UNIVERSIDADE FEDERAL DE GOIÁS MINISTÉRIO DA EDUCAÇÃO E ...

UNIVERSIDADE FEDERAL DE GOIÁS

MINISTÉRIO DA EDUCAÇÃO E DESPORTOS

INSTITUTO DE CIÊNCIAS BIOLÓGICAS

PROGRAMA DE PÓS – GRADUAÇÃO EM BIOLOGIA

Sheyla Maria Rondon Caixeta Bonfim Quitinase de Paracoccidioides brasiliensis:

clonagem molecular e análise estrutural, filogenética, expressão e atividade

Orientadora:

Profa. Dra. Maristela Pereira

Dissertação de mestrado

Goiânia-Go, 2005

Quitinase de Paracoccidioides brasiliensis: clonagem molecular e análise estrutural, filogenética, expressão e atividade

_________________________________________________________________Sheyla Maria Rondon Caixeta Bonfim

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UNIVERSIDADE FEDERAL DE GOIÁS

INSTITUTO DE CIÊNCIAS BIOLÓGICAS

PROGRAMA DE PÓS – GRADUAÇÃO EM BIOLOGIA

Sheyla Maria Rondon Caixeta Bonfim

Quitinase de Paracoccidioides brasiliensis: clonagem

molecular e análise estrutural, filogenética,

expressão e atividade

Orientadora:


Dissertação desenvolvida no

Laboratório de Biologia Molecular, do

Departamento de Ciências Fisiológicas,

da Universidade Federal de Goiás,

como requisito parcial para obtenção de

Título de Mestre em Ciências

Biológicas, área de concentração

Bioquímica e Biologia Molecular.

Goiânia-Go, 2005



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BBAANNCCAA EEXXAAMMIINNAADDOORRAA

TITULARES:


Instituto de Ciências Biológicas, Universidade Federal de Goiás.

Profa. Dra. Célia Maria de Almeida Soares

Instituto de Ciências Biológicas, Universidade Federal de Goiás.

Profa. Dra. Maria José Soares Mendes-Giannini

Faculdade de Ciências Farmacêuticas/UNESP-Araraquara

SUPLENTES:

Prof. Dr. Cirano J. Ulhoa

Instituto de Ciências Biológicas, Universidade Federal de Goiás

Profa. Dra. Rosália Santos Amorim Jesuíno

Instituto de Ciências Biológicas, Universidade Federal de Goiás



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Aos meus filhos Mariana e Gabriel

Que eles também possam acreditar que seus

sonhos podem se tornar realidade e com

perseverança lutar para que eles se realizem!



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Ao meu marido Djaniro Júnior

Presença constante em todos os momentos, grande

companheiro e incentivador da realização deste

trabalho.



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Piensa que puedas y podrás

Piensa en grande y tus hechos crecerán

Piensa en pequeño y quedarás atrás


La batalla de la vida no siempre la gana el hombre

más fuerte

Tarde o temprano el hombre que gana es aquel que

cree poder hacerlo




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É maravilhoso: amar, viver, sorrir, sonhar!

É maravilhoso Senhor ter tão pouco a pedir, tanto a

agradecer!



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Michael Quoist

À Profa. Dra. Maristela Pereira

Meus sinceros agradecimentos pelo empenho, pelo

espírito de abertura com que coordenou o trabalho e

com quem aprendi a cultivar o amor à pesquisa por

seu exemplo de extrema dedicação!



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AGRADECIMENTOS

Agradeço a DEUS pela oportunidade de realizar este trabalho neste

momento, e por ter convivido com pessoas especiais com as quais eu

aprendi muito!

Aos meus pais Lázaro e Lívia por estarem sempre presentes em todos os

momentos!

À minha família: Djaniro Júnior, Mariana e Gabriel que sempre me

apoiaram e me incentivaram a seguir adiante na realização de um grande

sonho.

Ao meu irmão André, grande amigo e companheiro, pelo incentivo e pela

presença constante!

Às Profas. Célia, Fabrícia, Rosália e Úrsula pelos ensinamentos e preciosas

sugestões que vieram a enriquecer o trabalho.

Aos professores do Curso de Mestrado que contribuíram para o

aprimoramento da minha formação profissional e que sem dúvida

nortearam os rumos para o êxito deste trabalho.

Aos amigos Bruno, Lílian, Moniquinha e Nádia, com os quais dividi

momentos de alegrias e também de tristezas, que fizeram com que nossos

laços de amizade se estreitassem ainda mais!

