
patogênicos a tecidos do hospedeiro é considerada indispensável para o início colonização e futura
disseminação. A adesão implica que o patógeno reconheça carboidratos ou proteínas ligantes na
superfície da célula do hospedeiro (Patti et al. 1994). Moléculas do patógeno que atuam nesse
processo são denominadas adesinas e são, na maioria, glicoproteínas de parede celular (Huang et
al. 2003).
7
Figura 2. Representação esquemática de proteínas covalentemente ligadas à parede
celular. A figura mostra cinco diferentes tipos de ligações covalentes entre as proteínas
(PPCs) e os demais componentes da parede celular descritos para S. cerevisiae e C. albicans.
As setas mostram os locais de clivagem pelos tratamentos comumente usados na extração.
Adaptado de Pitach et al. (2008).
Em Paracoccidioides, algumas moléculas já foram descritas como ligantes de
componentes da matriz extracelular. A gp43 foi a primeira a ser identificada como ligante de
laminina (Vicentini et al. 1994). Estudos adicionais mostraram que em ensaios de afinidade de
ligação, a gp43 foi capaz de se ligar tanto a fibronectina quanto a laminina (Mendes-Giannini et al.
2006). Outras moléculas de adesão em Paracoccidioides também foram descritas. A GAPDH
(gliceraldeído-3-fosfato desidrogenase) mostrou-se capaz de se ligar a laminina, fibronectina e
colágeno tipo I (Barbosa et al. 2006), bem como a TPI (triose fosfato isomerase) que também se
liga aos componentes da matriz, tais como laminia e fibronectina (Pereira et al. 2007).
8
Castro et al. (2008) descreveram uma proteína pertencente à família das glicosil hidrolases,
PbDfg5p, presente na superfície celular de Paracoccidioides e que estaria relacionada a formação
e manutenção da parede celular de fungos. Em Paracoccidioides sua presença foi evidenciada por
imunoeletromicroscopia, bem como em extratos protéicos, da parede celular, obtidos através do
tratamento enzimático de leveduras com β-1,3 glucanase. A PbDfg5p recombinante apresentou
capacidade de se ligar a laminina, fibronectina, colágeno tipo I e IV, além de apresentar um
``motif´´ RGD (Arg-Gly-Asp) em sua sequência predita, característica comum de algumas
adesinas.
A descrição de proteínas de superfície celular em Paracoccidioides é de extrema
importância para a compreensão da patogênese deste fungo, uma vez que essas moléculas podem
participar de processos essenciais tais como: síntese e manutenção da parede celular,
reconhecimento e adesão às células do hospedeiro, captação de nutrientes e escape do sistema
imune do hospedeiro. Além disso, essas moléculas podem atuam como antígenos de superfície e
funcionar como potenciais alvos para desenvolvimento de novas drogas antifúngicas e marcadores
moleculares (Pitarch et al. 2002).
1.4. Vias de secreção em Eucariotos
Em células eucarióticas, a via clássica de secreção envolve o reconhecimento de uma
sequência sinal na região N-terminal de proteínas a serem secretadas, resultando na translocação
através da membrana do retículo endoplasmático (RE) via vesículas com posterior
encaminhamento ao Complexo de Golgi. Após sofrerem modificações, essas proteínas são
transportadas do aparato de Golgi ao meio extracelular, via uma complexa rede de vesículas que
se fundem a membrana plasmática, liberando extracelularmente seu conteúdo proteico por um
mecanismo denominado de exocitose (Schatz & Dobberstein 1996). Entretanto, tem sido descrito
a existência no meio extracelular de inúmeras proteínas, sem a sequência sinal e funcionalmente
9
ativas sugerindo a existência de rotas de exportação não clássica (Cleves et al. 1996; Nombela et
al. 2006; Chaves et al. 2009; Cuervo et al. 2009; Nickel & Rabouille 2009).
Um repertório de mecanismos hipotéticos para transportar proteínas sem peptídeo sinal
através da membrana e parede celular foi descrito para C. albicans por Nombela et al. (2006)
conforme ilustra a Figura 3. A via clássica de secreção, a qual envolve o retículo endoplasmático
(ER) e o Complexo de Golgi são mostrados na letra (a); a afinidade de algumas proteínas, sem
peptídeo sinal, poderia levar a adesão e/ou internalização em vesículas secretórias (b); ou serem
exportadas através de endossomos (c). Outros potenciais mecanismos de secreção, pelos quais as
proteínas sem peptídeo sinal poderiam ser transportadas ao meio extracelular, incluem transporte
passivo (d); transporte através da membrana (flip-flop), como tem sido envolvido em Leishmania
(e); translocação (f) ou reconhecimento de substrato específico (g).
Estudos em S. cerevisiae, visando identificar mecanismos de secreção não clássicos e
genes envolvidos nesse processo, descreveram a expressão heteróloga de uma proteína
representativa de mamíferos, galectina-1 (Fig. 3, h), em mutantes para o transportador Ste6,
envolvido no transporte não clássico de -fator (Fig. 3, i) ao meio extracelular. Os dados
mostraram que a secreção de galectina-1 foi diminuída, mas não completamente eliminada, no
mutante, sugerindo que o transportador Step6 não seja essencial para a secreção de galectina-1, e
que existe um novo mecanismo de secreção não clássico atuando em S. cerevisiae, independente
de Set6 (Cleves et al. 1996). O estudo mostrou, entretanto, que genes NCE (non-classical export)
como NCE101 e NCE102 são relacionados ao transporte não convencional de galectina-1 em S.
cerevisiae. A existência de um repertório de mecanismos alternativos, explicaria porque Set6 não
é requerido para secreção de proteínas quando a via clássica de secreção é bloqueada (Cleves et al.
1996).
10
Figura 3. Representação esquemática da via clássica e não-clássica de secreção de
proteínas através da parede celular em leveduras. (a) via clássica de secreção, (b-g)
mecanismos alternativos de secreção não clássicos, (h) mecanismo envolvido na secreção de
galactina-1 em S. cerevisia mutante para Set6, (i) mecanismo não clássico de secreção
envolvendo o transportador Set6 de -fator em S. cerevisiae. Adaptado de Nombela et al.
(2006).
Em fungos, tem sido relada a existência de exportação de vesículas intactas através da
parede celular (Rodrigues et al. 2007; Albuquerque et al. 2008; Nosanchuk et al. 2008; Rodrigues
et al. 2008; Casadevall et al. 2009; Oliveira et al. 2009). Essas vesículas extracelulares são
descritas como exossomos, e seriam responsáveis pela secreção de um grande range de moléculas,
tais com: proteínas, lipídeos, polisacarídeos e pigmentos, e diferentes estudos sugerem que a
exportação dessas vesículas requer a participação de componentes da via clássica (Yoneda &
Doering 2006; Panepinto et al. 2009). Essa hipótese foi posteriormente suportada por um estudo
independente, onde os autores mostraram que células leveduriformes de C. neoopformans
expostas à Brefeldina A (BFA), um inibidor da via clássica de secreção, resultou em um defeito na
formação da cápsula, devido à inibição da secreção de polissacarídeos que integram a cápsula
deste fungo (Hu et al. 2007). Análises proteômicas do conteúdo de vesículas extracelulares em
Cryptococcus neoformans e Histoplasma capsulatum suportam a hipótese que o transporte através
11
da parede celular via vesículas é um mecanismo geral para transportar macromoléculas
relacionadas à virulência, em fungos, e que desempenham um papel importante na interação
patôgeno-hospedeiro (Albuquerque et al. 2008; Rodrigues et al. 2008). (Silverman et al. 2010)
descreveram uma via geral de secreção em Leishmania, baseada em exossomos, responsável pela
exportação de proteínas e liberação de moléculas no interior de macrófagos infectados. Mais
recentemente, foi descrito em Paracoccidioides vesículas extracelulares transportando moléculas
antigênicas que foram reconhecidas por soro de pacientes com PCM (Vallejo et al. 2011).
Análises proteômicas dessas vesículas, em Paracoocidioides, revelaram um conteúdo proteico
(Vallejo et al. 2011) e lipídico (Vallejo et al. 2012).
1.5. Proteínas extracelulares em fungos patogênicos
A capacidade de fungos patogênicos em desenvolver respostas multifacetadas para uma
ampla variedade de estressores presentes no ambiente do hospedeiro é de extrema importância na
virulência e patogênese (Ranganathan & Garg 2009). Essas respostas incluem uma gama de
moléculas que facilitam a adesão, invasão, inativação das defesas do hospedeiro e alteração ou
destruição das células do hospedeiro. Muitos deles são fatores extracelulares, secretados ou
associados à parede celular (Nombela et al. 2006; Holbrook et al. 2011). Proteínas extracelulares
são conhecidas por desempenhar funções importantes, tais como: captação de nutrientes,
comunicação célula a célula, e detoxificação do ambiente (Bonin-Debs et al. 2004). Muitas
proteínas são transportadas para a superfície celular para serem integradas a estrutura da parede
celular ou exportadas ao meio extracelular para captação de nutrientes ou defesa (Nombela et al.
2006). Mais especificamente, proteínas secretadas por micro-organismos patogênicos parecem
desempenhar papéis importantes na virulência (Tjalsma et al. 2004).
Proteínas extracelulares secretadas por fungos patogênicos são moléculas importantes
na interação patógeno/hospedeiro e tem a função de cumprir diversos papéis biológicos para
12
garantir sobrevivência e multiplicação das células fúngicas em organismos hospedeiros. Umas
das funções é a captação de nutrientes, tais como carbono e nitrogênio, a partir da digestão de
potenciais substratos presentes no meio extracelular. Dentre as mais bem descritas hidrolases
secretadas por fungos estão as α e amilases, celulases, lipases, pectinases e peptidases
(Archer & Wood 1995).
Micro-organismos patogênicos se utilizam de proteínas secretadas não apenas para
obtenção de nutrientes como também para virulência e sobrevivência sob as condições hostis
do hospedeiro. O fungo entomopatogênico Magnaporthe grisae utiliza serino proteases
secretadas em resposta ao estresse nutricional de nitrogênio extracelular (Donofrio et al. 2006).
Em Histoplasma capsulatum foram identificadas enzimas extracelulares, relacionadas à defesa
contra estresse oxidativo e chaperonas, no meio extracelular bem como no interior de
macrófagos infectados, sugerindo que estas moléculas participam da fase inicial da infecção
(Holbrook et al. 2011).
Poucas proteínas extracelulares são conhecidas em Paracoccidioides. A primeira a ser
descrita foi a glicoproteína secretada de 43 kDa (gp43), maior antígeno descrito para este
fungo. A principal função desta proteína na patogênese da PCM está relacionada aos
mecanismos de evasão durante a instalação da infecção primária (Flavia Popi et al. 2002),
estimula a formação de granulomas in vitro (Vigna et al. 2006) e apresenta determinantes
antigênicos de células T, os quais induzem uma resposta protetiva ao fungo (Calich & Kashino
1998). Estudos utilizando macrófagos primários, oriundos de medula óssea tratados com
epítopos de gp43 mostram que esta glicoproteína apresenta capacidade de inibir a resposta
inflamatória e a função dos macrófagos de liberar óxido nítrico (NO) e peróxido de hidrogênio
(H2O2), podendo atuar como um mecanismo de defesa do fungo frente ao sistema imune (SI)
do hospedeiro (Konno et al. 2009).
13
Outra proteína descrita como sendo secretada por Paracoccidioides é uma serina-tiol
protease extracelular. A atividade desta proteína foi detectada em filtrados de cultura de
Paracoccidioides, apresentado capacidade para clivar proteínas associadas à membrana basal
como laminina e fibronectina (Carmona et al. 1995), sugerindo a participação desta proteína na
adesão e invasão às células do hospdeiro
Uma aspartil-protease secretada foi identificada e o transcrito codificante para esta
proteína foi regulado positivamente durante a transição dimórfica de micélio para levedura. A
atividade desta aspartil-protease foi detectada em sobrenadante de cultura e na parede celular e
de leveduras de Paracoccidioides (Bastos et al. 2007; Tacco et al. 2009). Uma serino-protease
secretada da família das subtilisinas foi identificada e apresentou níveis de transcritos
aumentados em leveduras de Paracoccidioides isoladas de camundongos (Costa et al. 2007).
Esta serino protease também apresentou níveis de transcritos induzido durante incubação de
leveduras de Paracoccidioides com sangue e plasma humanos (Bailão et al. 2006; Bailão et al.
2007). A expressão de transcritos e de moléculas protéicas de serino-protease foi detectada com
níveis aumentados na privação de nitrogênio, os autores sugerem um papel importante desta
protease na aquisição desse elemento (Parente et al. 2010).
Inúmeros trabalhos têm relatado a existência de proteínas sem peptídeo sinal no secretoma
de diferentes organismos, sugerindo que estas proteínas possam ser secretadas por vias
alternativas de secreção e denominadas de proteínas `moonlights´ (Nombela et al. 2006; Chaves et
al. 2009; Cuervo et al. 2009; Nickel & Rabouille 2009). Em Paracoccidioides, a presença de
proteínas com localização predita no citoplasma em outros compartimentos celulares foi descrita
para a gliceraldeído-3-fosfato-desidrogenase - GAPDH (Barbosa et al. 2006) e para a triosefosfato
isomarase - TPI (Pereira et al. 2007) ambas localizadas no citoplasma e na parede celular.
Recentemente foi mostrado que a enolase de Paracoccidioides (PbENO) pode ser encontrada não
14
apenas no citoplasma e sim nos extratos proteicos da parede celular e no meio extracelular
(Nogueira et al. 2010).
A enzima formamidase foi encontrada em vesículas secretadas por H. capsulatum
(Albuquerque et al. 2008), e em Paracoccidioides, esta enzima (PbFMD) mostrou-se reativa
aos anticorpos presentes em soros de pacientes com PCM (Borges et al. 2005). Os autores
detectaram atividade enzimática da PbFMD em extratos cell free de Paracoccidioides, e
sugerem que ela possa ser secretada por mecanismos não-clássicos, uma vez que não possui
peptídeo sinal, e participar no processo de interação do fungo com as células do hospedeiro.
1.6. Brefeldina A, um inibidor da via de secreção
Brefeldina A (BFA) é uma lactona da classe dos macrolídeos produzida por organismos
fúngicos, tais como Penicillium brefeldianum (Nebenfuhr et al. 2002). A Figura 4 mostra a
estrutura química da BFA, a qual possui a fórmula molecular C16H24O4 .
Figura 4. Estrutura química da Brefeldina A.
BFA inibe reversivelmente o transporte de proteínas do retículo endoplasmático (RE) para
o Golgi e induz o transporte retrógrado de proteínas a partir do aparelho de Golgi para o retículo
endoplasmático (Figura 5). O mecanismo de ação da BFA consiste na capacidade de bloquear a
ação de um fator de troca de nucleotídeo guanina (GEF) denominado BIG2 (Charych et al. 2004).
15
Os fatores de troca de nucleotídeo guanina podem ser classificados em famílias de acordo com a
similaridade da sequência de aminoácidos que apresentam em sua estrutura, e de acordo com o
tipo de proteína ligadora de GTP que eles ativam (Zheng & Quilliam 2003). BIG2 ativa uma
subfamília de GTPase de eucariotos denominada ARF (do inglês: ADP-ribosylation factor)
(Morinaga et al. 1997; Togawa et al. 1999). A ativação de ARF é requerida para o brotamento das
vesículas no RE (do inglês: budding), o que permite o tráfego de proteínas via exocitose e
endocitose (Yahara et al. 2001; Charych et al. 2004). A Figura 5 mostra o mecanismo geral de
ação da BFA, bloqueando a ativação de ARF, efeito que impedirá a formação das vesículas
secretórias no RE, interferindo no processo de transporte de proteínas secretórias (Jackson &
Casanova 2000). BFA tem sido usada para estudar processos celulares que envolvam transporte
celular e se vias de secreção (Domozych, 1998; Klausner, et al 1992).
Figura 5. Mecanismo de ação da Brefeldina A. Brefeldin A liga-se a um
domínio ARF-GDP-Sec7 pra formar um complexo quaternário estável,
bloqueando assim o ciclo de ativação de ARF (ARF-GTP). Adaptado de
Jackson & Casanova (2000).
16
1.7. Interação do patógeno com as células imunes do hospedeiro
Células fagocíticas do sistema imune inato (fagócitos), como macrófagos, células
dendríticas e netrófilos, agem com o objetivo de eliminar micro-organismos patogênicos da
corrente sanguínea e dos tecidos do hospedeiro. Uma vez reconhecidos, os patógenos são
fagocitados pelos fagócitos, formando vesículas denominadas fagossomos. Posteriormente, ocorre
a fusão do fagossomo maduro com lisossomos, formando assim o fagolisossomo, onde enzimas
lisossomais e intermediários reativos de nitrogênio, como o óxido nítrico (NO), espécies reativas
de oxigênio (ROS) e o peróxido de hidrogênio (H2O2) são produzidos para a destruição do micro-
organismo. O fagolisossomo corresponde a um ambiente extremamente hostil ao patógeno, devido
à deprivação de certos nutrientes, acompanhada de um aumento na acidificação do meio vacuolar
(Janeway & Medzhitov 2002). A combinação desses fatores, normalmente é suficiente para matar
e degradar o micro-organismo fagocitado. Entretanto, patógenos têm desenvolvido estratégias para
subverter o ataque dos fagócitos (Haas 2007; Seider et al. 2010). Leveduras de H. capsulatum, um
fungo intracelular patogênico, são capazes de inibir a acidificação do fagossomo após serem
fagocitados pelos macrófagos (Strasser et al. 1999; Webster & Sil 2008). C. albicans inibe a
maturação do fagossomo, prevenindo a produção de NOS e ROS, permitindo assim o escape, via
formação de hifas (Fernandez-Arenas et al. 2009; Wellington et al. 2009; Seider et al. 2011).
Ambos patógenos, H. capsulatum e C. albicans, bloqueiam a fusão do fagossomo com lisossomos,
e utilizam desses mecanismos para escapar do SI e se estabelecer no hospedeiro. Mecanismos
semelhantes são descritos para outros patógenos intracelulares, tais como: Leishmania (Silverman
& Reiner 2012), 2012), C. neoformans (Oliveira et al. 2010) e Mycobacterium tuberculosis
(Schnappinger et al. 2006). Na maioria das vezes, essas interações envolvem vesículas
extracelulares (exossomos) ou proteínas secretadas pelos patógenos, as quais modulam a resposta
imune do hospedeiro criando um ambiente permissivo ao estabelecimento da infecção. Em
Leishmania, sabe-se que o parasita é dependente da secreção de moléculas efetoras no início e na
17
manutenção da infecção. Estudos de caracterização funcional dos exossomos em condições
semelhantes à infecção (37ºC, pH ácido) mostraram que os exossomos liberados durante choque
térmico (37ºC) e pH neutro foram enriquecidos de atividade quinase, enquanto que exossomos
produzidos em pH ácido apresentam maior atividade fosfatase, indicando que o conteúdo das
vesículas secretadas por Leishmania durante a infecção, poderiam estar envolvidas em vias de
sinalização (Silvreman et al 2012 a ; Hassini et a 2010). Nesse sentido, Silverman et al 2012
demonstraram a presença de exossomos e proteínas exossomais de Leishmania, no citoplasma de
macrófagos cultivados in vitro. Além da glicoproteína gp63, principal antígeno e fator de
virulência descrito em Leishmania, outras proteínas exossomais de Leishmania foram
identificadas no citoplasma de células fagocítcas, tais como: proteínas de choque térmico, Hsp 70
e 90, e um fator de elongação 1- (EF1-) (Nadan et al 2002; Silverman et al 2010ª). Esses dados
levaram Silverman & Reiner (2011) a propor um modelo, no qual os exossomos secretados por
Leishmania tem um papel importante na liberação de moléculas efetoras, as quais induzem um
ambiente permissivo à infecção. A Figura 6 mostra o modelo proposto por Silverman & Reiner
(2011), onde o conteúdo das vesículas secretadas por promastigotas, EF1- e gp63, é liberado no
citoplasma de macrófagos, resultando na ativação de múltiplas proteínas tirosina-fosfatases do
hospedeiro, incluindo SHP1 e PTP1B, as quais atuam (desfosforilado), ou seja, inibindo alvos da
via de sinalização IFN-/Jak-STAT1. Essa inibição resulta em bloqueio da função microbicida
(liberação de NO, ROS e TNF-) e indução da resposta anti-inflamatória (liberação de IL-10)
pelos macrófagos. Sabe-se que a IL-10 é uma potente citocina anti-inflamatória envolvida na
supressão da RI durante a infecção por Leishmania. Nesse sentido, o aumento na liberação de IL-
10 e o bloqueio da função microbicida, como resultado da exposição de macrófagos aos
exossomos, sugere um papel importante dessas vesículas na patogênese (Silerman & Reiner
2012).
18
Macrófago
Resposta anti-inflamatóriaIL-10
Função Microbicida NO, ROS e TNF-α
Figura 6. Modelo da participação dos exossomos secretados por Leishmania na
liberação de moléculas efetoras, as quais induzem um ambiente permissivo à
infecção. Adaptado de Silverman & Reiner (2012).
Os mecanismos envolvidos no estabelecimento de Paracoccidioides em hospedeiros
humanos são ainda pouco conhecidos. Konno et al. (2009) demonstraram que gp43 reduz a
função de macrófagos in vitro, confirmando os achados de Almeida et al. (1998), onde a adição de
anticorpos policlonais anti-gp43 induziram uma redução no índice de fagocitose de macrófagos
infectados com leveduras de Paracoccidiodies. Esses dados sugerem que a gp-43 possa estar
envolvida na aderência e captação do fungo pelos macrófagos. Paracoccidioides é ativamente
fagocitado por células mononucleares, incluindo macrófagos, podendo ser encontrado no
citoplasma de células gigantes em tecidos de pacientes com PCM, e possui a capacidade de
multiplicar-se no interior dessas células (Moscardi-Bacchi et al. 1994).
Nesse trabalho, identificamos o secretoma de ambas às formas de Paracoccidioides,
micélio e levedura, e descrevemos um papel importante das proteínas extracelulares na adesão e
internalização de células leveduriforme pelos macrófagos primários. Este estudo representa uma
análise global das proteínas que participam da interação do fungo com células do hospedeiro,
através da descrição de proteínas secretadas ao meio extracelular, bem como de proteínas que
constituem a superfície celular de Paracoccidioides, Pb01.
19
2- JUSTIFICATIVA
Em fungos patogênicos, proteínas de superfície celular bem como proteínas
extracelulares fazem parte das estratégias utilizadas pelo micro-organismo para estabelecer-se
no hospedeiro. Essas estratégias incluem: processo de reconhecimento, adesão e invasão dos
tecidos, escape do sistema imune, disseminação, captação de nutrientes, comunicação célula-
célula entre outros. Essas moléculas caracterizam-se por integrarem o ponto inicial de contato
entre a célula e o ambiente, contribuindo para a compreensão da interação fungo-hospedeiro.
Sendo assim, apresentam um papel importante na virulência e patogênese fúngica.
As análises proteômicas utilizadas neste trabalho permitiram identificar as proteínas de
superfície celular bem como proteínas secretadas pelo fungo ao meio extracelular. Os
resultados obtidos podem contribuir para estudos futuros que visam à descrição de alvos
moleculares alternativos para o desenvolvimento de novos antifúngicos ou marcadores
moleculares para diagnóstico ou vacinas
20
1.3. OBJETIVO
O presente trabalho teve como objetivo a identificação de proteínas de superfície celular
e extracelulares do fungo Paracoccidioides, Pb01. Foram propostos os seguintes objetivos
específicos:
- Estabelecimentos de uma metodologia de subfracionamento e extração de proteínas de
superfícies celular para uso em sistemas 2-DE;
-Identificação das proteínas de superfície celular de levedura de Paracoccidioides,
Pb01;
- Caracterização do perfil proteômico das proteínas secretadas pelas formas
leveduriforme e miceliana do fungo Paracoccidioides, Pb01;
- Análise comparativa das proteínas diferencialmente expressas entre as formas
leveduriforme e miceliana do fungo;
- Avaliar o papel das proteínas secretadas na interação do fungo com o hospedeiro.
21
4- MANUSCRITO
Analysis of the Secretomes of Paracoccidioides Myceliaand Yeast CellsSimone Schneider Weber, Ana Flavia Alves Parente, Clayton Luiz Borges, Juliana Alves Parente,
Alexandre Melo Bailao, Celia Maria de Almeida Soares*
Laboratorio de Biologia Molecular, Instituto de Ciencias Biologicas, Universidade Federal de Goias, Goiania, Goias, Brazil
Abstract
Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability ofpathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment isimportant for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. Toanalyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used aproteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionizationquadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spotswere detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoformswere indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silicoanalyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. Wealso investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. Theaddition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by bothParacoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatantto the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, theproteins detected in the fungal secretome were also identified within macrophages. These results indicate thatParacoccidioides extracellular proteins are important for the fungal interaction with the host.
Citation: Weber SS, Parente AFA, Borges CL, Parente JA, Bailao AM, et al. (2012) Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells. PLoSONE 7(12): e52470. doi:10.1371/journal.pone.0052470
Editor: Robert A. Cramer, Geisel School of Medicine at Dartmouth, United States of America
Received August 9, 2012; Accepted November 13, 2012; Published December 18, 2012
Copyright: � 2012 Weber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work performed at Universidade Federal de Goias was supported by the following grants: Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq) (558923/2009-7, 563398/2010-5, 477962/2010-6 and 473277/2011-5), Financiadora de Estudos e Projetos (FINEP) (0107055200), Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPES) (PNPD 024979/09-5) and Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG). SSW is therecipient of a CNPq PhD fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
The Paracoccidioides genus represents the causative agent of
paracoccidioidomycosis (PCM), one of the most frequent systemic
mycoses that affect rural populations in Latin America [1]. The
genus comprises four phylogenetic lineages (S1, PS2, PS3 and
Pb01-like) [2,3]. The phylogenetic analysis of many Paracoccidioides
isolates has resulted in the differentiation of the genus into two
species, P. brasiliensis, which represents a complex of three
phylogenetic groups and P. lutzii, which includes the Pb01-like
isolate [4,5].
Paracoccidioides grows as a yeast form in the host tissue and in
culture at 36uC, while it grows as mycelium in the saprobic
condition and in culture at room temperature (18–23uC) [6]. As
the dimorphism is dependent on temperature, when the mycelia/
conidia are inhaled into the host lungs, the transition of the
mycelia to the pathogenic yeast phase occurs [7].
The ability of the pathogenic fungi to develop a multifaceted
response to the wide variety of stressors found in the host
environment is of extreme importance for the virulence and
pathogenesis [8]. Many of those molecules are extracellular
factors, which are either secreted or associated with the fungal
cell wall. The secreted proteins perform important functions, such
as the provision of nutrients, cell-to-cell communication, and
detoxification of the environment and the killing of potential
competitors [9–11].
In eukaryotic cells, the classical secretory pathway of proteins is
driven by a canonical N-terminal signal peptide. This classical
pathway involves the recognition of a signal sequence in the
proteins to be exported, which results in their translocation across
the endoplasmic reticulum (ER) membrane and delivery to the
Golgi apparatus [12]. Functional proteins lacking predicted signal
peptides are secreted into the extracellular medium, thereby
suggesting the existence of unconventional mechanisms of protein
secretion in eukaryotes [9,13,14]. A repertoire of hypothetical
mechanisms for driving proteins that lack an N-terminal secretion
signal through the plasma membrane to the outside of the cell has
been described for Candida albicans and Saccharomyces cerevisiae.
These mechanisms include: passive transport, translocation,
substrate-specific recognition and the affinity of some proteins to
secretory vesicles, which lead to adhesion or internalization in
endosomal sub compartments [9]. In this later mechanism, which
is described as nonconventional export, the formation of the
exosomes is required, which involves vesicles derived from
membrane invagination (endosomes) resulting in the release of
internal vesicles to the extracellular environment [15–17]. Studies
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have demonstrated that several fungi produce extracellular vesicles
containing key molecules associated with virulence, stress response
and vesicular transport [18–20]. The extracellular vesicles
produced by Cryptococcus neoformans and Histoplasma capsulatum are
used for the delivery of molecules associated with pathogenesis to
the extracellular space. This group of molecules includes well-
known virulence factors, such as enzymes associated with capsule
synthesis in C. neoformans, laccase, acid phosphatase, heat shock
proteins and several antioxidant proteins, including superoxide
dismutase, thioredoxin and catalases. These proteins are recog-
nized by the sera of patients with cryptococcosis and histoplas-
mosis, thereby suggesting that these proteins are produced during
human infection [18,19]. Additionally, the co-incubation of
cryptococcal extracellular vesicles with murine macrophages
results in a dose-dependent stimulation of nitric oxide production
by phagocytes and an increase in extracellular tumor necrosis
factor alpha (TNF-a), interleukin-10 (IL-10) and transforming
growth factors b (TGF-b) levels [21]. These findings indicate that
the extracellular vesicles of C. neoformans are biologically active and
can stimulate macrophage function, thereby activating these
phagocytic cells to enhance their antimicrobial activity. Taken
together, these data suggest that fungal secretory vesicles possess
the potential to influence the interaction between C. neoformans and
the host cell.
Our group had described extracellular proteins in Paracoccidiodes.
Proteins lacking predicted signal peptides, such as enolase, have
been shown to be secreted by Paracoccidioides into the extracellular
medium [22]. Additionally, formamidase activity has been
detected in Paracoccidiodes cell-free extracts [23]. An aspartyl
protease has been reported in Paracoccidioides culture supernatants
[24], and a serine protease, which depicted increased levels of
transcript during nitrogen starvation, has also been identified in
Paracoccidioides culture supernatants, thereby indicating the poten-
tial function of this protein in fungus nitrogen acquisition [25].
The Paracoccidioides serine protease transcript is induced in yeast
cells infecting murine macrophages [25] and during the incubation
of yeast cells with human plasma [26], thereby suggesting that the
protein plays a putative role in Paracoccidioides interaction with the
host cell.
In a recent study, it has been shown that extracellular vesicles in
Paracoccidioides yeast cells, Pb18, carry antigenic molecules (a-
galactopyranosyl epitopes) that are recognized by the sera of PCM
patients [20]. The vesicle and vesicle-free fractions were used to
identify the extracellular proteome via LC/MS [27]. Eighty-five
proteins were identified exclusively in the vesicle fractions
compared with 140 proteins that were detected solely in the
vesicle-free fractions, for which 120 sequences displayed overlap in
both fractions. The authors described 75 extracellular proteins
that were common to Paracoccidiodes, Pb18, and at least two of the
other analyzed fungal species.
In the current study, we identified the most abundant
constituents of the extracellular proteome in mycelia and
Paracoccidioides, Pb01, yeast cells. We also identified proteins
differentially secreted by mycelia and yeast cells and investigated
the influence of protein export inhibition on the phagocytic ability
of macrophages in an attempt to further understand the role that
extracellular proteins play in the establishment and pathogenesis of
the PCM.
Materials and Methods
2.1. Microorganism and culturing conditionsP. lutzii, Pb01, (ATCC MYA-826) was used in the experiments
conducted in this study. The cells were cultured in Fava Netto’s
medium [0.3% (w/v) proteose peptone, 1% (w/v) peptone, 0.5%
(w/v) meat extract, 0.5% (w/v) yeast extract, 4% (w/v) glucose,
and 0.5% (w/v) NaCl, pH 7.2] at 36 C and 22uC, for yeast cells
and the mycelia phase, respectively. Yeast cell viability and growth
were evaluated in triplicate every 6 hours. A trypan blue dye
exclusion test was performed to ensure over 95% cell viability
during the incubation process.
2.2. Preparation of extracellular protein extractsThe yeast and mycelia extracellular proteins were prepared by
inoculating 50 mg/mL of wet weight cells in Fava Netto’s liquid
medium, and the cells were maintained while shaking (200 rpm)
for 24 hours at 36uC and 22uC, respectively. After incubation, the
cells were removed by centrifugation at 10,000 g for 30 min at
4uC. The culture supernatant was sequentially filtered through
0.45 mm-pore and 0.22 mm-pore membrane filters. Culture
filtrates were concentrated and subsequently washed three times
with ultrapure water via ultracentrifugation through a 10-kDa
molecular weight cut off in ultracel regenerated membrane
(Amicon Ultra centrifugal filter, Millipore, Bedford, MA, USA).
The protein concentrations were determined via Bradford assay
[28]. For proteomic analysis, three biological replicates of protein
samples obtained from three different experiments were per-
formed for yeast cells and mycelia. The three yeast and mycelia
cell-free supernatant samples were assessed for the presence of
Paracoccidioides DNA via PCR, which if positive indicated fungal
cellular lyses in the samples as described below.
2.3. The Polymerase Chain Reaction (PCR) analysisThe genomic DNA isolated from Paracoccidiodes mycelia and
yeast cells was obtained according to standard protocol [29]. The
PCR reactions were performed with cell-free supernatant (2 mL)
and genomic DNA samples as follow: 40 cycles of 94uC for 30 s,
55uC for 30 s, and 72uC for 1 min. A 1622-bp PCR product was
generated using sense S2 (59- ATGGGTCTCAAGGGAATTC-
39) and antisense At2 (59-CATCCCCTACTTCATTC-39) oligo-
nucleotides for the gene encoding formamidase (GenBank
accession number AY163575). The PCR amplicons were detected
via 1% (w/v) agarose gel electrophoresis with ethidium bromide
staining. PCR sensitivity was assessed using Paracoccidioides Pb01
genomic DNA (at five dilutions) as a template (50 ng to 1 pg).
2.4. Two-dimensional gel electrophoresis (2-DE)For the first dimension, each sample containing 500 mg of total
protein was treated with a 2D-Clean-up Kit (GE Healthcare,
Uppsala, Sweden) according to the manufacturer’s instructions.
The amount of protein (500 mg) was diluted to 200 mL of the final
sample volume and corresponded to 30 g of wet weight cells
(Section 2.2). The proteins samples were diluted in 250 mL of
rehydration solution containing 7 M urea, 2 M thiourea, 2% (w/v)
3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS), 0.002% (w/v) dithiothreitol (DTT), 0.5% (v/v) ampho-
lyte 3–11 and trace amounts of bromophenol blue [30]. These
samples were loaded onto a 13 cm ImmobilineTM DryStrip gel
with a pH linear range of 3–11 in a Multiphor-II Electrophoresis
System (GE Healthcare, Uppsala, Sweden). The samples were
separated according to their isoelectric points at 20uC with a
current of 50 mA/strip. The following program was applied: 30 V
for 14 hours; 500 V for 500 Vh (step); 1 kV for 800 Vh (gradient);
8 kV for 11.3 kVh (gradient) and 8 kV for 2.9 kVh (step). After
isoelectric focusing, the strips were equilibrated twice for 40 min in
equilibration buffer [50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/
v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue]
containing 18 mM DTT and 135 mM iodoacetamide [31]. The
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second dimension (SDS-PAGE) was performed with 12% poly-
acrylamide gels using a vertical system (GE Healthcare) and
standard Tris/glycine/SDS buffer for one hour at 150 V and 250
V until the end of the run at 12uC. The gels were stained with
Coomassie brilliant blue (PlusOne Coomassie Tablets PhastGel
Blue R-350, GE Healthcare) according to the manufacturer’s
instructions.