Às colegas e colaboradoras da equipe: Aline, Cristielly, Ludier Kesser,

Glaciane, Lidiane, Patrícia Kott e Patrícia Zambuzzi.



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A todos os colegas do Laboratório de Biologia Molecular pela colaboração

e ensinamentos.

À Dona Zildete sempre carinhosa, prestativa e zelosa com todos os colegas

que fazem parte do grupo de trabalho do LBM.

A todos que contribuíram para a realização deste trabalho.

Muito Obrigada!



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SUMÁRIO

RESUMO.....................................................................................................................14

ABSTRACT................................................................................................................15

I. INTRODUÇÃO.....................................................................................................17

I.1. O FUNGO Paracoccidioides brasiliensis...............................................................17

I.2. A PARACOCCIDIOIDOMICOSE.......................................................................18

I.2.1. CONSIDERAÇÕES GERAIS.................................................................18

I.2.2. EPIDEMIOLOGIA..................................................................................19

I.2.3. PATOGÊNESE E PATOGENIA.............................................................19

I.2.4. TRATAMENTO......................................................................................21

I.3. PAREDE CELULAR..............................................................................................22

I.3.1. ESTRUTURA DA PAREDE CELULAR...............................................22

I.3.2. DIMORFISMO CELULAR.....................................................................23

I.4. ENZIMAS HIDROLÍTICAS DE FUNGOS.........................................................24

I.5. QUITINASES..........................................................................................................25

I.5.1. ASPECTOS GERAIS..............................................................................25

I.5.2. CLASSIFICAÇÃO..................................................................................27

I.5.3. CARACTERÍSTICAS.............................................................................32

I.5.3.1. ESTRUTURA.........................................................................32

I.5.3.2. REGULAÇÃO........................................................................33

I.5.3.3. ATIVIDADE...........................................................................34

I.5.3.4. INIBIDORES..........................................................................36

I.5.4. APLICAÇÕES...........................................................................37



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II. JUSTIFICATIVA...............................................................................................38

III. OBJETIVOS.......................................................................................................40

IV. MANUSCRITO.................................................................................................42

V. PERSPECTIVAS................................................................................................91

VI. REFERÊNCIAS BIBLIOGRÁFICAS......................................................93

VII. ANEXOS..........................................................................................................117 ANEXO I. PRODUÇÃO CIENTÍFICA DO MESTRADO...........................118

ANEXO II. NORMAS DA REVISTA...........................................................120



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LISTA DE ABREVIATURAS

[α-32P]-dCTP Deoxicitosina trifosfato marcada com fósforo 32 na posição α

bp: base pair - pares de bases

cDNA Ácido desoxirribonucléico complementar

AMPc Adenosina monofosfato cíclico

DNAse Desoxirribonuclease

EST Expressed Sequence Tags

GlcNac N-Acetilglicosamina

gp43 Glicoproteína de 43 kDa

Kb Kilobases

Kda Kilodaltons

L Litro

MAPK Mitogen-activated protein kinase- Proteína quinase ativada por mitógeno

Mpb Mega pares de bases

Mg Miligrama

mL Mililitro

mm Milímetro

mM Milimolar

mRNA Ácido ribonucleico mensageiro

ng Nanograma

ORF Open Reading Frame- Quadro aberto de leitura

Pb Paracoccidioides brasiliensis

PBS Phosphate Buffer Solution

PCM Paracoccidioidomicose

PCR Polimerase Chain Reaction- Reação da Polimerase em Cadeia

pH Potencial hidrogeniônico

SDS Dodecil Sulfato de Sódio

Ug Micrograma

uL Microlitro



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RESUMO

Um cDNA codificante de uma quitinase (Pbcts1) foi clonado por

rastreamento de uma biblioteca de células leveduriformes de

Paracoccidioides brasiliensis . O cDNA consiste de 1888 pares de bases e

codifica uma ORF de 1218 pares de bases correspondente à uma proteína de

45 kDa com 406 resíduos de aminoácidos. Pbcts1 é composta de duas

assinaturas no domínio catalítico da família 18 e parece pertencer à classe

fungo/bactéria. Análise de Southern Blott indicou que Pbcts1 está em cópia

única. Análise filogenética de Pbcts1 e outras quitinases apontam a

possibilidade de parálogos de várias quitinases estarem agrupados, baseado

em funções especializadas as quais refletem diversos e múltiplos papéis

desempenhados por quitinases de fungos. Análises computacionais revelaram

que Pbcts1 apresenta uma estrutura complexa de domínios que podem

implicar em multifuncionalidade. Embora Pbcts1 não apresente domínio de

ligação à quitina (CBD) e regiões ricas em serina/ treonina/ prolina, o domínio

imunoglobulina tipo C (Igc1) propicia a interação com a cadeia de quitina

durante a catálise. Atividade glicosil hidrolase foi avaliada e os resultados

demonstraram que P. brasiliensis é capaz de produzir e secretar estas enzimas

principalmente durante a transição levedura para micélio. Desta maneira, P.