2.5. Image analysisThe 2-D gel images were obtained using an Image Scanner III
(GE Healthcare). 2D gel spot detection, matching and intensity
calculations were performed with Image Master 2D Platinum v7.0
(GE Healthcare). Three independent samples were prepared for
each fungal phase to ensure reproducibility. The final set contained
six gel images. Software matching between the images was
performed, and the matching process was thoroughly assessed via
visual inspection. The volume percentage of the spots was used for
statistical calculations and the determination of overexpressed
proteins.
2.6. 2D-gel statistical analysisTo compare the differences in protein expression between
Paracoccidioides mycelia and yeast cells the ANOVA test was applied
considering statistically significant p-value #0.05. To compare the
proteins with multiple isoforms, the sum of the percentage of the
volumes (in relation to the total proteins) of each isoform was first
obtained in triplicate. Next, the sum of the percentage of volumes
for the proteins was used for statistical analysis, which was
performed to determine the significant differences in expression
profiles between Paracoccidioides mycelia and yeast proteins. All
statistical calculations were performed using the software STA-
TISTICA version 7.0. (Statsoft Inc., 2005). The spectrometry
analyses were performed on spots displaying significant alterations
($ fold change of 2.5) between yeast and mycelia samples and
spots displaying similar expression levels (common spots).
2.7. Mass spectrometry analysisSpots of interest were manually excised and digested as
previously described [32,33]. Briefly, the gel pieces were
resuspended in 100 mL acetonitrile (ACN) and dried in a speed
vacuum. The gel pieces were then reduced with 10 mM DTT and
alkylated with 55 mM iodoacetamide. The supernatant was then
removed, and the gels were washed with 100 mL ammonium
bicarbonate by vortexing for 10 min. The supernatant was
removed, and the gel pieces were dehydrated in 100 mL of a
solution containing 25 mM ammonium bicarbonate/50% (v/v)
ACN, vortexed for 5 min, and centrifuged. This step was then
repeated once. Next, the gel pieces were dried in a speed vacuum
and 12.5 ng/mL trypsin (sequencing grade modified trypsin,
Promega, Madison, WI, USA) solution was added followed by a
rehydration step performed on ice at 4uC for 10 min. The
supernatant was removed, 25 mL of 25 mM ammonium bicar-
bonate was added and the supernatant was then incubated at
37uC for 16 hours. Following digestion, the supernatant was
placed in a clean tube. Next, 50 mL 50% (v/v) ACN and 5% (v/v)
trifluoroacetic acid (TFA) were then added to the gel pieces. The
samples were vortexed for 30 min, sonicated for 5 min, and the
solution was then combined with the aqueous extraction above.
The samples were dried in a speed vacuum, the peptides were
solubilized in 10 mL ultrapure water, and the samples were
subsequently purified in ZipTipH Pipette Tips (ZipTipsH C18
Pipette Tips, Millipore, Bedford, MA, USA). Two microliters of
each peptide sample were deposited onto a matrix-assisted laser
desorption ionization quadrupole time-of-flight mass spectrometry
(MALDI-Q-TOF MS) target plate and dried at room temperature.
Next, the peptide mixture was covered with 2 mL of matrix
solution (10 mg/ml a-cyano-4-hydroxyciannamic acid matrix in
50% (v/v) ACN and 5% (v/v) trifluoroacetic acid). The mass
spectra were recorded in the positive reflectron mode on a
MALDI-Q-TOF mass spectrometer (SYNAPT, Waters Corpora-
tion, Manchester, UK).
The search against the NCBI non-redundant database using the
MS/MS data was performed using Mascot software v. 2.4 (http://
www.matrixscience.com) (Matrix Science, Boston, USA). The
Mascot MS/MS ion search parameters were as follows: tryptic
peptides with one missed cleavage allowed; fungi taxonomic
restrictions; fixed modifications: carbamidomethylation of Cys
residues; variable modifications: oxidation of methionine; and an
MS/MS tolerance of 0.6 Da. The identified proteins were
described in functional categories according to the MIPS
Functional Catalogue Database (http://fsd.riceblast.snu.ac.kr).
For the identification of the MS spectra using the NCBI database,
we included the analysis of post-translational modifications (PTMs)
for multiple identified proteins/isoforms. We included variable
modifications in the search as follows: the acetylation of lysine and
the phosphorylation of serine/tyrosine/tryptophan. All proteins/
isoforms that presented matches with predicted modified peptides
were selected for manual spectral analysis.
2.8. In silico analysesA number of programs available online were used for the
characterization of the identified extracellular proteins. The
identified proteins were analyzed using SignalP 3.0 software
(http://www.cbs.dtu.dk/services/SignalP/) for the prediction of
signal peptides. For cases when the signal sequence was detected,
the protein was considered to be secreted via a classical pathway.
For the prediction of secreted proteins by non-classical pathways,
the software SecretomeP 2.0 (http://www.cbs.dtu.dk/services/
SecretomeP/) was employed. Using the prediction methods, a
score ranging between 0 and 1 was assigned to each protein in
which a score equal or higher than 0.5 was considered indicative
of secretion.
The Fungal Secretome Database (FSD) (http://fsd.riceblast.
snu.ac.kr) was used to analyze our results. The FSD provides a
summary of putative secretory proteins by in silico analysis based
on prediction programs for 158 fungal/oomycete genomes,
including Paracoccidioides Pb01 [34]. Each protein sequence
identified in this study was also analyzed for adhesin function
using the Faapred web server (http://bioinfo.icgeb.res.in/faap/
query.html) and for GPI anchor properties using big-PI Fungal
Predictor software (http://mendel.imp.univie.ac.at/gpi/fungi/
gpi_fungi.htm). The Faapred web server allows for the prediction
of adhesins in the fungal proteomes using an accurate method with
scores higher than or equal to 0.8 [35]. The big-PI Fungal
Predictor is a sensitive prediction tool used for the recognition of
C-terminal motifs capable of GPI lipid anchoring in fungal
sequences [36].
The ORF sequences of the identified proteins of Paracoccidioides
Pb01 yeast cells were submitted for Blast analysis using the NCBI
website (http://www.ncbi.nlm.nih.gov/) against orthologues pre-
viously reported for Paracoccidioides Pb18 [27] as well the secretomes
of the pathogenic fungi Histoplasma capsulatum [10,18], Cryptococcus
neoformans [19] and Aspergillus fumigatus [37].
2.9. Enzymatic activity assayTo determine the activity of formamidase (FMD), ammonia
formation was assessed as previously described [23]. Protein
extracts were obtained as described in Section 2.2. Next, 1 mg of
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total protein extract was added to 200 mL of 100 mM formamide
substrate solution in 100 mM phosphate buffer containing 10 mM
EDTA, pH 7.4. The samples were incubated at 37uC for 30 min.
Next, 400 mL phenol-nitroprusside and 400 mL alkaline hypo-
chlorite were added to the samples. The samples were then
incubated for 6 min at 50uC, and the absorbance was measured at
625 nm. The amount of ammonia released for each sample was
determined using a standard curve. One unit (U) of FMD activity
was defined as the amount of enzyme required to hydrolyze 1
mmol formamide per mg total protein per minute (mmol/mg/min).
The levels of superoxide dismutase (SOD) and glutathione S-
transferase (GST) activity were measured using commercially
available kits (Superoxide dismutase Assay Kit and Glutathione-S-
Transferase (GST) Assay Kit, Sigma-Aldrich, Co., St. Louis, MO,
respectively). The SOD Assay Kit includes Dojindo’s highly water-
soluble tetrazolium salt (WST-1), which produces a water-soluble
formazan (WST-1 formazan) dye upon reduction with a superox-
ide anion. The reaction product (WST-1 formazan) absorbs at
440 nm, and it is proportional to the amount of superoxide anion.
The levels of SOD activity, which were defined as inhibition rate
%, were quantified by measuring the decrease in the color
development at 440 nm. The GST Assay Kit employs 1-Chloro-
2,4-dinitrobenzene (CDNB) to produce GS-DNB by conjugation
of the thiol group of glutathione (GSH). The reaction product (GS-
DNB) absorbs at 340 nm, and the rate of increase in the
absorption is directly proportional to the GST activity of the
sample. The GST-specific activity is defined as mmol of GS-DNB
per mg of total protein per minute (mmol/mg/min). The
enzymatic activity results represent the mean of three independent
determinations, and statistical comparisons were performed using
the Student’s t test. The samples with p-values #0.05 were
considered statistically significant.
2.10. Brefeldin A treatments and Paracoccidioidesinternalization by macrophages
Brefeldin A (BFA, Sigma), was used to evaluate the role of
conventional protein secretion on Paracoccidioides internalization by
macrophages. Stock solutions (1 mg/mL) of Brefeldin A (BFA)
were prepared in methanol and stored at 220uC until use. The
yeast cells were cultivated in Fava Netto’s liquid medium in either
the absence or presence of BFA (6 mg/mL) for 6, 12 and 24 hours.
The cell-free supernatant samples were obtained as described
above, reduced to an equal final volume (1 mL), and processed for
one-dimensional electrophoresis (SDS-PAGE). Next, 30 mL of
each cell-free supernatant sample was loaded onto a 12% SDS
PAGE gel, and the proteins were separated via electrophoresis.
The gels were run at 150 V for approximately 2 hours and
visualized using Coomassie brilliant blue staining.
Bone marrow-derived macrophages (BMM) were obtained as
previously described from 6 to 8-week-old BALB/c male mice
[38]. The macrophages were generated by extracting bone
marrow cells from the femurs of BALB/c male mice, and the
cell cultures were incubated at 37uC with 5% CO2 in RPMI
medium (RPMI 1640, Vitrocell, Brazil) supplemented with 10%
(v/v) fetal bovine serum and GM-CSF (Recombinant Murine
Granulocyte Macrophage Colony Stimulating Factor, PeproTech-
Brasil FUNPEC, Brazil) at 10 ng/mL for 8–10 days, which
prompts differentiated macrophages to adhere to the plastic-
bottom plates.
To determine the number of adhered/internalized fungi cells by
BMM, the differentiated macrophages were quantified and plated
at 56105 cells per well on glass coverslips in 6-well culture plates
and infected with Paracoccidioides yeast cells at a 1:5 ratio
macrophage: yeast. The cells were co-cultivated for 12 hours at
37uC in 5% CO2 to allow for fungi adhesion and/or internali-
zation. The supernatants were then aspirated, the monolayer was
gently washed twice with PBS 1X to remove any non-adhered/
internalized yeast cells, and the samples were processed for
microscopy. The glass coverslips were fixed with methanol and
stained with Giemsa (Sigma). A total of 200 macrophages were
quantified to determine the average number of adhered/internal-
ized fungal cells [39].
The number of viable fungi after co-cultivation with macro-
phages was determined by quantifying the number of colony
forming units (CFUs). The cells were rinsed twice with PBS 1X to
remove any non-internalized yeast cells. The wells were washed
with distilled water to promote macrophages lysis, and the
suspensions were collected in individual tubes. Fifty microliters
of cell homogenate was plated in BHI medium supplemented with
5% (v/v) fetal bovine serum and incubated at 37uC in 5% CO2 for
15 days. The number of CFUs was expressed as the mean value 6
the standard deviation. A portion of this macrophage homogenate
was processed for immunoblot analysis. The experiments were
performed in triplicate, and the statistical analyses were performed
using the Student’s t test.
BFA treatment was performed to evaluate the role of
extracellular proteins in the adhesion/internalization of Paracoc-
cidioides by macrophages. Paracoccidioides yeast cells were cultivated
in Fava Netto’s liquid medium in either the absence (control) or
presence of BFA (6 mg/mL) for 24 hours prior to the infection of
macrophages. The effect of Brefeldin on macrophages was
evaluated by adding BFA posteriorly to the co-culture of
macrophages with fungal cells (not in the preliminary culture of
Paracoccidioides cells) as an additional control (data not show). To
test the biological activity of the secreted proteins released by
Paracoccidioides yeast cells, we evaluated whether the addition of
concentrated culture supernatant influenced the adhesion/inter-
nalization of Paracoccidioides by macrophages. We added 30 mL of
extracellular protein extract of Paracoccidioides yeast cells (2 mg/mL)
to the macrophage and fungal cell co-culture. The number of
adhered/internalized yeast cells by BMM and the survival rate
(viable fungi after co-cultivation) were determined. The results
were compared with the control (absence of extracellular proteins
and BFA). All animal work was conducted in accordance with the
international rules for animal experimentation. The animal
protocol was approved by the Universidade Federal de Goias
ethical committee of animal treatment (Number: 192/2011).
2.11. Sample preparation and nanoUPLC-MSE acquisitionIn vitro cultured macrophages were infected with Paracoccidioides
Pb01 yeast cells, and co-cultivated for 12 hours at 37uC in 5%
CO2. The infected and non-infected macrophages (BMM) were
incubated in distilled water to promote macrophages lysis and
centrifuged at 10,0006 g for 5 min to separate the macrophage
cytosol from the pellet containing host nuclei, cytoskeleton, and
Paracoccidioides yeast cells. The macrophage lysate supernatant was
sequentially filtered through a 0.22 mm-pore-size membrane filter,
washed three times with ultrapure water by ultracentrifugation
using a 10-kDa molecular weight cut off in ultracel regenerated
membrane (Amicon Ultra centrifugal filter, Millipore, Bedford,
MA, USA), and concentrated to a final volume of 1 mL. The
protein concentrations were determined via Bradford assay [28].
To analyze the expression profile of the Paracoccidioides secreted
proteins with in vitro cultured macrophages, the samples (obtained
as described above) were analyzed using nanoscale liquid
chromatography coupled with tandem mass spectrometry. Sample
aliquots (100 mg) were prepared for nanoLC-MS/MS as previ-
ously described [40]. The digested peptides were further separated
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via nanoUPLC-MSE and analyzed using a nanoACQUITYTM
system (Waters Corporation, Manchester, UK). The MS data
obtained via UPLC-MS were processed and examined using the
ProteinLynx Global Server (PLGS) version 2.4 (Waters Corpora-
tion, Manchester, UK). For protein identification and quantifica-
tion level analysis, the observed intensity measurements were
normalized with the identified peptides of the digested internal
standard.
2.12. Immunoblot analysis of proteins secreted byParacoccidioides in macrophages
For immunoblot analysis, the same samples used for nanoUPLC
mass spectrometry analysis were probed using triosephosphate
isomerase and enolase antibodies [22,41]. Thirty micrograms of
protein sample was loaded onto a 12% SDS-PAGE gel and
separated by electrophoresis. The gels were run at 150 V for
approximately 2 hours. The proteins were transferred from the
gels to nitrocellulose membranes at 30 V for 16 h in a buffer
containing 25 mM Tris-HCl (pH 8.8), 190 mM glycine and 20%
(v/v) methanol. The gels were stained with Ponceau red to verify
complete protein transfer. Next, each membrane was incubated in
blocking buffer (1X PBS, 1.4 mM KH2PO4, 8 mM Na2HPO4,
140 mM NaCl, 2.7 mM KCl (pH 7.3), 5% (w/v) nonfat dried
milk, and 0.1% (v/v) Tween 20) for 2 h. The membranes were
washed with buffer (1X PBS and 0.1% (v/v) Tween 20) and
incubated with primary antibodies for 2 h at room temperature.
The primary antibodies were used at a dilution of 1 antibody/
40000 buffer (v/v). The primary polyclonal antibody employed
was either anti-enolase [22] or anti-triosephosphate isomerase
[41]. The membranes were then washed in blocking buffer three
615 min. The membranes were incubated with the appropriate
conjugated secondary antibody, either a horseradish-peroxidase
conjugated anti-rabbit or anti-mouse IgG, at a 1/5000 (v/v) ratio,
and the blots were developed using an enhanced chemiolumines-
cence detection system (ECL, GE Healthcare).
Results
3.1. Validation of the extracellular protein extractionmethod
Viability analysis was performed with the yeast cells. Using
trypan blue staining, we observed yeast cell viability over 95%
during a 24-h cultivation in liquid medium (Figure 1A). To
standardize sample acquiring, PCR analysis was performed to
assess for the Paracoccidioides formamidase gene. The sensitivity of
the PCR assay was assessed using FMD specific primers with a
serial dilution of the genomic DNA sample (ranging from 50 ng to
1 pg). The smallest quantity of DNA that was amplified by PCR
was 5 pg (Figure 1C, lane 4). This result implies that technique is
able to amplify the FMD gene with only 5 pg DNA present in the
cell-free supernatant. As shown in Figure 1D, the FMD gene was
not detected in the yeast and mycelia cell-free supernatants. These
data both contribute to the hypothesis that the cell lyses failed to
influence the extracellular protein profiles of the extracts obtained
for proteomic analysis and validate the extraction method of
extracellular proteins expressed by Paracoccidioides. Additionally,
none of the secreted had motifs for GPI-anchor (data not shown),
as evidenced by in silico, suggesting that the presence of the
proteins in the secretome was not due to the accidental release
from the fungal surface.
3.2. Analysis of secreted proteins in Paracoccidioides Pb01yeast cells and mycelia
In this study, we applied a proteomic strategy to identify
proteins constitutively secreted by Paracoccidioides Pb01 and
analyzed the proteins preferentially secreted by yeast cells and
mycelia. Figure 2 depicts the representative two-dimensional gel of
both phases performed in biological triplicates. Proteins were
distributed in a molecular mass range from 14.10 to 105.25 kDa
and an experimental pI value ranging from 3.76 to 10.18 was
observed. The figure displays the presence of constitutive and
differentially expressed proteins in both secretomes.
Figure 3 depicts the graphic summation of the proteomic
analysis, which revealed an average of 356 spots for the mycelia
and 388 spots for the yeast secretome, which were matched and
assessed for statistical significance using an ANOVA test to
compare the differences in protein expression between the two
fungal phases. After performing the statistical analysis, it was
determined that 123 and 146 spots (i.e., proteins) were differen-
tially secreted by mycelia and yeast cells, respectively (Figure 3A).
Coomassie G-250 Blue Stained maps were used for MS
identification of the individual protein spots. A total of 160
proteins/spots were identified unambiguously (displayed in
Table S1), which displays all proteins and isoforms identified in
the secretome analysis. The identified proteins/isoforms were
analyzed using several prediction programs. While not all secreted
proteins/isoforms have canonical signal peptides, 20 of the 160
(12.5%) contained a potential N-terminal secretion signal (Mean S
score $0.5, Table S1). The proteins/isoforms that presented the
signal peptide consensus included enzymes such as glycosyl
hydrolase, beta glucosidase, enoyl-CoA hydratase, aminopeptidase
and disulfide isomerase. Using the Secretome P algorithm, 84 of
160 (52.5%) proteins/isoforms were predicted to be secreted
molecules, including aconitase, enolase, fructose-biphosphate
aldolase, 2-methylcitrate synthase, glyceraldehyde-3-phosphate
dehydrogenase, dipeptidyl-peptidase, thioredoxin-like proteins
and peroxisomal catalase.
Regarding the proteins identified in the secretomes, fungal
adhesin prediction analysis using the Faapred web server revealed
that approximately 21% of the Paracoccidioides secretome (33
proteins/isoforms) was predicted to be adhesin-like proteins, such
as enolase, glyceraldehyde-3-phosphate dehydrogenase, and
triosephosphate isomerase (Table S1). All identified proteins/
isoforms were grouped into Enzyme Classification (EC) according
to guidelines of the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (NC-IUBMB) as
listed in Table S1. More than half of the Paracoccidioides Pb01
secretome comprises enzyme-like proteins (64%), and the most
prevalent enzyme classes found were oxidoreductases (17%),
transferases (17%), and hydrolases (14%) as depicted in Figure S1.
3.3. Constitutive proteins in the secretome ofParacoccidioides Pb01
In this report, we described the proteins constitutively present
in the secretome of Paracoccidioides Pb01 mycelia and yeast cells.
For this analysis, the isoforms of each protein were combined to
perform the statistical analysis. Twenty eight proteins were
constitutive to mycelia and yeast secretomes (Figure 3B). Most of
these proteins were grouped into the functional category of
metabolism, energy and protein fate. The enzymes in the energy
category, including enolase, fructose-bifophosphate aldolase,
glyceraldehyde-3-phosphate dehydrogenase and phosphogycerate
kinase, were abundant in both secretomes as depicted in Table 1.
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3.4. Extracellular proteins upregulated in ParacoccidioidesPb01 mycelia
A comparative analysis was performed between the extracellular
protein profiles of mycelia and yeast cells. Based on protein
categorization, 30 proteins were preferentially secreted by mycelia
compared with yeast cells (Figure 3B and Table 2). The proteins
identified are implicated in a variety of biological processes, such
as cell rescue, cell defense, cell virulence, metabolism, energy, cell
cycle, DNA processing, protein fate and protein synthesis (Table 2).
Proteins that play a role in cell rescue, defense and virulence
include the heat shock protein SSC1, disulfide isomerase,
glutathione reductase, peroxisomal catalase, thioredoxin reductase
and the TCTP family of proteins. Enzymes involved in the
metabolism of amino acids were abundant in the mycelia
secretome. Some of the proteins expressed in the mycelia
secretome were significantly upregulated compared with the yeast
cells, including the Cofilin/tropomyosin-type actin-binding family
of proteins (fold change of 1911.74), peroxisomal catalase (fold
change of 61.29), glutamate carboxypeptidase (fold change of
51.03), aconitase (fold change of 56.98) and heat shock protein
SSC1 (fold change of 35.47) (Table 2). Interestingly, several
proteins were detected exclusively in the mycelia secretome,
including detoxification proteins such as thioredoxin reductase and
disulfide-isomerase tigA, proteins that play a role in the
metabolism of amino acids, and proteins involved in energy
production. The proteins expressed exclusively in the secretome of
mycelia (compared with the yeast phase) are summarized in
Table S2.
3.5. Extracellular proteins upregulated in ParacoccidioidesPb01 yeast cells
When comparing the secretome of mycelia and yeast cells, 24
proteins were identified that are preferentially secreted by yeast
cells into the extracellular environment as depicted in Table 3
and Figure 3B. The differentially expressed proteins displayed
fold changes ranging from 2.72 to 70672.37 when compared
with mycelia. Most of these proteins play a role in cell rescue,
cell defense, cell virulence, cell cycle, DNA processing, and
energy production (Table 3). The proteins that play a role in cell
rescue, defense and virulence, including heat shock protein 60
(fold change of 6.20), Hsp90 binding co-chaperone (fold change
of 20.97), disulfide isomerase Pdi1 (fold change of 5.76),
superoxide dismutase (fold change of 2.72), thioredoxin-like
protein (fold change of 8.40) and hsp70-like protein (fold change
of 7.91), were more abundant in the yeast secretome. The 2-
methylcitrate synthase protein was significantly expressed in the
yeast phase secretome with a fold change of 70672. Interestingly,
several proteins were detected exclusively in the yeast phase,
including glutathione S-transferase, energy production-associated
enzymes such as pyruvate kinase and phosphoglycerate mutase,
proteins that play a role in cell cycle and DNA processing, and
proteins involved in cellular transport such as the vesicular-fusion
protein, SEC17. The proteins expressed exclusively in the yeast
Figure 1. Validation of the extracellular protein extraction method. A- The viability of Paracoccidioides yeast cells incubated in Fava Netto’sliquid medium (dark gray square) and the incubation of yeast cells in Fava Netto’s liquid medium containing 6 mg/mL Brefeldin A (light gray square).Viability was assessed using trypan blue staining. The error bars represent the standard deviation of three biological replicates. B- The growth ofParacoccidioides yeast cells in liquid medium in either the absence (dark line) or presence of 6 mg/mL Brefeldin A (light gray line). Culture growth wasevaluated by quantifying the number of yeast cells per mL. The error bars represent the standard deviation of three biological replicates. C- PCRsensitivity for the formamidase gene was assessed using Paracoccidiodes Pb01 genomic DNA (at five dilutions) as a template (50 ng to 1 pg). Lanes: 1250 ng; 2 25 ng; 3 250 pg; 4 25 pg; 5 21 pg; 6 - negative control (without genomic DNA). The formamidase PCR amplicons were assessed via 1%agarose gel electrophoresis and stained with ethidium bromide. D- The yeast and mycelia cell-free supernatant samples (2 mL) were assessed for thepresence of Paracoccidiodes DNA via PCR using oligonucleotides specific for the formamidase gene.doi:10.1371/journal.pone.0052470.g001
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secretome (compared with the mycelia) are summarized in
Table S2.
3.6. Enzymatic activity correlates with proteomic dataTo validate the significance of the proteomic results, enzymatic
assays were performed with extracellular extracts for formami-
dase (FMD), superoxide dismutase (SOD) and glutathione S-
transferase (GST). The differences observed with the 2D-gel
analysis correlate with the enzymatic assays (Figure 4). The
protein levels and enzymatic activity of FMD was increased in
the mycelia secretome compared with the yeast phase as depicted
in Table 2 and Figure 4, panel A. SOD and GST presented
higher levels of activity in the yeast parasitic phase (Table 3 and
Figure 4, panels B and C, respectively). For all the analyzed
enzymes, the activity levels correlated with the proteomics data
(Tables 2 and 3).
3.7. Protein isoforms and post-translational modificationsThe 160 proteins/isoforms identified in the secretome of
Paracoccidioides Pb01 are encoded by 86 different genes. Protein
isoforms were identified for 37 of these gene products as depicted
in Table S1. This observation suggests that many secreted proteins
undergo post-translational modification. Table S3 presents the
predicted post-translational modifications for the proteins identi-
fied in the mycelia and yeast secretomes. Hsp-70 like protein, heat
shock SSC1, and 2-methylcitrate synthase represent the molecules
displaying the highest number of identified isoforms (seven, eight
and nine, respectively). The presence of the same protein
displaying differential relative migration with 2D analysis may
be associated with post-translational modification (PTM). To
predict the putative PTMs, which explain the high isoform
frequency, the most common S-T phosphorylation and lysine
acetylation was included as a variable modification in the
MASCOT search. The predicted PTMs were confirmed via
manual spectral analysis as previously described [33]. In spite of
the limitations associated with the MASCOT search for the PTM
study, this strategy has been used previously to perform such
analysis in organisms [42]. We observed an increase in peptide
matches and sequence coverage when using those search criteria.
Table S3 presents the predicted post-translational modifications
for proteins in the mycelia and yeast secretomes. A total of 116
isoforms were analyzed using such criteria, and peptides displaying
phosphorylation, acetylation or both PTMs were detected for 44
isoforms (Table S3). Serine/threonine phosphorylation was ob-
served for 31 isoforms, while acetylation was observed for 30
isoforms (Table S4). The spectral analysis of isoform 46 of the heat
shock protein, SSC1, revealed twelve phosphorylation and six
acetylation sites. Many isoforms identified in the secretome
displayed similar molecular weights and different pIs shifts. In
contrast, a series of isoforms differed in both molecular weight and
pI. This observation may be due to other PTMs, such as
glycosylation, which were not analyzed because of the high
complexity spectrum of the structures that glycan branches assume
[43]. Further studies using analytical tools more appropriate for
PTM analysis are required to confirm these putative findings.
3.8. Conventional and Non-conventional proteinsecretion by Paracoccidiodes
The in silico analysis indicated that 65% of the Paracoccidioides
secretome included proteins/isoforms with known classical or non-
classical secretion signals and that are actively exported to the
extracellular space. Using the SignalP program, we found that
12.5% of the extracellular proteins/isoforms displayed putative
signal peptide sequences (Table S1). In contrast, 52.5% of the
identified extracellular proteins/isoforms were predicted, using the
SecretomeP program, to be secreted by mycelia and yeast via a
Figure 2. Proteins detected in the secretome of yeast cells and mycelia of Paracoccidiodes via 2D-gel analysis. Protein profile generatedafter the separation of the secreted fraction of proteins by yeast cells (A) and mycelia (B) using 2D-eletrophoresis (first dimension: IEF pH range 3 –11non-linear, second dimension: 12% (w/v) SDS-PAGE) and visualized using Coomassie brilliant blue staining. The 2-D gel images of three biologicalreplications of each phase were compared to identify the differential expression levels of proteins using Image master 2D Platinum software. Theprotein spots that were identified via MS/MS are numbered and listed in Table S1. The pH gradient is shown above the gel, and the molecular massprotein standards (kDa) are indicated to the left of the gels.doi:10.1371/journal.pone.0052470.g002
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non-classical mechanism. Proteins/isoforms that were not pre-
dicted to be secreted by yeast and mycelia represented 56 of the
160 identified spots (35%) (Table S1). These data are in agreement
with those reported by the Fungal Secretome Database (FSD),
which describes that 58% of the 9,136 proteins expressed by the
Paracoccidioides Pb01 genome are predicted to be Class NS (non-
classically secreted), while 14% display a classical secretion signal
(Class SP) and 28% of the proteins have not been classified
(Figure S2).
3.9. Blocking canonical protein secretion inParacoccidioides Pb01 yeast cells influences fungaladhesion/internalization by macrophages
Experiments were performed to investigate the effect of protein
secretion on the adhesion/internalization of Paracoccidioides yeast
cells by macrophages. As demonstrated in Figure 1A, yeast cell
viability was over 90% during 24-hour incubation in the presence
of the protein secretion inhibitor, Brefeldin A (BFA). Additionally,
the growth rate of yeast cells in the presence of BFA was not
altered during the incubation as depicted in Figure 1B. The yeast
cell protein profile upon incubation with BFA was analyzed via
SDS-PAGE using equal volumes of each extract (30 mL). As
depicted in Figure 5A, BFA reduced the levels of proteins secreted
by yeast cells starting from 12 hours with a stronger effect after
24 hours of incubation. Therefore, treatment with BFA at
24 hours was used for the adhesion/internalization experiments
involving Paracoccidioides Pb01 and macrophages.
The number of internalized/adhered Paracoccidioides cells by
macrophages was significantly decreased after yeast treatment with
BFA for 24 hours compared with the control as shown in Figure 5,
panel B. Additionally, the yeast cell treatment with BFA resulted in
a decreased recovery of viable yeast cells in macrophages,
(Figure 5, panel C). This finding reflects the consequence of the
decrease in the number of internalized/adhered Paracoccidioides
cells by macrophages with the addition of BFA, thereby suggesting
that secretion inhibition decreases the association of macrophages
with yeast cells. In contrast, the addition of concentrated culture
supernatant containing Paracoccidioides extracellular proteins result-
ed in an increase in the number of internalized/adhered fungal
cells by macrophages, as well as in increase in the recovery of
viable yeast cells into macrophages (Figure 5, panels B and C,
respectively).
The Brefeldin’s effect on macrophages, which was evaluated by
adding BFA to the co-cultivation, failed to cause a significant
difference in the average number of internalization/adherence
yeast cells by macrophages (data not shown). Figure S3 depicts
microscopy analysis of Paracoccidioides adhesion and internalization
by macrophages, which was used to determine the average
number of internalized/adhered fungi cells in the control (panels A
and B), in the presence of BFA (panels C and D) and in the
presence of concentrated culture supernatant (panels E and F).
The BFA treatment reduced the number of yeast cells adhered/
internalized by macrophages, while the addition of extracellular
proteins increased the rate of phagocytosis.
3.10. Paracoccidioides Pb01 yeast cell secreted proteinsidentified in macrophages
A nanoLC-MS/MS-based proteomics approach was employed
to identify Paracoccidioides Pb01 yeast cell secreted proteins in in vitro
cultured macrophages. A total of 18 Paracoccidioides proteins were
identified in the cytoplasm of infected macrophages (Table 4),
including enolase, heat shock proteins, translation elongation
factors, DNA damage checkpoint proteins, and peptidyl-propyl
cis-trans isomerase D. No Paracoccidioides proteins were identified in
the negative control.
3.11. Immunoblot analysis of macrophage lysatesWe aimed to determine whether Paracoccidioides expresses
constituents of the extracellular proteome during infection in
cultured macrophages by examining the secretion of candidate
proteins via immunoblot analysis. The immunoblot analysis of
macrophage lysates revealed that Paracoccidioides yeast cells secrete
enolase (PbEno) and triosephosphate isomerase (PbTpi) in infected
macrophages (Figure Suplementary 4, line 2, panels A and B,
respectively). The pre-incubation of Paracoccidioides yeast cells with
Brefeldin A, a protein secretion inhibitor, displayed a lack of
PbEno and PbTpi expression in macrophages (Figure Suplemen-
tary 4, line 1, panels A and B, respectively).This finding is most
likely due to the reduced number of internalized/adhered
Paracoccidioides cells by macrophages during BFA treatment as
Figure 3. Graphic summation of proteomics analysis. A- Thecomparative analysis using Image master 2D Platinum software displaysthe analyzed spots and the preferentially expressed spots in myceliaand yeast secretomes, while the mass spectrometry analyses displaysthe identified differentially expressed spots. B- The Venn diagramshows the number of identified proteins via MS/MS. The preferentiallysecreted extracellular proteins include those with statistically significantalteration in the mycelia and yeast secretome, while the constitutiveproteins refer to those with similar expression patterns.doi:10.1371/journal.pone.0052470.g003
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Table 1. Constitutive proteins secreted by Paracoccidioides Pb01 yeast and mycelia.