brasiliensis deve ser capaz de usar quitina como fonte de carbono. Em adição,

Pbcts1 apresenta um domínio Aamy, indicando a possibilidade de atividade

alfa amilase. A presença de um sinal endocítico na proteína deduzida sugere

que ela pode ser secretada por um mecanismo de exportação vesicular não

clássico. A expressão de Pbcts1 em micélio, levedura, durante diferenciação

de micélio para levedura e em células leveduriformes obtidas de ratos

infectados, sugere a relevância desta molécula em P. brasiliensis, elegendo

PbCTS1 como um atrativo alvo de drogas.



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ABSTRACT

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening

a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The

cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding

to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1

is composed of two signature family 18 catalytic domains and seems to

belong to fungal/bacterial class. Southern blot analysis indicated that

Pbcts1 is a single copy. Phylogenetic analysis with PbCTS1 and other

chitinases point to the possibility of paralogous of several chitinases to be

grouped based in specialized functions, which may reflect the multiple and

diverse roles played by fungi chitinases. Computer-based sequence analysis

revealed that PbCTS1 presents a complex structure of domains that could

imply in multi functionality. Although PbCTS1 does not seem to present

chitin-binding domains (CBD) and serine/threonine/proline-rich regions

(STP), the immunoglobulin C-Type domain could propitiate interaction

with the chitin chain during catalysis. Glycosyl hydrolase activity was

evaluated and the results demonstrated that P. brasiliensis is able to

produce and secrete these enzymes mainly during transition from yeast to

mycelium. In this way, P. brasiliensis should be able to use chitin as a

carbon source. In addition, PbCTS1 presents an Aamy domain, indicating

the possibility of alpha amylase activity. The presence of an endocytic

signal in the deduced protein suggests that it could be secreted by a

vesicular nonclassical export pathway. The Pbcts1 expression in mycelium,

yeast, during differentiation from mycelium to yeast and in yeast cells

obtained from infected mice suggests the relevance of this molecule in P.

brasiliensis electing PbCTS1 as an attractive drug target.



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I. INTRODUÇÃO

I.1. O FUNGO Paracoccidioides brasiliensis

Paracoccidioides brasiliensis é um fungo dimórfico patogênico humano

causador da micose sistêmica denominada paracoccidioidomicose (PCM). Sabe-se que

o fungo adapta-se bem em solos úmidos, com pH ácido e ricos em vegetação e matéria

orgânica, em regiões de temperatura moderada com curto período de seca, onde pode

crescer sob a forma miceliana e produzir conídeos (Restrepo 1985).

O fungo cresce como micélio à 23ºC - 25ºC no meio ambiente e como levedura

à 35ºC - 37ºC nos tecidos do hospedeiro (Kanetsuna et al. 1972) caracterizando o

dimorfismo celular. Esse dimorfismo pode ser reproduzido em laboratório através do

cultivo do fungo nas respectivas temperaturas (Salem-Izaac etg al. 1997).

O fungo já foi isolado de animais como o morcego Artibeus lituratus (Grosse

et al. 1965), o macaco Saimiri sciureus (Johnson et al. 1977), o pingüim Pygoscelis

adeliae (Gezuele et al. 1989) e o tatu Dasypus novemcinctus (Silva-Vergara et al. 1999).

A relação entre o fungo e o meio ambiente não está totalmente definida, apesar do seu

isolamento de solo do Brasil (Silva-Vergara et al. 1998), da Argentina (Negroni et al.

1966) e da Venezuela (Albornoz et al. 1971). P. brasiliensis isolados do tatu têm sido

analisados quanto à virulência (Peracoli et al. 1999), antigenicidade e aspectos

moleculares (Sano et al. 1999) evidenciando a similaridade entre eles. Embora o P.

brasiliensis tenha sido isolado do solo, do trato digestivo e das fezes de alguns animais,

seu habitat natural ainda permanece desconhecido (Lacaz et al. 1999) devido à

dificuldade de um número maior de isolamentos.