General Informationnumber (NCBI) 1 Protein description
Number of isoformsin Paracoccidioidessecretome 2
Amountof isoformabundances 3
ANOVA(p-value) 4
SignalPScore $0.5 5
SecretomePScore $0.5 6
1. CELL RESCUE, DEFENSE and VIRULENCE
gi|295659787 heat shock protein Hsp88 1 1.24 0.3645 NO NO
gi|295659837 heat shock protein SSB1 1 2.77 0.0779 NO 0.8618
gi|295669402 Mn superoxide dismutase 1 1.82 0.3780 NO 0.8823
2. METABOLISM
2.1. Amino Acid Metabolism
gi|295667902 aminomethyltransferase 1 1.52 0.2302 NO 0.6367
gi|295658698 fumarylacetoacetase 4 1.50 0.7047 NO 0.7492
gi|295658947 O-acetylhomoserine (thiol)-lyase 1 1.00 0.9268 NO 0.7930
gi|225683737 spermidine synthase 1 1.93 0.0864 NO NO
2.2. Nucleotide Metabolism
gi|295674697 adenosine kinase 2 0.21 0.0646 NO NO
2.3. Secundary Metabolism
gi|295666938 nucleoside diphosphate kinase 1 1.01 0.5725 NO NO
2.4. Phosphate Metabolism
gi|295662360 mannitol-1-phosphate 5-dehydrogenase 3 0.42 0.0766 NO NO
2.5. C-Compound and Carbohydrate Metabolism
gi|295667790 beta-glucosidase 1 1.63 0.2116 0.999 0.8279
gi|295663469 glycosyl hydrolase 1 1.77 0.1350 0.996 0.8631
gi|295665168 TOS1 1 1.00 0.7772 0.999 0.9249
3. ENERGY
3.1. Glycolysis and Gluconeogenesis
gi|295672732 enolase 4 0.67 0.0832 NO 0.5000
gi|295671120 fructose-bisphosphate aldolase 5 398.84 0.0549 NO 0.6628
gi|295658119 glyceraldehyde-3-phosphatedehydrogenase
2 0.76 0.4655 NO 0.9120
gi|295669690 phosphoglycerate kinase 1 1.05 0.3639 NO 0.6626
gi|225678203 NmrA-like family protein 1 1.01 0.5624 NO 0.5898
3.3. Tricarboxylic-acid Pathway
gi|295673937 malate dehydrogenase 1 1.36 0.0834 0.764 0.8163
gi|295668473 dihydrolipoyl dehydrogenase 3 17.92 0.0541 0.571 NO
4. CELL CYCLE AND DNA PROCESSING
gi|295664474 cell division cycle protein 1 1.24 0.2116 NO NO
gi|295658863 Cofilin/tropomyosin-type actin-binding family protein
2 1.91 0.0093 NO NO
gi|295672736 DNA damage checkpoint protein rad24 2 0.86 0.0693 NO NO
5. PROTEIN FATE (folding, modification, destination)
gi|295663907 peptidyl-prolyl cis-trans isomerase A2 1 1.06 0.4941 0.975 0.8963
gi|295672668 peptidyl-prolyl cis-trans isomerase B 2 191.86 0.0920 0.928 NO
gi|295662699 peptidyl-prolyl cis-trans isomerase cypE 1 1.12 0.2723 NO 0.8712
gi|295672447 peptidyl-prolyl cis-trans isomerase H 3 144.1 0.0755 NO 0.7266
gi|295665666 Grp1p protein 1 1.06 0.6021 NO 0.9061
6. UNCLASSIFIED PROTEINS
gi|295667926 conserved protein 1 1.01 0.7060 0.972 0.9280
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showed in Figure S3. The negative control represents the non-
infected macrophage lysate which indicated that those antibodies
did not target mouse proteins (Figure Suplementary 4, line 3).
3.12. Comparative analysis between ParacoccidioidesPb01 extracellular proteins with orthologue proteinsfound in pathogenic fungi secretomes
We compared the ORFs sequences of the extracellular proteins
identified in Paracoccidioides Pb01 yeast cells with the orthologues
previously reported in the secretome of Paracoccidioides Pb18 [27],
H. capsulatum [10,18], C. neoformans [19] and A. fumigatus [37] as
demonstrated in Table S5 and Figure S5. P. brasiliensis (Pb18) and
P. lutzii (Pb01) displayed specific variations in the yeast phase
secretome. A total of 63 proteins/isoforms detected in the
secretome of yeast cells in Pb01 (57%) have also been described
for the secretome of Pb18. Among the common proteins, several
enzymes were identified including those involved in the metab-
olism of carbohydrates and lipids and heat shock proteins.
Additionally, 47 proteins/isoforms, from a total of 110, were not
common to Pb01 and Pb18, thereby suggesting differences in gene
expression/secretion between the two species of Paracocidioides.
Proteins exclusively identified in the secretome of Pb01 yeast cells
(compared with Pb18) included glutathione reductase and gluta-
thione S transferase. Eighty eight identified extracellular proteins
in Paracoccidioides Pb01 (80%) had orthologues that have been
previously described in fungal secretomes (Table S5 and Fig-
ure S5), while 22 extracellular proteins were exclusively found in
the Pb01 yeast cells. We found that 59% of the extracellular
proteins identified in Pb01 (65 proteins/isoforms) had orthologues
in H. capsulatum (Suplementary Figure 5). Among these proteins, 45
proteins/isoforms were also found in Pb18 (Table S5). Concerning
to C. neoformans and A. fumigatus the small number of proteins
described in the secretomes may account to the reduced number
in orthologues to Pb01.
Discussion
In the current study, the secretome profile of Paracoccidioides
Pb01 was described for the first time. Using a proteomic analysis,
160 proteins/isoforms were identified. Additionally, the proteomic
analysis revealed that the secretome consisted of 20 extracellular
proteins/isoforms of the classical secretory pathway and 84
extracellular proteins/isoforms secreted via non-classical secretory
pathways. Secretion involves a vesicular transport mechanism,
thereby suggesting that an exosome-like structure represents a
conserved mechanism for the release of molecules by fungal cells
[17–19]. Moreover, recent results have demonstrated vesicle-
dependent secretion in Paracoccidioides [27].
A total of 28 proteins, which correspond to 47 isoforms (29.37%
of the total identified proteins/isoforms) were described as
constitutively (no difference in protein levels) secreted by
Paracoccidioides Pb01 yeast and mycelia. This group includes
proteins that are involved in cellular processes such stress response,
metabolism, energy and protein fate. Two heat shock proteins/
isoforms were constitutively secreted by mycelia and yeast cells.
Heat shock proteins have also been reported to be involved in the
increase of the export of proteins via non-classical mechanisms in
eukaryotic cells [44–46]. Therefore, we hypothesize that heat
shock proteins may be involved in protein exportation by
Paracoccidioides.
We analyzed the Paracoccidioides Pb01 yeast cells and mycelia
secretomes and performed a comparative analysis of the changes
in protein profiles between the two phases. In the present study,
without any enrichment strategies, 118 and 110 protein/isoforms
(Table S1) were detected in Paracoccidioides mycelia and yeast,
respectively. The proteomic analysis resulted in the identification
of 160 non-redundant proteins/isoforms, of which 98 are
differentially expressed in the two phases.
Formamidase was found to be preferentially secreted by
mycelia. This result was consistent with the enzymatic activity
assay results, as formamidase activity was significantly higher in
mycelia compared with yeast cells. Formamidase was also detected
in H. capsulatum extracellular vesicles [18]. Proteins involved in
amino acid metabolism, such as serine hydroxymethyl transferase,
were preferentially secreted by mycelia. In microorganisms [47]
and plants [48], this enzyme plays an important role in conferring
tolerance to salinity stress by generating amino acids, such as
serine, and ammonium quaternary compounds, such as glycine
betain. These molecules protect secreted proteins and other
molecules against salinity stress by acting as non-enzymatic
chaperones [48]. In Paracoccidioides, this enzyme may play role in
the protection against environmental stress during the mycelia
phase.
Additionally, proteins that play a role in cell defense, such as
peroxisomal catalase, glutathione reductase, thioredoxin reductase
and translationally-controlled tumor protein (TCTP), were iden-
tified in the Paracoccidioides mycelia secretome. The increased
secretion of enzymes involved in cell defense may reflect a need for
increased protection against stress. The mycelia phase of
Paracoccidioides occurs in the soil and can be exposed to many
environmental stresses, such as high temperature and solar
radiation and reactive oxygen species that can emerge from
external sources [49]. Additionally, reports have implicated the
involvement of TCTP in the prevention of cell death by
modulating the anti-apoptotic activity of Mcl-1 [50]. The results
Table 1. Cont.
General Informationnumber (NCBI) 1 Protein description
Number of isoformsin Paracoccidioidessecretome 2
Amountof isoformabundances 3
ANOVA(p-value) 4
SignalPScore $0.5 5
SecretomePScore $0.5 6
gi|295657286 conserved protein 1 1.00 0.7950 NO 0.8050
1NCBI database general information number (http://www.ncbi.nlm.nih.gov/).2Number of identified isoforms of protein in Paracoccidioides, Pb01 secretome.3The average of amount of values of abundances of all identified isoforms used to statistical test.4ANOVA – statistically significant differences are considered with p,0.05 (*).5Secretion prediction according to Signal P 3.0 server, the number corresponds to signal peptide probability (http://www.cbs.dtu.dk/services/SignalP/).6Secretion prediction according to Secretome P 2.0 server, the number corresponds to neural network that exceeded a value of 0.5 (NN-score $0.50) (http://www.cbs.dtu.dk/services/SecretomeP/).doi:10.1371/journal.pone.0052470.t001
The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 10 December 2012 | Volume 7 | Issue 12 | e52470
Ta
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The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 11 December 2012 | Volume 7 | Issue 12 | e52470
Ta
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The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 12 December 2012 | Volume 7 | Issue 12 | e52470
Ta
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3.
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The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 13 December 2012 | Volume 7 | Issue 12 | e52470
suggest the secretion of proteins/enzymes required to protect the
saprobe phase from environmental insults.
Glutathione S-transferase (GST) was found to be preferentially
secreted by Paracoccidioides Pb01 yeast compared with the mycelia
secretome. GST represents a group of detoxification enzymes,
which are involved with protection against oxidative stress and the
detoxification of xenobiotics and heavy metals [51,52]. In
agreement with the proteomic data, the GST activity was
significantly higher in the yeast cell secretome compared with
that of the mycelia. Additionally, superoxide dismutase was
preferentially secreted by Paracoccidioides Pb01 yeast cells. The H.
capsulatum extracellular superoxide dismutase (SOD3) has been
shown to be preferentially expressed by yeast cells compared with
the expression by non-pathogenic mycelia [10]. SOD3 specifically
protects against extracellular reactive oxygen species and facilitates
H. capsulatum pathogenesis by detoxifying host-derived reactive
oxygen species [53]. Proteomic analysis results displayed eight
isoforms of 2-methylcitrate synthase in Paracoccidioides yeast, which
were predicted to be exported via a non-classical secretory
pathway. The statistical analyses revealed 2-methylcitrate synthase
was secreted at significant levels (fold change of 70672.37). This
protein was also previously demonstrated in the extracellular
vesicles of H. capsulatum and Paracoccidioides Pb18 [18,27], although
its function at the extracellular environment had not been
established. DNA damage checkpoint protein (14-3-3 protein
family) was also preferentially expressed by the Paracoccidioides Pb01
yeast secretome. The 14-3-3 protein regulates a diverse range of
cell signaling pathways by forming protein-protein interactions
and modulating the protein function in eukaryotic cells [54].
Moreover, 14-3-3 protein modulates vesicular trafficking and
exocytosis, as displayed in different experimental systems [55,56].
This protein was also detected in the extracellular environment by
the proteomic analysis of various fungi, such as C. neoformans [19],
H. capsulatum [10,18] and Paracoccidioides, Pb18 [27]. Proteins that
function in cell rescue, defense and virulence were more abundant
in the yeast secretome, which included proteins involved in stress
response and detoxification, such as heat shock proteins, disulfide
isomerase Pdi1, glutathione S-transferase Gst3 and superoxide
dismutase. During the infection process by the pathogenic yeast,
the release of reactive oxygen species (ROS) by the immune
effector cells plays an important role in killing microbes [57].
Therefore, the response to stress is likely to be an important
virulence attribute of this pathogenic fungus.
Proteomics studies can be heavily influenced by the growth
conditions. As example, Botrytis cinerea was shown to secrete a wide
array of enzymes and there are significant changes in the relative
abundance and composition of secreted enzymes in a substrate
dependent manner [58]. We have used a carbohydrate-and
peptide rich medium in our studies that could explain the
abundance of some glycolysis and TCA-cycle proteins in the
secretomes of yeast and mycelia. Although this consideration, the
yeast and mycelia phase grown in the same medium secrete
differentially a wide array of proteins/enzymes, suggesting that
those molecules are likely tailored to benefit the saprobe and
pathogenic yeast phase.
Despite its limitations, 2D gel electrophoresis remains a powerful
method for global inspection of post-translational modification, as it
is associated with shifts in protein molecular mass and charge.
Among the secreted proteins identified in Paracoccidioides Pb01
mycelia and yeast cells, 37 appeared in more than one spot on the
2D-gel, thereby totaling 116 identified isoforms (72.5% of the total).
We speculated that some of the pI or molecular mass changes were
caused by PTM. Spectral analysis combined with a PTM
MASCOT search revealed that 39% of the detected isoforms
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The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 14 December 2012 | Volume 7 | Issue 12 | e52470
harbor serine/tyrosine phosphorylation and/or lysine acetylation
sites. Eight of the nine isoforms of 2-methylcitate synthase were
found to be phosphorylated, which correlates with the decrease in
pI. We also identified 30 acetylated proteins out of 116 isoforms, and
all isoforms of the hsp70-like proteins harbored at least five
acetylated peptides. The pI shifts in those molecules correlates with
those observed in proteins derived from breast cancer cell lines, in
which the pI shifts were consistent with the number and type of
modifications [59]. The PTM may be associated with novel
functions of intracellular proteins located outside of the fungal cells.
However, the impact of these modifications on pathogenesis,
environmental adaptation, nutrient uptake and morphological
maintenance/changes has not yet been clarified and would require
a more comprehensive global PTM analysis and more detailed
investigation of the impact of each modification on the function of
each protein modification [42].
Figure 4. Enzymatic activity analysis validates the secretomedata for Paracoccidioides mycelia and yeast cells. Activity assayresults of (A) formamidase (FMD), (B) superoxide dismutase (SOD) and(C) glutathione S-transferase (GST) assessed for mycelia and yeastprotein extracts. FMD activity was assessed by measuring the levels ofammonia released using a standard curve. The SOD and GST Assay Kitwere used to determine SOD and GST enzymatic activity, respectively.The student’s t test was used for statistical comparisons, and theobserved differences were statistically significants (p#0.05). The errosbars represent the standard deviation of three biological replicates.doi:10.1371/journal.pone.0052470.g004
Figure 5. Blocking the conventional protein secretion pathwayleads to a decrease in Paracoccidioides yeast cell phagocytosis.A- The protein profile of the cell-free supernatant samples reveals theeffect of blocking the protein secretion pathway on yeast cells.Paracoccidioides yeast cells were cultivated in Fava Netto’s liquidmedium in either the absence (control) (lanes 1, 3 and 5) or presence ofBrefeldin A (BFA) at 6 mg/mL (lanes 2, 4 and 6) for 6, 12 and 24 hours,respectively. The cell-free supernatant samples were prepared (asdescribed in the Materials and Methods section), reduced to equal finalvolumes (1 mL), and processed for one-dimensional electrophoresis(SDS-PAGE). Thirty microliters of each sample was separated via SDS-PAGE and visualized using Coomassie brilliant blue staining. Thenumbers on the left side correspond to the molecular mass standard. B-The average number of internalized/adhered Paracoccidioides cells bymacrophages was determined. Macrophages were infected withParacoccidioides yeast cells, which were pre-cultivated previouslywithout BFA (control), in the presence of BFA or the presence ofconcentrated culture supernatant containing extracellular proteins (EP).The adhered/internalized cells were analyzed as described in thematerials and methods section. C- The number of viable yeast cells afterphagocytosis by macrophages was evaluated by counting the numberof colony forming units (CFUs). The results are representative oftriplicate biological samples. Statistical significance (* p#0.05) wasdetermined by comparing the results with the control group.doi:10.1371/journal.pone.0052470.g005
The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 15 December 2012 | Volume 7 | Issue 12 | e52470
We investigated the function of conventional protein secretion
in the Paracoccidioides macrophage interaction. The addition of
BFA to the culture medium led to a decrease in secreted
proteins, significantly inhibited the macrophage/fungus interac-
tion (assessed by the adherence and internalization of yeast cells)
and caused a reduction in viable cell recovery from macrophag-
es. Consequently, it may be hypothesized that the inhibition of
macrophage functions might facilitate the survival of the
pathogen within the phagocytic cell [39,60]. The Paracoccidioides
yeast cells is known to both be phagocytosed by and multiply
inside macrophages [61]. The involvement secreted proteins in
the uptake of Paracoccidioides by macrophages has been described
[61]. The gp43, the main antigenic molecule secreted by
Paracoccidioides, participates during the initial steps of attachment
of the fungus to macrophages was demonstrated when polyclonal
antibodies directed to some gp43 epitopes induced a marked
decrease in the phagocytic indexes of macrophages challenged
with Paracoccidioides yeast cells [61]. These data attest the function
of gp43 extracellular proteins in the adherence of the fungus and
uptake by macrophages. Similarly, we observed that the addition
of BFA significantly inhibited the yeast cells internalization by
macrophages. In contrast, the addition of concentrated culture
supernatant significantly increased the interaction of macrophag-
es with yeast cells, thereby suggesting that the secreted proteins
released by Paracoccidioides yeast cells exert some biological
activity. A similar ability has been described for Leishmania-
secreted vesicles during infection, which deliver effectors that
mediate parasite invasion favoring survival in the host [62,63].
Furthermore, Leishmania exosomes and exosomal proteins have
been detected in the cytoplasmic compartment of infected
macrophages [64].
Specialized secretion systems are used by human pathogens to
export virulence factors into host target cells [64–66]. In this
report, we present evidence that Paracoccidioides also releases
effector proteins in macrophages; however, this mechanism
remains elusive. We identified 18 secreted proteins in infected
macrophages, including the protein orthologues heat shock
protein 10, heat shock protein 70, DNA damage checkpoint
protein (14-3-3 protein) and elongation factor 1 alpha (EF-1a),
which have been previously described as exosomal proteins
released into infected macrophages by Leishmania [63,64], thereby
suggesting that these proteins may be effectors present in the host
cytosol after the infection by macrophages has been established.
Moreover, it has been described that the Leishmania protein, EF-
1a, accesses the host cell cytosol and activates multiple host
protein-tyrosine phosphatases (PTPs), which negatively regulate
interferon-c (INF-c) signaling, thereby preventing effective
expression of the macrophage microbicidal arsenal including
TNF-a and nitric oxide (NO) production. Additionally, heat-
shock results in an increase in exosomal vesicles release into the
host cell by Leishmania [67]. Taken together, these results suggest
that Paracoccidioides extracellular proteins may be important for
fungal survival within macrophages. Therefore, we hypothesized
that proteins secreted by Paracoccidioides may facilitate the survival
of the fungus within the host, at least during the initial phase of
infection, thereby reinforcing the concept that secreted proteins
released by pathogenic fungi play a crucial role in pathogenesis
and virulence [11,68]. We hypothesized that the Paracoccidioides
exoproteome may modulate both signaling pathways and
function of the macrophages, thereby creating an environment
permissive for early infection as has been described for other
intracellular pathogens, such as Leishmania [63,67,69]; H.
capsulatum [70,71]; C. neoformans [72]; Candida glabrata [65] and
C. albicans [73].
We found that Paracoccidioides secrete various types of proteins,
including enzymes, heat shock proteins and many conventional
cytosolic factors. Although we cannot completely rule out cellular
lyses, the secretome samples were assessed to ensure that cell
Table 4. Paracoccidiodes, Pb01 yeast cells secreted proteins in macrophages by nanoLC-MS/MS.
General InformationNumber (NCBI)1 Protein description Protein Score
Matchingpeptides Coverage (%)
gi|295666197 2 methylcitrate dehydratase 1.515.417 6 20.88
gi|226291035 ATP synthase subunit alpha 2.842.065 10 27.34
gi|295658821 ATP synthase subunit beta 5.217.957 20 54.39
gi|295661300 DNA damage checkpoint protein rad24 1.295.329 6 40.57
gi|295672736 DNA damage checkpoint protein rad24 1.222.672 5 36.16
gi|295671178 elongation factor 1 alpha 8.897.567 13 33.26
gi|295668925 elongation factor 1 gamma 1 2.384.459 9 25.06
gi|146762537 enolase 14668.68 12 45.43
gi|295658119 glyceraldehyde 3 phosphate dehydrogenas 3201.45 8 41.95
gi|226278527 10 kDa heat shock protein 20459.57 5 45.63
gi|60656557 heat shock protein 1483.16 18 20.57
gi|295658865 heat shock protein 60 8.995.715 23 47.56
gi|295671569 heat shock protein SSC1 2.446.586 13 22.5
gi|295659116 Hsp70-like protein 8.972.324 22 47.09
gi|295658218 malate dehydrogenase 1.135.156 10 28.79
gi|295673937 malate dehydrogenase 4.400.604 13 65.88
gi|295666938 nucleoside diphosphate kinase 14429.06 5 38.82
gi|295668481 peptidyl-prolyl cis-trans isomerase D 1.868.925 8 27.08
1NCBI database general information number (http://www.ncbi.nlm.nih.gov/).doi:10.1371/journal.pone.0052470.t004
The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 16 December 2012 | Volume 7 | Issue 12 | e52470
lyses did not influence the extracellular protein profiles. Some of
the proteins here described have been detected in the extracel-
lular environment in many fungi by different groups
[9,10,19,27], thereby supporting the suggestion that they are
indeed exported from intact cells. Interestingly, cytosolic
enzymes, such as enolase and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), exert both enzymatic activity and
alternative extracellular functions in Paracoccidioides. These
enzymes have been found in the cytoplasm and at the cell wall
of the Paracoccicioides yeast cells [22,74]. The cell wall-associated
GAPDH binds to extracellular matrix components and mediates
the attachment and internalization of the Paracoccidioides yeast
cells to host tissues, thereby potentially playing a role in the
establishment of disease [74]. Paracoccidioides recruits plasminogen
and activates the plasminogen fribrinolytic system in a process
mediated by the cell wall-localized enolase, which potentially
plays a role in the establishment of PCM [22]. Therefore,
cytosolic enzymes may represent ‘moonlighting’ proteins func-
tioning in the extracellular environment [13,75].
Additionally, it is important to note that 63 of the 110 identified
proteins/isoforms in the Pb01 yeast secretome displayed ortholo-
gues in the Pb18 secretome. The difference in the number of genes
between Pb01 and Pb18 [5] may explain the variations in the
protein profiles. This may also be because they were sub-cultured
using different media (Fava-Netto for Pb01 and modified YPD
medium for Pb18). Additionally, the application of varying
proteomic methodologies to analyze the extracellular proteins of
Pb01 and Pb18 may give rise to the variations found in the
secretomes between the two members of the Paracoccidioides genus.
The fact that members of the phylogenetic groups of Paracoccidioides
produce varying combinations of extracellular yeast phase proteins
is not unexpected; the pools of extracellular proteins secreted by
different Histoplasma strains are also distinct from one another [76].
In conclusion, the results suggest that different strains may utilize
different strategies for survival and pathogenesis.
Conclusion
We have identified abundant proteins expressed in Paracoccid-
ioides yeast and mycelia secretomes. Several extracellular proteins
have also been described in the secretome of other organisms using
different methodologies, which is important for the validation of
our findings. Strikingly, many proteins do not use the classical
secretory pathways, and many proteins most likely exert other
activity once secreted. Moreover, the data clearly indicate that
Paracoccidioides Pb01 predominantly uses non-classical targeting
mechanisms to direct protein export. Our findings display the
potential role that extracellular proteins play in fungus survival in
the host and may lead to the description of molecules that function
as virulence factors.
Supporting Information
Figure S1 Enzyme Class (EC) of identified proteins.According to the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (NC-IUBMB), the
identified enzyme-like proteins were grouped into six classes
(Ligase, Isomerase, Lyase, Hydrolase, Transferase and Oxidore-
ductase).
(TIF)
Figure S2 Prediction of protein secretion in Paracoccid-ioides. All identified proteins were submited for in silico analysis
using the SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/)
and SecretomeP 2.0 (http://www.cbs.dtu.dk/services/
SecretomeP/) programs. The results obtained for the Paracoccid-
ioides mycelia and yeast secretomes were grouped into classes and
compared with the data provided in the Fungal Secretome
Database (FSD) (http://fsd.riceblast.snu.ac.kr). Class SP includes
proteins that were predicted by SignalP 3.0, Class NS represents
proteins secreted via a non-conventional pathway, which were
predicted by SecretomeP 2.0, and NC represents proteins that
were not classified.
(TIF)
Figure S3 Microscopy of adhered/internalized Paracoc-cidioides yeast cells by macrophages (magnification1600X). The yeast cells adhered and internalized (panel A) or
internalized (panels A–F). The arrows indicate internalized yeast
cells, and the asterisk indicates adhered yeast cells. The glass
coverslips, which were cultivated in the absence of BFA (panels A
and B), the presence of BFA (panels C and D) or the presence of
concentrated culture supernatant (panels E and F) as described in
the Materials and Methods section, were fixed with methanol,
stained by Giemsa and visualized via electronic microscopy.
(TIF)
Figure S4 Immunoblot analysis of Paracoccidioides-secreted proteins inside macrophages. The tested samples
included the lysate of infected macrophages with Paracoccidioides
yeast cells treated previously with Brefeldin A (line 1), the lysate of
infected-macrophages with Paracoccidioides yeast cells (line 2) and
the lysate of non-infected macrophages as a negative control (line
3). A- Immunoblot analysis of secreted proteins in infected
macrophages probed with enolase antibody (47 kDa). B-Immunoblot analysis of triosephosphate isomerase (29 kDa). C-Membranes stained with Ponceau red showing the protein profile
of the tested samples (lines 1–3).
(TIF)
Figure S5 Comparative analysis between Paracoccidi-oides Pb01 extracellular proteins and orthologous pro-teins found in pathogenic fungi secretomes. The bar graph
showing the proteins identified in the secretome of Paracoccidioides
Pb01, Paracoccidioides Pb18 [27], Histoplasma capsulatum [10,18],
Cryptococcus neoformans [19] and Asperillus fumigatus [37] of various
pathogenic fungi. The gray bars represent the number of
extracellular orthologous proteins that overlap between Paracocci-
diodies Pb01 yeast cells and the analyzed species. The white bars
represent the extracellular proteins that not do overlap with
Paracoccidioides Pb01 yeast cells.
(TIF)
Table S1 Secreted proteins/isoforms by Paracoccidioi-des Pb01. 1 Spots numbers indicated in Figure 2. 2 NCBI
database general information number (http://www.ncbi.nlm.nih.
gov/). 3 Molecular Mass in kDa (theoretical/experimental).4 Isoelectric point (theoretical/experimental). 5 Mascot MS/MS
score for fragmentation data (http://www.matrixscience.com).6 Number of matched peptides (MS/MS). 7 Protein Expression in
M: mycelia phase; Y: Yeast phase and C: protein common to the
two fungal phases or protein with no differential expression.8 ANOVA test - statistically significant differences are considered
with p,0.05 (*); protein found at just one fungal phase (**).9 Secretion prediction according to Signal P 3.0 server. The
number corresponds to signal peptide probability (Score $0.5)
(http://www.cbs.dtu.dk/services/SignalP/). 10 Secretion predic-
tion according to Secretome P 2.0 server, the number corresponds
to neural network that exceeded a value of 0.5 (NN-score $0.50)
(http://www.cbs.dtu.dk/services/SecretomeP/). 11 Enzyme Clas-
sification recommended by Nomenclature Committee of the
The Secretome of the Paracoccidioides Phases
PLOS ONE | www.plosone.org 17 December 2012 | Volume 7 | Issue 12 | e52470
International Union of Biochemistry and Molecular Biology (NC-
IUBMB). N adhesin-like proteins predicted by Faapred web server
(http://bioinfo.icgeb.res.in/faap/query.html).
(DOC)
Table S2 Exclusive proteins secreted by Paracoccidioi-des Pb01 yeast and mycelia. 1 Spots numbers indicated in
Figure 2. 2 NCBI database general information number (http://
www.ncbi.nlm.nih.gov/). 3 Number of identified isoforms of
protein in each Paracoccidioides, Pb01 phase secretome. 4 The
average of amount of values of abundances of all identified
isoforms. 5 Protein Expression in M: mycelia phase; Y: Yeast
phase. 6 Secretion prediction according to Signal P 3.0 server, the
number corresponds to signal peptide probability (http://www.
cbs.dtu.dk/services/SignalP/). 7 Secretion prediction according to
Secretome P 2.0 server, the number corresponds to neural
network that exceeded a value of 0.5 (NN-score $0.50) (http://
www.cbs.dtu.dk/services/SecretomeP/).
(DOC)
Table S3 Predicted post-translational modifications ofidentified protein isoforms in mycelia and yeast secre-tomes. 1 Protein Expression in M: mycelia phase; Y: Yeast phase
and C: protein common or protein with no differential expression.2 PTM – Post translational modifications: acetyl (k) – lysine
acetylation; phospo (S-T-Y) – serine, threonine and tyrosine
phosphorylation. 3 Values returned by MASCOT search tool
using none or specific variable modifications.
(DOC)
Table S4 Spectral identification of post-translationalmodification in identified protein/isoforms. 1 PTM – Post
translational modifications: acetyl (k) – lysine acetylation; phospo
(S-T-Y) – serine, threonine and tyrosine phosphorylation.2 Peptide localization in protein sequence. 3 Theoretical peptide
molecular mass without any PTM. 4 Obtained experimental mass
by spectral analysis.
(DOC)
Table S5 Comparative analysis of secreted proteins/isoforms by Paracoccidioides Pb01 yeast cells withothers pathogenic fungal secretomes. 1 Spots numbers
indicated in Figure 2. 2 NCBI database general information
number of Paracoccidioides Pb01 (http://www.ncbi.nlm.nih.gov/).3 Accession number of orthologues present in the Paracoccidioides
Pb18 secretome (Vallejo et al, 2012). 4 Accession number of
orthologues present in the Histoplasma capsulatum secretome
(Albuquerque et al, 2008; Holbrook et al, 2011). 5 Accession
number of orthologues present in the Cryptococcus neoformans
extracelular vesicles (Rodrigues et al, 2008). 6 Accession number of
orthologues present in the Aspergillus fumigatus secretome (Warten-
berg et al, 2011).
(DOC)
Author Contributions
Conceived and designed the experiments: CMAS. Performed the
experiments: SSW AFAP. Analyzed the data: SSW AFAP CLB JAP
AMB CMAS. Contributed reagents/materials/analysis tools: CMAS.
Wrote the paper: CMAS SSW.
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1. RESULTADOS DE TRABALHOS EM DESENVOLVIMENTO
5.1. Proteoma da superfície celular de Paracocidioides, Pb01
5.1.1. Padronização da extração de proteínas da parede celular
O primeiro passo realizado com o intuito de analisar o proteoma da parede celular de
Paracoccidioides, Pb01 foi padronizar a extração das proteínas de parede celular. Essa é uma
etapa difícil, pois PPCs são pouco abundantes, possuem baixa solubilidade, são na sua maioria
de natureza hidrofóbica e altamente glicosiladas.
A padronização da extração das PPCs do fungo Paracoccidioides, Pb01 foi baseada
nos dados da literatura para o modelo S. cerevisiae e C. albicans com algumas modificações,
uma vez que o modelo estrutural da parede celular é aplicável para outros Ascomicetos,
incluindo o Paracoccidioides, um fungo dimórfico capaz de crescer na forma de levedura e
micélio (Kapteyn et al. 1996; Pitarch et al. 2002; de Groot et al. 2004; Pitarch et al. 2008).
A metodologia padronizada para extração de PPCs em Paracoccidioides, Pb01, foi
validada para utilização em análises proteômica. O protocolo padronizado é descrito abaixo e
as etapas são ilustradas na Figura 7.
A Figura 7 mostra o fluxograma da metodologia padronizada, a qual permite obter
três diferentes frações protéicas da superfície celular de Paracoccidioides. A Fração 1, extraída
com detergentes e agentes redutores, contém as proteínas que se associam a superfície celular
de forma não covalentes ou por pontes dissulfetos. A Fração 2, extraída com NaoH, representa
proteínas de parede celular sensíveis ao tratamento com álcalis (ASL-PPCs) incluindo as
proteínas de parede celular com repetição interna (PIR-PPCs); enquanto que a Fração 3
representa proteínas com âncoras de glicosilfosfatidilinositol (GPI) ligadas à parede (GPI-
PPCs). O HF-piridina cliva especificamente ligações fosfodiéster, pela qual as GPI-PPCs estão
43
ligadas às cadeias de -1,6-glicana (Kapteyn et al. 1996; de Groot et al. 2004), permitindo o
isolamento da proteína sem a âncora GPI.
PROTOCOLO DE EXTRAÇÃO DE PROTEÍNAS DA PAREDE CELULAR
1. As células foram cultivadas em ambas as fases do fungo (levedura e micélio) em
meio Fava-Neto líquido por 72 h. Posteriormente a cultura foi centrifugada (10.000 rpm/10
min/4C) e o sedimento obtido foi lavado cinco vezes com tampão de lise (Tris-HCl 10
mM pH 7,4; 1 mM PMSF).
2. Na sequência, as células foram maceradas com nitrogênio líquido até pó fino e
branco e resuspensas em tampão de lise e centrifugadas (10.000 rpm/10 min/4C).
3. O sedimento obtido após centrifugação foi lavado da seguinte forma: 5X com água
destilada gelada estéril, 5X com NaCl 5% (p/v), 5x com NaCl 2% (p/v) e 5x com NaCl 1%
(p/v).
4. Após as lavagens, o sedimento foi tratado com tampão de extração (Tris-HCl 50
mM pH 7,8; SDS 2% (p/v); EDTA 100 mM e Mercaptoetanol 40 mM, DTT 10 mM) por
10 minutos à 100C.
5. O sobrenadante da centrifugação constitui a primeira fração (Fração 1). O
sedimento resistente a extração com SDS foi lavado 5X com acetato de sódio 0,1 M pH
5,5.
6. Posteriormente, o sedimento obtido por esse processo foi dividido em dois: (I) O
primeiro foi tratado com NaOH 30 mM durante 18h à 4°C para obtenção da segunda
fração (Fração 2). (II) O segundo digerido com fluoreto hidrogenado de piridina (HF-
piridina) durante 24 h à 0C para fornecer a terceira fração (Fração 3).
44
Figura 7. Representação esquemática das etapas da extração de proteínas da
parede celular padronizada para Paracoccidioides. O protocolo permite a obtenção
de três frações da parede celular denominadas Fração 1, Fração 2 e Fração 3.
Adaptado de Pitarch et al. (2008).