A caracterização genômica de diferentes isolados do fungo tem sido possível

através da utilização da técnica de eletroforese em gel de pulso alternado PGFE (“Pulse

Field Gel Electroforesis”) (Feitosa et al. 2003). Alguns isolados apresentam um padrão

de quatro bandas cromossomais variando entre 2-10 Mpb, embora outros apresentem

variações entre 23.0-27.6 Mpb. Ainda, a microfluorimetria por microscopia confocal

indicou o conteúdo de DNA de P brasiliensis como sendo de 45.7 a 60,9 Mpb (Cano et

al. 1998). Montoya et al. (1999) estimaram que o tamanho estaria entre 23-31 Mpb,

indicando a presença de 10.000 a 15.000 genes dispostos em 4-5 cromossomos,



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evidenciando que significativas diferenças têm sido relatadas em relação ao tamanho do

genoma apresentado pelo fungo.

O fungo foi inicialmente caracterizado por Almeida (1930) como sendo do

gênero Paracoccidioides e da espécie brasiliensis. Atualmente o fungo P. brasiliensis é

classificado como pertencente ao reino Fungi, ao filo Ascomycota, à ordem Onygenales,

à família Onygenaceae, ao gênero Paracoccidioides e à espécie brasiliensis (San-Blas et

al. 2002).

I.2. A PARACOCCIDIOIDOMICOSE

I.2.1. CONSIDERAÇÕES GERAIS

Inicialmente descrita por Adolpho Lutz em 1908, a PCM causa a morte em

homens adultos, trabalhadores da área rural, em idade produtiva, de camada social de

baixa renda e pacientes com deficiência imunológica (Coutinho et al. 2002). Essa

doença é mais freqüente em indivíduos do sexo masculino, sendo que os baixos índices

de casos na população feminina são atribuídos ao efeito inibitório do hormônio 17 β-

estradiol na transição micélio-levedura. A apresentação clínica e o curso da infecção

dependem do paciente e do isolado e o aparecimento de lesões secundárias nas mucosas,

pele, linfonodos e glândulas adrenais é freqüente (Restrepo-Moreno 1993).

A infecção no hospedeiro humano geralmente ocorre pela inalação de

microconídeos, os quais alcançam o epitélio alveolar pulmonar onde se transformam na

forma parasitária de levedura podendo disseminar-se para outros órgãos (Restrepo et al.

2001).



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I.2.2. EPIDEMIOLOGIA

A PCM é uma micose endêmica com alta prevalência na América Latina

(Franco et al. 1987) onde se estima que cerca de dez milhões de pessoas estejam

infectadas, embora somente cerca de 2% apresentam predisposição para desenvolver a

doença (McEwen et al. 1995). Dados epidemiológicos indicam uma distribuição que se

extende desde o México até a Argentina (San-Blas et al. 2002). A distribuição da PCM

é heterogênea e os países que se destacam por apresentar o maior número de casos

relatados são o Brasil, Colômbia e a Venezuela. No Brasil, as regiões Sul, Sudeste e

Centro-Oeste apresentam maior incidência, apresentando o estado de Goiás os maiores

números de casos (Wanke & Londero 1994).

Coutinho et al. (2002) avaliaram 3.181 óbitos por PCM no Brasil no período de

1980 a 1995. Esta micose mostrou grande magnitude, destacando-se como a oitava

causa de mortalidade entre as doenças predominantemente crônicas e como a detentora

da maior taxa de mortalidade entre as micoses sistêmicas. A taxa de mortalidade média

anual foi de 1.45/milhão de habitantes, sendo que a região Sul apresentou maior taxa

seguida da região Centro-Oeste, a qual apresentou tendência à ascensão. O estudo

mostrou que a taxa de mortalidade pode ser considerada como indicador para definir a

doença como importante agravo de saúde no Brasil. A doença prevaleceu como

endêmica nas áreas não metropolitanas, onde a proporção homens/mulheres foi de 562

homens/100 mulheres acometidos. O grupo etário entre 30-59 anos foi o mais atingido,

seguido dos pacientes com 60 anos ou mais. A taxa de mortalidade predominou em

indivíduos do sexo masculino, com 84,75% dos óbitos. Blotta et al. (1999) relataram

que a incidência de PCM na infância está em torno de 5% e o tipo adulto de PCM

totaliza 90% dos casos relatados.

I.2.3. PATOGÊNESE E PATOGENIA

A PCM é uma doença granulomatosa caracterizada pela formação de

aglomerados de células epiteliais com áreas centrais de necrose e agregados de

leucócitos polimorfonucleares. Evidências indicam que o granuloma está intimamente

relacionado à resposta imune do hospedeiro e deve representar uma resposta tecido-



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específica ao fungo na tentativa de destruí-lo e prevenir a sua multiplicação (De Brito et

al. 1994).

A inalação de propágulos do fungo pelo hospedeiro humano é a principal via de

entrada do parasita (Lacaz et al. 1991) propiciando o início da infecção pelos pulmões e a

disseminação para outros órgãos através dos linfonodos e da corrente sanguínea. No

homem, micélios ou conídeos se convertem em leveduras, que se reproduzem por

brotamentos múltiplos ou simples, nos tecidos infectados, sendo fagocitados por

macrófagos (Brummer et al. 1989). A fase leveduriforme sintetiza moléculas antigênicas

que são capazes de interagir com o sistema imune do hospedeiro e contribuir para a

patogênese da doença. Dessa maneira, o processo de transição é o primeiro requerimento

para o estabelecimento da PCM no hospedeiro (Lacaz et al. 1991).

A maioria dos indivíduos infectados desenvolve a forma assintomática ou

subclínica, a qual progride para doença com uma diversidade de formas clínicas em função

de fatores do hospedeiro, níveis de virulência das linhagens e condições ambientais (San-

Blas et al. 2002). Duas formas da doença são reconhecidas: a forma sub-aguda ou aguda e a

forma crônica. As formas disseminada sub-aguda e pulmonar aguda têm sido descritas

como a forma juvenil encontrada em crianças. As formas disseminada e pulmonar crônica

têm sido encontradas em adultos jovens (Franco et al. 1987).

As manifestações clínicas da PCM são classificadas em duas formas polares

principais (Franco et al. 1994). A forma polar positiva é caracterizada pela presença de

lesões generalizadas, pelo elevado título de anticorpos específicos aos antígenos de P.

brasiliensis, imunidade celular enfraquecida e presença de reação inflamatória

granulomatosa, contendo muitos fungos viáveis. Na forma polar negativa, as lesões são

localizadas, o título de anticorpos específicos ao P. brasiliensis é baixo ou ausente, a

imunidade celular é preservada e as lesões granulomatosas são compactas e com baixo

número de fungos. Ambas formas clínicas da doença estão associadas com extensas

seqüelas mediadas por infecções sistêmicas, incluindo lesões e uma resposta anormal

mediada por células.

Assim, as formas severas da doença com intenso envolvimento de vários

órgãos com lesões progressivas conduzem à morte, podendo ser acompanhadas por

perda gradual de respostas imunes celulares específicas e títulos altos de anticorpos

específicos. As formas moderadas da doença, apresentando poucas lesões localizadas,

conduzem à cura e paralelamente mantêm as respostas imunes e baixos níveis de

anticorpos específicos (Fava- Netto 1995).



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I.2.4 TRATAMENTO

O tratamento de PCM é prolongado, sendo que os pacientes recebem terapia

entre um e dois anos, e na ausência de terapia a doença pode ser fatal.

Alguns antifúngicos estão disponíveis comercialmente, com atuação a nível de

envelope celular (parede e membrana plasmática) e particularmente de esteróis que

compõem a membrana do fungo: ergosterol e a sua biossíntese. As drogas antifúngicas

correntes têm sido os polienos e os azoles.

A anfotericina B tem sido o principal polieno administrado sistematicamente

para tratar infecção visceral. Ela atua através da ligação ao ergosterol prejudicando as

funções da membrana, causando o extravazamento de conteúdos celulares e

apresentando alta toxicidade para células de mamíferos, principalmente nefrotoxicidade

(Odds et al. 2003).

Avanços recentes foram obtidos no desenvolvimento da terceira geração de

azoles (Tkacz & Didomenico 2001). Os azoles, posaconazole, ravuconazole e

voriconazole, apresentam como principal efeito a inibição da demetilação do lanosterol

em ergosterol durante o mecanismo de biossíntese. Voriconazole e ravuconazole são

relacionados ao fluconazol, que é um agente antifúngico com desejáveis propriedades

farmacológicas (Hahn et al. 2000), e que de acordo com testes de susceptibilidade,

demonstrou ser mais eficaz in vivo do que in vitro (Kurita et al. 2003). Atualmente o

cetoconazol e o itraconazol têm sido as drogas de escolha.