Uma questão importante para a validação da extração é a garantia de que, as amostras
obtidas incluam apenas proteínas de superfície celular e não estejam contaminadas com
proteínas solúveis ou citoplasmáticas do fungo. Nesse sentido foram selecionadas duas
amostras da etapa de lavagens do sedimento (etapa 3 do protocolo acima descrito). A primeira
amostra corresponde ao sobrenadante da primeira lavagem com NaCl 5 %, e a segunda amostra
45
ao sobrenadante da última lavagem com NaCl 1 %. Conforme mostra a Figura 8, a última
lavagem não continha proteínas solúveis em NaCl 1 %, garantindo que o sedimento estava livre
de contaminação com proteínas citoplasmáticas.
Figura 8. Validação do método de extração das
proteínas da parede celular. SDS-PAGE do
sobrenadante da primeira lavagem com NaCl 5 % e
na última lavagem com NaCl 1 % corado com Prata.
5.1.2. Análise proteômica da superfície celular de Paracoccidioides,
Pb01
Após obtenção dos extratos proteicos da parede celular, as amostras foram validadas
para análise proteômica. Foram realizadas eletroforeses bidimensionais (2D-PAGE). As
amostras foram aplicadas em fitas immobiline pH 3-11 (Ge Healthcare Biosciences). A
focalização isoelétrica foi realizada em sistema IPGphor e as fitas reduzidas com 2% DTT (p/v)
e alquiladas em 2,5% (p/v) de iodoacetamina em tampão de equilíbrio (Uréia 6 M pH 6,8; 30%
(v/v) de glicerol; 2% (p/v) SDS). A segunda dimensão foi realizada em gel de poliacrilamida
12% (p/v) e os géis corados com prata.
46
A Figura 9 mostra os mapas protéicos obtidos para a Fração 1 e Fração 2 da parede
celular de levedura de Paracoccidioides, Pb01.
Figura 9. Mapa protéico da superfície celular de levedura de Paracoccidioides, Pb01 (A) Fração 1
da parede celular, contendo proteínas associadas a parede celular por ligações não-covalentes e/ou
dissulfeto, extraídas com detergente e agentes redutores. (B) Fração 2 da parede celular, a qual contêm
proteínas sensíveis ao tratamento com álcalis (ASL-PPCs). Os números correspondem à identificação
dos spots. Os valores a direita indicam o padrão do marcador de peso molecular in kDa.
Posteriormente, os spots foram cortados do gel e digeridos com tripsina, segundo o
protocolo de digestão em gel padronizado em nosso laboratório (Parente et al. 2011). As
proteínas foram identificadas por espectrometria de massas (MALDI-Q-TOF). A Tabela 1 e a
Tabela 2 mostram as proteínas identificadas na Fração 1 e Fração 2 da parede celular de
levedura de Paracoccidioides, Pb01, respectivamente. As análises proteômicas da Fração 3
estão em desenvolvimento.
Foram identificadas um total de 40 proteínas associadas à parede celular, extraídas
pelo tratamento com agentes redutores, constituindo o que denominamos Fração 1 da parede
celular. Na Fração 2, constituída por proteínas sensíveis ao pH alcalino, identificamos 22
47
proteínas denominadas ASL-PPCs. A Figura 10 mostra a classificação funcional das proteínas
identificadas de acordo com MIPS Functional Catalogue Database (FunCatDB).
Figura 10. Classificação funcional das proteínas identificadas na superfície celular de
leveduras de Paracoccidioides, Pb01. As proteínas identificadas foram agrupadas em
categorias funcionais de acordo MIPS Functional Catalogue Database (FunCatDB).
48
Tabela 1 - Proteínas associadas à parede celular de Paracoccidioies (Fração 1)
Nr. spot
1
Accesso (NCBI)
2
Proteína MM
(kDa) T 3
pI (pH) T
4
PMF Score
Cobertura de
sequência
MSMS Ion
Score
Número de peptídeos
identificados Sequência dos peptídeos
1. ENERGIA
1.1. Glicólise e Gliconeogênese
24 gi|146762537 Enolase 47.412 5.67 106 71% 112 4
R.SGETEDVTIADIVVGLR.A R.GNPTVEVDVVTETGLHR.A R.AIVPSGASTGQHEACELR.D R.IEEELGSNAVYAGDKFR.A
47 gi|295671120 fructose-bisphosphate aldolase 39.721 6.09 97 40% 55 2 R.DKNSPIILQVSQGGAAFFAGK.G
R.SIAPSYGIPVVLHTDHCAK.K
56 gi|295671120 fructose-bisphosphate aldolase 39.721 6.09 84 74% 97 3 K.TGVIVGDDVLR.L
R.SIAPSYGIPVVLHTDHCAK.K R.LHPELLSK.H
7 gi|295658119 glyceraldehyde-3-phosphate
dehydrogenase 36.619 8.26 147 88%
4 gi|295658119 glyceraldehyde-3-phosphate
dehydrogenase 36.619 8.26 183 86%
5 gi|295658119 glyceraldehyde-3-phosphate
dehydrogenase 36.619 8.26 164 80%
41 gi|295669690 phosphoglycerate kinase 45.311 6.48 100 81% 32 1 K.ASGGQVILLENLR.F
18 gi|295670663 triosephosphate isomerase 27.159 5.39 86 83% 113 4
K.FFVGGNFK.M R.VLLREDDEFVAR.K
K.VATTEQAQEVHASIR.K K.ISPEAAENIR.V
1.2. Ciclo do Ácido Tricarboxílico
23 gi|295658218 malate dehydrogenase 34.671 6.36 188 65% 190 5
R.LFGVTTLDVVR.A K.RLFGVTTLDVVR.A
K.AKDGAGSATLSMAYAGFR.F R.AETFTQEFTGQKDPSK.A
K.GIVEPTYIYLSGVDGGEAIKR.E
1 gi|295673937 malate dehydrogenase 36.022 8.99
92 3 R.VTQLALYDIR.G
R.DALKDSEIVLIPAGVPR.K K.DQGINFFASNVK.L
49
49 gi|295673937 malate dehydrogenase 36.022 8.99
49 1 R.DALKDSEIVLIPAGVPR.K
6 gi|295673937 Malato desidrogenase 36.022 8.99 147 66% 51 2 K.ASQGEKDVIEPTFVDSPLYK.D
K.LGPNGVEEILPVGNVSEYEQK.L
25 gi|225678324 succinyl-CoA ligase subunit alpha 34.921 8.54 82 68%
1.3. Complexo Piruvato Desidrogenase
33 gi|295673931 pyruvate dehydrogenase 52.714 6.45 55 37%
1.4. Transporte de elétrons
48 gi|295666219 ATP synthase D chain, mitochondrial 19.653 7.88 103 73% 109 4
R.GQTAASLAAFK.K K.NQAVVDEIEK.H
K.AIEAFEAQAMK.G K.ARPDILEK.T
31 gi|295657771 ADP ATP carrier protein 33.633 9.81
39 4 R.LDHKYNGIMDCFSR.T
R.YFPTQALNFAFR.D K.YSSSFDAAR.Q R.SFFKGAGANILR.G
59 gi|295658821 ATP synthase subunit beta 55.181 5.28 176 82% 251 9
R.GASVTDTGAPIMIPVGPGTLGR.I R.IMNVTGDPIDER.G K.VVDLLAPYAR.G
K.AHGGYSVFTGVGER.T K.VALVFGQMNEPPGAR.A
R.IPSAVGYQPTLAVDMGGMQER.I R.IPSAVGYQPTLAVDMGGMQER.I
R.GISELGIYPAVDPLDSK.S R.FLSQPFTVAQVFTGIEGK.L
32 gi|226291035 ATPase alpha subunit 60.404 9.12 124 65%
9 gi|295658923 citocrome b c1 complex subunit 2 49.014 9.10 102 39% 116 3 K.WVGQLCR.D
R.LVHGGKPLQISDIGQGIEK.V K.GLAANPTAQALDSAHNVAFHR.G
8 gi|295658923 cytochrome b-c1 complex subunit 2 49.014 9.10 117 38%
1.5. Fermentação
43 gi|295674635 alcohol dehydrogenase (ADH) 38.002 7.55 113 74%
1.6. Metabolismo de energia de reserva
37 gi|295671152 phosphoglucomutase 83.658 6.59 74 51%
50
1.7. Oxidação de ácidos graxos
42 gi|295666179 2-methylcitrate synthase 51.516 9.02 207 53% 71 4
R.GEDVIGEVTVASTIGGMR.G K.ASYWEPTFDDSISLLAK.I K.SMVWEGSVLDANEGIR.F K.SGQVVPGYGHGVLR.K
2. RESGATE CELULAR, DEFESA E VIRULÊNCIA (RDV)
20 gi|295658865 heat shock protein 62.266 5.51 70 49% 72 2 K.AVTLQDKFENLGAR.L K.TIDDELEVTEGMR.F
14 gi|295659116 heat shock protein 70 70.919 5.08 203 57%
10 gi|295671569 heat shock protein SSC1 73.821 5.92 124 70% 82 3 K.FTDPECQR.D
R.VVNEPTAAALAYGLER.E R.VVNEPTAAALAYGLER.E
11 gi|295671569 heat shock protein SSC1 73.821 5.92
12 gi|295671569 heat shock protein SSC1 73.821 5.92
13 gi|295673716 hsp70-like protein 68.856 5.39 170 51% 58 2 K.NQYAANPQR.T
K.DNNLLGKFELTGIPPAPR.G
28 gi|17980998 Y20 protein 21.646 6.09 65 64%
30 gi|17980998 Y20 protein 21.646 6.09 107 64% 100 3 K.IAIVFYSLYGHIQK.L R.YGNFPGQWK.A
R.GGSPWGAGTYAGADGSR.Q
15 gi|295666534 Woronin body major protein 24.480 7.27 231 81% 83 3 M.GYYDDEGHYHSFR.R -
MGYYDDEGHYHSFR.R + Oxidation (M) R.QLHEESSFISHPSSSVVVQSMLGPVYK.T
3. FATE
29 gi|295664272 mitochondrial-processing peptidase
subunit beta 53.088 5.84 165 50% 71 2
R.LSFNVTEAEVER.A K.ITEKDVMSFAQR.K
26 gi|295672668 peptidyl-prolyl cis-trans isomerase B 22.815
83 53%
4. CICLO CELULAR E PROCESSAMENTO DO DNA
19 gi|295668877 Actin 41.825 5.63 155 64% 216 7
R.AVFPSIVGRPR.H R.HHGIMIGMGQK.D
R.VAPEEHPVLLTEAPINPK.S R.GYSFSTTAER.E K.SYELPDGQVITIGNER.F
K.QEYDESGPSIVHR.K K.QEYDESGPSIVHR.K
5. SÍNTESE PROTEÍCA
3 gi|295672792 60S acidic ribosomal protein P0 lyase 33.767 5.02 79 59%
51
22 gi|225681738 elongation factor 1-alpha 28.326 9.53 70 40% 117 3 R.VETGVIKPGMVVTFAPANVTTEVK.S K.SVEMHHQQLTAGNPGDNVGFNVK.N
K.MIPSKPMCVEAFTEYPPLGR.F
6. TRANSPORTE CELULAR
16 gi|295666381 GTP-binding nuclear protein GSP1/Ran 24.082 6.90 146 73% 74 3 K.AKTITFHR.K R.VCENVPIVLCGNKVDVK.E
K.NLQYYDISAK.S
21 gi|295659414 outer mitochondrial membrane protein
porin 30.265 8.98 75 47% 125 5
K.AANDLLNKDFYHTSAANLEVK.S K.SKAPNGVTFHVK.G
K.APNGVTFHVK.G K.GLKAEILTQYLPSSQSK.G
K.LNLYFKQPNIHAR.A
7. PROTEÍNAS NÃO CLASSIFICADAS
2 gi|295668410 conserved hypothetical protein 53.660 6.02 70 62%
1 Número dos spots indicado na Figura 9
2 Número de acesso no banco de dados (NCBI)
3 Massa Molecular (MM) teórica (T) em kDa
4 Ponto isoelétrico (pI) teórico (T)
52
Tabela 2 - Proteínas de superfície celular de Paracoccidioides (Fração 2)
Nr. spot
1
Accesso (NCBI)
2
Proteína MM
(kDa) T 3
pI (pH) T
4
PMF Score
Cobertura de sequência
MSMS Ion
Score
Número de peptídeos
identificados Sequência dos peptídeos
1. ENERGIA
1.1. Glicólise e Gliconeogênese
9 gi|146762537 Enolase 47412 5.67 57 41%
28 gi|295672732 enolase 43.859 8.93 67 65% 129 4
R.SVYDSRGNPTVEVDVVTETGLHR.A R.GNPTVEVDVVTETGLHR.A
K.NVNSVIGPAIIK.E K.KPYVLPVPFQNVLNGGSHAGGR.L
29 gi|295672732 enolase 43.859 8.93 55 70%
27 gi|295672732 enolase 43.859 8.93 115 48% 108 4
R.AIVPSGASTGQHEACELR.D R.AIVPSGASTGQHEACELRDGDQSK.W
K.LGANAILGVSLAIAK.A K.KPYVLPVPFQNVLNGGSHAGGR.L
47 gi|295672732 enolase 43.859 8.93
85 3 R.GNPTVEVDVVTETGLHR.A R.AIVPSGASTGQHEACELR.D
K.KPYVLPVPFQNVLNGGSHAGGR.L
14 gi|295671120 fructose 1,6-biphosphate
aldolase 1 39.721 6.09 76 35%
41 gi|295671120 fructose 1,6-biphosphate
aldolase 1 39.721 6.09 73 39% 175 3
K.NKPVYLVFHGGSGSTK.A K.NKPVYLVFHGGSGSTKAEFK.E K.DYLMSAVGNPEGEDKPNKK.Y
1.2. Ciclo do Ácido Tricarboxílico
54 gi|295669158 aspartate aminotransferase 47.193 9.25 162 51% 118 3 R.DDQGKPYVLPSVR.A
R.HFLKEGNGIVLSQSFAK.N K.NMGLYGER.V
1.3. Complexo Piruvato Desidrogenase
21 gi|226294995 dihydrolipoyl dehydrogenase 55.431 8.02 70 24%
53
1.4. Transporte de elétrons
53 gi|295658923 cytochrome b-c1 complex
subunit 2 49.014 9.10 156 59%
52 gi|295673096 NADH-cytochrome b5 reductase 37.197 8.90 117 76% 51 2 R.FEFEDPESVSGVHVSSAVLTK.Y
K.ELEKLENTYPR.R
2. RESGATE CELULAR, DEFESA E VIRULÊNCIA (RDV)
2 gi|295673162 disulfide isomerase Pdi1 59.305 4.80 158 49% 80 3 K.YEQLAQLYADNPEFAAK.V
K.IDATANDVPEEIQGFPTVK.L K.LFAAGSKDKPFDYQGLR.T
1 gi|295673162 disulfide isomerase Pdi1 59.305 4.80 174 63% 265 9
K.ALAPEYETAATQLK.E // R.DNFPFGATNDAK.L // R.DNFPFGATNDAK.L // K.MLKPIAEK.Q //
K.AFGAHAGNLNLK.A // K.ADKFPAFAIQDPVNNK.K // K.KYPFDQELK.I //
K.IDATANDVPEEIQGFPTVK.L // R.TIQGLADFVR.D
6 gi|295659787 heat shock protein Hsp88 80.684 4.92
86 2 R.FIAGPIIQR.Y // R.YTDKVEAER.A
45 gi|295659116 hsp70-like protein 68.856 5.39 86 28% 100 2 K.DNNLLGKFELTGIPPAPR.G
K.FELTGIPPAPR.G
3. FATE
20 gi|152031181 kex protein 36.850 7.16
21 1 -.PTGAVSYLSTTRK.L
4. CICLO CELULAR E PROCESSAMENTO DO DNA
13 gi|295664474 cell division cycle protein 90.565 4.98
81 2 R.AHFEEAMQMAR.R // R.AHFEEAMQMAR.R
7 gi|295661300 DNA damage checkpoint protein
rad24 29.738 4.68 61 50%
5. SÍNTESE PROTEÍCA
56 gi|295671178 elongation factor 1-alpha 50.558 9.24 99 50% 63 2 K.SVEMHHQQLTAGNPGDNVGFNVK.N
K.MIPSKPMCVEAFTEYPPLGR.F
6. TRANSPORTE CELULAR
48 gi|295659414 outer mitochondrial membrane
protein porin 30.265 8.98 118 71% 146 5
M.PAPPAFSDIAK.A K.AEILTQYLPSSQSK.G K.LNLYFKQPNIHAR.A R.VNAQVEAGAK.A
K.YRLDPSSFAK.A
7. BIOGÊNESE DE COMPONENTES CELULARES
12 gi|295657091 tropomyosin-1 18.833 4.99 56 85%
54
7. PROTEÍNAS NÃO CLASSIFICADAS
58 gi|295668431 suaprga1 34.702 4.52 55 2 R.TFGDEKIR.V // R.IDQFQPR.E
1 Número dos spots indicado na Figura 9
2 Número de acesso no banco de dados (NCBI)
3 Massa Molecular (MM) teórica (T) em kDa
4 Ponto isoelétrico (pI) teórico (T)
55
6. DISCUSSÃO
Usando uma estratégia proteômica, nós descrevemos pela primeira vez o perfil do
secretoma do isolado Pb01 de Paracoccidioides. Um total de 160 proteínas/isoformas
extracelulares foi identificado, incluindo ambas as formas do fungo, micélio e levedura. Sendo
que, 20 proteínas/isoformas foram preditas de serem secretadas pela via clássica, enquanto que
84 secretadas por vias alternativas. A análise proteômica, entre micélio e levedura, revelou 28
proteínas, as quais correspondem a 47 proteínas/isoformas (29,37% do total de
proteínas/isoformas identificadas), como sendo secretadas constitutivamente, sem diferença no
nível de expressão entre ambas as fases. Nesse grupo, incluem proteínas envolvidas em
processos celulares como resposta ao estresse, metabolismo e energia. Duas proteínas de
choque térmico foram constitutivamente secretadas por Paracoccidioides. Proteínas de choque
térmico (HSPs) têm sido envolvidas em aumento da exportação de proteínas via mecanismos
não clássicos de secreção em células eucariotas (Rubartelli et al. 1990; Jackson et al. 1992;
Cleves et al. 1996). Portanto, nos supomos que HSPs possam estar envolvidas na exportação de
proteínas em Paracoccidioides.