Em 2001 -pela primeira vez em trinta anos- um membro de uma nova classe de

drogas terapêuticas antifúngicas, caspofungina ingressou na clínica. A caspofungina

pertence à classe das equinocandinas (anidulafungina, caspofungina e micafungina) que

inibem a síntese de polissacarídeos da parede celular como a β-1,3 glucana (Denning 2002).

As limitações terapêuticas das duas classes de drogas antifúngicas correntes,

polienos e azoles, os quais apresentam toxicidade e um limitado espectro de eficácia,

respectivamente, e o aumento de incidência de infecções fúngicas ameaçadoras

(Georgopapadakou & Tkacz 1995), bem como o surgimento de isolados clínicos

resistentes a drogas (Rex et al. 1994) tem se tornado preocupante. Entretanto, pela

impossibilidade de dar ampla cobertura a resistência a drogas e prover tratamentos mais

eficazes e seguros de infecções fúngicas, novas abordagens para identificação e

desenvolvimento das mesmas se faz necessário.



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I.3. PAREDE CELULAR

A parede celular está envolvida em vários processos vitais para a célula, tais

como crescimento vegetativo, morfogênese, divisão celular, secreção de

macromoléculas e proteção contra alterações osmóticas e tem servido como aparato

através do qual a informação extracelular é transmitida e integrada nos processos

intracelulares (Qadota et al. 1996).

A parede celular está em contato com células do hospedeiro e atua como

reservatório de moléculas como antígenos e enzimas, que têm um papel ativo durante a

infecção (Latgé 1999). Estudos sugerem que a parede celular é uma estrutura dinâmica

onde polímeros constitutivos são continuamente modificados e rearranjados durante a

biossíntese (Popolo et al. 2001); os mecanismos de biossíntese têm sido reconhecidos

como específicos alvos para drogas capazes de bloqueiar a síntese de componentes da

parede sem interferir em qualquer outro processo metabólico do hospedeiro.

I.3.1. ESTRUTURA DA PAREDE CELULAR

Os principais constituintes da parede celular de ambas as formas miceliana e

leveduriforme do fungo P. brasiliensis são quitina, glucanas e proteínas. A parede de

micélio tem uma grande concentração de proteínas (24 a 41%) quando comparada com

células leveduriformes (7 a 14%) (Kanetsuna et al. 1969). O principal polissacarídeo da

parede celular de leveduras de P.brasiliensis é a α-glucana, enquanto que em micélio

são as β-glucanas e as galactomananas (aproximadamente 6%). As α-glucanas de

leveduras contêm pequenas concentrações de ligações glicosídicas α-1,3 ou α-1,6,

sendo que as β-glucanas de micélio contêm ligações glicosídicas β-1,3. Estudos de

microscopia eletrônica têm mostrado que em micélio a parede celular apresenta uma

única camada contendo quitina e β-glucana (Carbonell & Rodriguez 1968) enquanto

que foram observadas três camadas em levedura; a camada interna é formada de β-

glucana e a externa de α-glucana (Carbonell 1972).

Esse estudo possibilitou a identificação de genes envolvidos no metabolismo

da parede celular de P. brasiliensis. Entre eles estão as sintases (β-1,3 glucana sintases,

α-1,3 glucana sintases e quitina sintases) e as enzimas envolvidas na remodelagem da



23

parede celular (manosiltransferases e hidrolases). A rede de laboratórios da região

Centro Oeste do Brasil desenvolveu o projeto Genoma EST entitulado"Genoma

Funcional e Diferencial de P. brasiliensis" (http:/www.biomol.unb.br/Pb) com o

principal objetivo de gerar informações sobre o transcriptoma das fases de micélio e

levedura deste fungo, onde 3938 ESTs e 1040 genes dos transcriptomas foram anotados

(Felipe et al. 2003).

A parede celular apresenta um papel essencial na patobiologia de P.

brasiliensis. As mudanças morfogenéticas estão diretamente associadas com o ciclo de

vida deste fungo gerando um rearranjo molecular (Da Silva et al. 1994). Os

polissacarídeos da parede celular α-1,3-glucana e β-1,3-glucana têm sido sugeridos

como possíveis fatores de virulência durante a transição dimórfica de P. brasiliensis,

pois um baixo conteúdo de α-1,3-glucana na parede celular de leveduras tem sido

correlacionado com baixa virulência (San-Blas et al. 1994). Em adição, o cultivo, in

vitro, de isolados de P. brasiliensis por um longo período de tempo leva à perda da

virulência e a um baixo conteúdo de α-1,3 glucana na parede celular (San-Blas et al.

1994). A α-1,3 glucana presente na superfície pode atuar como uma camada de

proteção contra mecanismos de defesa do hospedeiro devido à incapacidade de células

fagocíticas do hospedeiro em digerirem a α-1,3 glucana (San-Blas et al. 1994).

A transição dimórfica do P. brasiliensis ocorre simultaneamente com

mudanças na composição da parede celular, envolvendo alterações estruturais nos

polímeros de carboidratos (San-Blas et al. 1994) e reorganização dos lipídeos de

membrana, especialmente glicoesfingolipídeos (Toledo et al. 1999). Quando o fungo

adota a forma leveduriforme, o aumento do conteúdo de quitina é observado na parede

celular seguido por alteração na estrutura anomérica da glucana de um polímero de β-

1,3 para α-1,3 (San-Blas & Niño Vega 2002).

I.3.2. DIMORFISMO CELULAR

O termo dimorfismo refere-se à habilidade apresentada por alguns fungos

patógenos como H. capsulatum, Blastomyces dermatitidis e P. brasiliensis de

apresentarem mudanças reversíveis entre as formas miceliana e leveduriforme . Em P.

brasiliensis este processo de diferenciação é reversível e dependente da regulação da



24

enzima glucana sintase levando à produção de β-1,3 glucanas na fase miceliana e α-1,3

glucanas na fase leveduriforme (San-Blas 1993).

Embora a base molecular do dimorfismo de P. brasiliensis ainda permaneça

desconhecida, vários genes expressos diferencialmente nas fases leveduriforme e

miceliana deste fungo (Costa et al. 2002, Felipe et al. 2005). A identificação de genes

diferencialmente expressos necessários para a transição dimórfica é de suma

importância para o melhor entendimento a cerca dos eventos decorrentes do

estabelecimento da infecção e doença (Hensel et al. 1996).

No intuito de entender os mecanismos de controle morfogenético,

pesquisadores têm estudado a estrutura da parede celular, processos metabólicos e

cascatas de sinalização, além de utilizar métodos clássicos para detecção enzimática e

quantificação bioquímica de uma grande variedade de metabólitos. Estudos têm

revelado que os mecanismos de sinalização celular que regulam a patogenicidade são

controlados por AMPc (Adenosina monofosfato cíclico) e MAPK (Proteína quinase

ativada por mitógeno), visto que o AMPc exógeno inibe a transição levedura-micélio,

mantendo a forma patogênica do fungo (Borges-Walmsley et al. 2000). Drogas

inibidoras do mecanismo de sinalização cálcio/calmodulina foram capazes de inibir a

transição micélio-levedura de P. brasiliensis, sendo que a calmodulina se liga ao cálcio

alterando sua conformação e participando na diferenciação do fungo P. brasiliensis

(Carvalho et al. 2003).

I.4. ENZIMAS HIDROLÍTICAS DE FUNGOS

As enzimas hidrolíticas glucanases, quitinases e N-acetyl-β-D-

glicosaminidases (NAGs) têm sido associadas à patogenicidade e virulência (Kurokawa

et al. 1998), podendo estar associadas também à manutenção da estrutura da parede

celular (Gow et al. 1993) e morfogênese de fungos durante o crescimento apical, divisão

e diferenciação celulares (Isaac & Gokhale 1982), bem como na liberação de esporos

(Papavizas 1985) e elongamento de hifas (Kuranda & Robbins 1991). As enzimas

hidrolíticas são responsáveis pelo afrouxamento da estrutura da parede celular (Wessels

1988). A maioria das hidrolases caracterizadas apresentam atividade de quitinase, de

glucanase e também de transglicosilase estando envolvidas na remodelagem da parede

celular durante o crescimento e morfogênese, bem como contribuindo para a digestão e



25

utilização de quitina exógena como fonte de nutrientes orgânicos para produção de

energia e biossíntese.

De acordo com a nomenclatura sugerida por Sahai et al. (1993), as enzimas

quitinolíticas são divididas em dois principais tipos: as quitinases (EC 3.2.1.14) que

podem ser endoquitinases ou exoquitinases e NAGs (EC 3.2.1.30). Endoquitinases são

definidas como enzimas que catalizam a hidrólise randômica de ligações β-1,4 de N-

acetilglicosamina (GlcNac) produzindo produtos solúveis como quitotetraose,

quitotriose e diacetilquitobiose. Exoquitinases podem ser sub-divididas em duas sub-

categorias: quitobiosidases que catalizam a liberação progressiva de diacetilquitobiose

do final não redutor da cadeia de quitina (Harman et al. 1993) e NAG que hidroliza

quitobiose ou libera N-β-D-acetilglicosamina do polímero de quitina.