A análise comparativa revelou significantes mudanças nos perfis de proteínas
secretadas por micélio e levedura de Paracoccidiodies. Do total de 160 proteínas/isoformas
descritas, foram identificadas 84 proteínas/isoformas como sendo diferencialmente secretadas.
Formamidase foi descrita como sendo preferencialmente secretada pela fase miceliana
de Paracoccidiodies. Esse dado foi confirmado pelo ensaio de atividade enzimática, o qual
revelou uma atividade enzimática significativamente maior em micélio, quando comparada
com levedura. Formamidase foi também detectada em vesículas extracelulares de H.
capsulatum (Albuquerque et al. 2008). Proteínas envolvidas no metabolismo de aminoácidos,
como a hidroximetiltransferase, foram preferencialmente identificadas no secretoma de
micélio. Em micro-organismos (Waditee-Sirisattha et al. 2012) e plantas (Hasegawa et al.
56
2000), essa enzima desempenha um papel importante conferindo tolerância ao estresse causado
pela salinidade, gerando serina e compostos quaternários da amônia, como a glicina betaína.
Essas moléculas protegem as proteínas secretadas contra a salinidade, atuando como uma
chaperona não enzimática (Endoh et al. 2004). Em Paracoccidiodies, essa enzima poderia ter
um papel na proteção contra o estresse ambiental, durante a fase miceliana.
Adicionalmente, proteínas que desempenham papel na defesa contra estresse, tais
como catalase peroxisomal, glutationa redutase, tioredoxina redutase e proteína de tumor
controlada transcricionalmente (TCTP), foram identificadas no secretoma de micélio. O
aumento da secreção de proteínas envolvidas com defesa celular, em micélio, pode refletir a
necessidade de aumentar a proteção contra o estresse ambiental. A fase miceliana de
Paracoccidiodies desenvolve-se no solo, e pode estar exposta a inúmeros estresses ambientais,
como altas temperaturas, radiação solar e espécies reativas de oxigênio que podem emergir de
amostras externas (Bucková et al. 2010). Estudos tem associado o envolvimento de TCTP na
prevenção da morte celular pela modulação da atividade anti-apoptótica de Mcl-1 (Liu et al.
2005). Esses resultados sugerem a secreção de proteína e enzimas requeridas para a proteção,
da fase não patogênica, contra estressores ambientais.
Glutationa S-transferase (GST) foi encontrada preferencialmente secretada por células
leveduriformes de Paracoccidiodes, quando comparamos com o secretoma de micélio. GST
representa um grupo de enzimas de detoxificação, as quais são envolvidas na proteção contra o
estresse oxidativo e com a detoxificação de xenobióticos e metais pesados (Hayes & Strange
1995; Nebert & Dalton 2006). Em concordância com os dados proteômicos, a atividade
enzimática da GST foi significativamente maior em levedura. Adicionalmente, superóxido
dismutase (SOD) foi preferencialmente encontrada no secretoma da fase patogênica. Em H.
capsulatum, uma superóxido dismutase extracelular (SOD3) foi preferencialmente expressa
pelas células leveduriformes (Holbrook et al. 2011). A SOD3 protege especificamente contra
57
espécies reativas de oxigênio extracelular e facilita a patogênese por H. capsulatum, pela
detoxificação de ROS derivados do hospedeiro (Youseff et al. 2012).
Análise proteômica resultou na descrição de oito isoformas de 2-metilcitrato sintase no
secretoma de levedura de Paracoccidiodes, a qual foi predita de ser secretada por via não
clássica. A análise estatística mostrou um nível alto de expressão (70.672,37 vezes) em
levedura, dessa em proteína em relação ao micélio. A 2-metilcitrato sintase foi previamente
demonstrada em vesículas extracelulares de H. capsulatum (Albuquerque et al. 2008) e no
isolado Pb18 de Paracoccidiodes (Vallejo et al. 2012), embora a função extracelular dessa
proteína ainda não ter sido descrita. A proteína 14-3-3 (DNA damage checkpoint) foi
preferencialmente secretada por levedura de Paracoccidiodes, Pb01, a qual regula uma
diversidade de vias de sinalização, através da modulação da função de proteínas e pela
formação de interações proteína-proteína em células eucarióticas (Yang et al. 2006). Além
disso, a proteína 14-3-3 modula o tráfego de vesículas e exocitose, como mostrado me
diferentes sistemas experimentais (Morgan & Burgoyne 1992; Roth et al. 1999). Essa proteína
foi também detectada no ambiente extracelular pela análise proteômica de vários fungos, como
C. neoformans (Albuquerque et al. 2008), H capsulatum (Albuquerque et al. 2008; Holbrook et
al. 2011), e Paracoccidiodes, Pb18 (Vallejo et al. 2012).
Proteínas envolvidas em Resgate Celular, Defesa e Virulência (RDV) foram mais
abundantes no secretoma de levedura de Paracoccidiodes, Pb01, as quais incluem proteínas
envolvidas em defesa contra estresse e detoxificação, como HSPs, dissulfito isomerase (Pdi1),
GST e SOD. Durante o processo de infectivo, a liberação de ROS pelas células efetoras imunes
do hospedeiro desempenha um papel importante na eliminação de patógenos (Imlay 2003).
Assim, a resposta ao estresse provavelmente seja um atributo de suma importância para a
virulência em fungos patogênicos.
58
Estudos proteômicos podem ser influenciados pela condição de crescimento. Como
exemplo, em Botrytis cinerea foi descrito mudanças significativas na abundância e composição
de enzimas extracelulares dependentes da condição de cultivo (Shah et al. 2009). Nós
utilizamos um meio rico em peptídeos e carboidratos que poderiam explicar a abundância de
algumas enzimas glicolíticas e do ciclo do TCA no secretoma de Paracoccidiodes, Pb01.
Embora exista essa consideração, ambas as formas de Paracoccidiodes, Pb01 foram crescidas
no mesmo meio e exibiram uma grande variedade de proteínas extracelulares diferencialmente
expressas, sugerindo que essas moléculas sejam adaptadas para beneficiar as fases miceliana e
leveduriforme.
Apesar de suas limitações, eletroforese em gel 2D continua a ser um poderoso método
para inspeção global de modificações pós-tradicionais (PTMs), uma vez que essas
modificações refletem mudanças na massa molecular e na carga das proteínas alvos. Dentre as
proteínas extracelulares identificadas no secretoma de micélio e levedura de Paracoccidiodes,
37 proteínas apresentarm mais que um spots nos mapas proteicos, o que totaliza 116 isoformas
identificadas, o que corresponde a 72,5% do total identificado (160 proteins/isoformas). Nos
especulamos que algumas mudancas nas massas e pontos isoelétricos (pI) poderiam ser
causados por PTM. Analise do espectro combinada com uma pesquisa por PTM no programa
Mascot, revelou que 39% das isoformas detecadas apresentavam fosforilação em
serina/treonina ou acetilação de lisina. Oito das nove isoformas da 2-metilcitrato sintase foram
encontradas fosforiladas, as quais correlacionam-se com diminuição nos pIs. Identificamos
também 30 proteínas acetiladas do taotal de 116 isoformas presentes no secretomas de
Paracoccidiodes. As HSP-70 apresentaram pelo menos 5 peptídeos acetilados em cada uma das
suas isoformas identificadas. Mudancas nos pIs dessas moléculas correlacionam-se com as
observadas em proteínas derivadas de linhagens celulares de cancer de mama, nos quais as
udanças nos pIs foram consistentes com o número e tipo de PTM observada (Zhu et al. 2005).
59
Modificações pós-traducionais podem ser associadas a novas funções e localização fora das
célula fungicas. Entretanto, o impacto dessas modificações na patogênese, adaptação ambiental,
captação de nutrientes e mudança/manutenção da morfologia da célula, ainda não estão
estabelecidas e requer uma invesigação mais detalhada do impacto dessas PTMs sobre cada
proteína modificada (Teutschbein et al. 2010).
Com o intuito de melhor entender a participação de proteínas extracelulares durante o
processo infectivo, nós investigamos a influência do bloqueio da via de secreção de proteínas
em Paracoccidiodes, Pb01 sobre interação das leveduras com as células imunes fagocíticas. A
adição de brefeldina A, ao meio de cultura das células leveduriformes, resultou na diminuição
da quantidade de proteínas secretadas e inibiu significativamente a interação dos macrófagos
com o fungo (avaliado pela adesão e internalização de células leveduriormes), e resultou em
redução da viabilidade das células fúngicas recuperadas após o co-cultivo. Tem sido descrito
que a inibição da função dos macrófagos poderia facilitar a sobrevivência de patógenos em
células imunes (Flavia Popi et al. 2002; Konno et al. 2009). Sabemos que leveduras de
Paracoccidiodes são fagocitadas e tem a capacidade de multiplicar-se no interior de
macrófagos (Moscardi-Bacchi et al. 1994; Almeida et al. 1998). Tem sido descrita também, a
participação de proteínas extracelulares na aderência e captação de células leveduriformes
pelos macrófagos (Almeida et al. 1998). A glicoproteína gp43, principal molécula antigênica
secretada por Paracoccidiodes, participa durante a etapa inicial de interação do fungo com os
macrófagos. Essa participação foi demonstrada quando a adição de anticorpos policlonais anti-
gp43 induziu uma redução marcante no índice de fagocitose de macrófagos co-cultivados com
leveduras de Paracoccidioides (Almeida et al. 1998). Similarmente, nós observamos uma
redução na adesão/internalização de células leveduriformes quando bloqueamos a via clássica
de secreção. Enquanto que, a adição de sobrenadante de cultura concentrado significativamente
aumentou a interação do fungo com os macrófagos, sugerindo que as proteínas secretadas por
60
Paracoccidioides exerçam alguma atividade biológica. Habilidade semelhante tem sido descrita
para vesículas secretadas por Leishmania durante a infecção (Silverman et al. 2010; Silverman
& Reiner 2012), as quais liberam efetores que medeiam a invasão do parasita, favorecendo a
sobrevivência no hospedeiro. Ainda em Leishmania, estudos mostraram exossomos e proteínas
exossomais no citoplasma de macrófagos infectados (Silverman et al. 2010).
Sistemas de secreção especializados têm sido usados por patógenos humanos para
exportar fatores de virulência até o interior de células do hospedeiro (Kaur et al. 2007;
Silverman et al. 2010; Seider et al. 2011). Nesse estudo, nós apresentamos evidências que
Paracoccidiodies também libera efetores no citoplasma de macrófagos, entretanto, o
mecanismo permanece não elucidado. Identificamos 18 proteínas secretadas por leveduras de
Paracoccidioides em macrófagos infectados, incluindo proteínas ortólogas, proteínas de
choque térmico de 10 e 70 kDa, proteína 14-3-3 e um fator de elongação 1 alfa (EF1-), as
quais foram previamente descritas como proteínas exossomais em Leishmania (Silverman et al.
2010; Silverman & Reiner 2012). Além disso, Silverman & Reiner (2012) descreveram que
EF1- é liberado, por células promastigotas de Leishmania, no citosol de macrófagos, ativando
inúmeras tirosinas-fosfatases do hospedeiro, o que resulta na inibição da função microbicida de
macrófagos. Adicionalmente, o choque térmico resulta em um aumento da liberação de
exossomos pela Leishmania (Hassani et al. 2011). Esses dados sugerem que proteínas
extracelulares de Paracoccidiodes podem ser importantes para a sobrevivência do fungo em
macrófagos. Portanto, nós acreditamos que proteínas secretadas por Paracoccidiodes facilitem
a sobrevivência do fungo no hospedeiro, pelo menos durante a fase inicial da infecção,
reforçando a ideia que proteínas secretadas por fungos patogênicos desempenham papel
importante na patogênese e virulência (Tjalsma et al. 2004; Nosanchuk et al. 2008). Nós
acreditamos ainda que, o exoproteoma de Paracoccidiodes pode modular ambos, vias de
sinalização e função de macrófagos, criando um ambiente permissivo para a infecção como tem
61
sido descrito para outros patógenos intracelulares, como Leishmania (Naderer & McConville
2008; Hassani et al. 2011; Silverman & Reiner 2012), H. capsulatum (Eissenberg et al. 1993),
C. neoformans (Tucker & Casadevall 2002), C. glabrata (Seider et al. 2011), e C. albicans
(Fernandez-Arenas et al. 2009).
Descrevemos que Paracoccidioides, Pb01 secreta inúmeros tipos de proteínas,
incluindo enzimas, proteínas de choque térmico e proteínas citosólicas convencionais. Embora
não possamos excluir completamente o papel da lise celular, a amostra usada para análise do
secretoma foi testada para garantir que a lise celular não influenciasse no perfil proteico.
Algumas proteínas extracelulares aqui descritas têm sido descritas no ambiente extracelular em
outros fungos por diferentes grupos (Nombela et al. 2006; Rodrigues et al. 2008; Holbrook et
al. 2011; Vallejo et al. 2012), o que suporta a hipótese que essas moléculas são secretadas pelas
células intactas. Interessantemente, enzimas citosólicas, como enolase e GAPDH, exercem
ambas, atividade enzimática e função extracelular alternativa em Paracoccidioides. Essas
enzimas têm sido encontradas no citoplasma e na parede celular de células leveduriformes de
Pb (Barbosa et al. 2006; Nogueira et al. 2010). A GAPDH associada à parede celular tem a
capacidade de ligar-se aos componentes da matriz extracelular e mediar a adesão e
internalização do fungo nos tecidos do hospedeiro, participando do estabelecimento da doença
(Barbosa et al. 2006). Paracoccidioides recruta plasminogênio e ativa o sistema fibrinolítico
através de um processo mediado pela enolase de superfície celular, a qual possivelmente
desempenha um papel no estabelecimento da PCM (Nogueira et al. 2010). Portanto, enzimas
citosólicas podem representar proteínas `moonlighting´ que atuam no ambiente extracelular
(Chaves et al. 2009; Jeffery 2009).
É importante notar que, 63 das 110 proteínas/isoformas identificadas no secretoma de
levedura de Paracoccidioides, isolado Pb01, apresentaram ortólogos no secretoma do Pb18. A
diferença no número de genes entre os dois isolados (Desjardins et al. 2011), pode explicar a
62
variação no perfil proteico. A variação encontrada pode também ser devido ao fato que, os
isolados foram cultivados em diferentes meios (Fava-Neto para o Pb01 e YPD modificado para
o Pb18). As diferentes metodologias proteômicas utilizadas para analizar o secretoma dos dois
membros do gênero Paracoccidioides também contribui para explicar as diferenças
encontradas. O fato de membros de grupos filogenéticos de Paracoccidioides produzirem
combinações variadas de proteínas extracelulares não é inesperado; cepas de H. capsultaum
também secretam diferentes perfis proteicos entre si (Holbrook & Rappleye 2008).
Os resultados de proteínas de superfície celular de Paracoccidiodies fazem parte de
um trabalho que ainda está em desenvolvimento. Consideramos que a padronização da extração
das proteínas de parede celular é um resultado importante, uma vez que essa etapa é de extrema
dificuldade devido à natureza hidrofóbica e a baixa abundância e solubilidade dessas proteínas.
Identificamos um grupo de proteínas associadas à parede celular e outro de proteínas sensíveis
ao tratamento com álcali. Esperamos que a completa descrição do proteoma da parede celular
de Paracoccidiodes nos fornece uma compreensão do papel dessas moléculas, as quais
integram a barreira entre o patógeno e o hospedeiro, a superfície celular.
Muitas proteínas identificadas na superfície celular foram também descritas no
secretoma de Paracoccidioides, levando-nos a supor que elas possam ter sido identificadas ao
cruzarem a barreira da superfície celular em direção ao meio externo, porém o mecanismo
ainda não está estabelecido.
63
7. CONCLUSÃO
Identificamos um grande número de proteínas secretadas por ambas as fases, micélio e
levedura, de Paracoccidioides, Pb01. Várias proteínas extracelulares identificadas têm sido
também descritas no secretoma de outros organismos utilizando diferentes metodologias, o que
é importante para a validação dos nossos resultados. Muitas proteínas secretadas não usam a
via clássica de secreção e devem apresentar outras funções no ambiente extracelular. Além
disso, os dados indicam claramente que Paracoccidiodes usa predominantemente mecanismos
não clássicos para exportar proteínas. Nossos achados descrevem um importante papel para as
proteínas extracelulares na sobrevivência do fungo no hospedeiro, podendo levar a descrição de
moléculas que atuam como fatores de virulência.
64
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