A quitina, um polissacarídeo de N-acetilglicosamina (GlcNac) unidas por

ligações glicosídicas tipo β 1-4, é um componente estrutural da parede celular de

fungos e de exoesqueleto de invertebrados, como insetos e crustáceos (Flach 1992),

sendo considerado o segundo mais abundante biopolímero natural depois da celulose

(Haki et al. 2003). A manutenção da parede celular envolve a ação coordenada de

síntese (quitina sintases) e hidrólise (quitinases), sendo que o equilíbrio deve ser

regulado para manter a plasticidade durante o crescimento celular e separação das

células mãe e células filhas (Martín-Cuadrado et al. 2003). As enzimas quitina sintase e

quitinase apresentam-se reguladas durante a transição levedura-micélio em C. albicans

como mostrado por Selvaggini et al. (2004).

I.5. QUITINASES

I.5.1. ASPECTOS GERAIS

As quitinases são enzimas que hidrolizam a quitina a oligossacarídeos através

da clivagem de ligações glicosídicas entre os carbonos C1 e C4 de GlcNac consecutivas

presentes no polímero de quitina. Essas enzimas estão amplamente dispersas na

natureza e têm sido encontradas em uma grande variedade de organismos, incluindo

plantas (Mitsunaga et al. 2004) bactérias (Yuli et al. 2004), fungos (Selvaggini et al.

2004), algumas classes de invertebrados, como insetos e parasitas (Ramalho-Ortigão et



26

al. 2003), vertebrados (Suzuki et al. 2001) e até mesmo em organismos que não

produzem quitina (Watanabe et al. 1999).

Em plantas, as quitinases atuam na defesa contra fungos patogênicos que

contêm quitina (John 1997), inibindo crescimento de fungos e gerando oligossacarídeos

que atuam como elicitores, moléculas-sinal que regulam o crescimento e

desenvolvimento em plantas (Kasprzewska 2003). Em Dioscorea opposita Thunb a

presença de uma seqüência de aminoácidos C-terminal envolvida no endereçamento

para vacúolos intracelulares, pode auxiliar no entendimento do mecanismo de defesa

associado à proteólise (Mitsunaga et al. 2004).

As bactérias produzem várias quitinases, provavelmente para hidrolizar a

diversidade de moléculas de quitina encontradas na natureza e utilizá-la como fonte de

carbono, sendo expressas diferencialmente em função da disponibilidade de nutrientes

no ambiente (Svitil et al. 1997). As quitinases de bactérias têm sido isoladas de

Enterobacter sp. (Park et al. 1997), Clostridium paraputrificum (Morimoto et al. 1997),

Streptomyces thermoviolaceus (Tsujibo et al. 1998), Streptomyces sp. (Patil et al. 2000),

Chromobacterium violaceum (Chernin et al. 1998), Bacillus sp. (Yuli et al. 2004),

Vibrio fuernisii e Vibrio cholerae (Li et al. 2004).

Em fungos, a quitinase atua na degradação da parede celular (Kitamura et al.

2002), atuando no crescimento de hifas, diferenciação em esporos, assimilação de

quitina e micoparasitismo (Gooday et al. 1997) como demonstrado em Gliocladium

virens, Aphanocladium album e Trichoderma spp. (Di Pietro et al. 1993).

Em insetos, a quitina associa-se a proteínas para formar a cutícula do

exoesqueleto e matriz peritrófica, a qual contém uma mistura de glicosaminoglicanos,

glicoproteínas e quitina (Lehane et al. 1996). A matriz peritrófica serve como barreira

contra patógenos (Shahabuddin et al. 1996), além de atuar na digestão em insetos

hematófagos (Shao et al. 2001) e também como substrato para a ligação do grupo heme

como ocorre em Aedes aegypti (Pascoa et al. 2002). As quitinases são importantes na

degradação da matriz peritrófica do intestino de insetos por permitir uma absorção

eficiente de nutrientes. Em crustáceos essa localização da quitinase permite a

degradação parcial do seu exoesqueleto durante o desenvolvimento, através de um

mecanismo de controle hormonal (Spindler-Barth 1993).

Estudos moleculares têm mostrado a presença de genes de quitinases

caracterizados de uma variedade de parasitas eucariotos incluindo Plasmodium

(Shahabuddin et al. 1999), o nematoda Brugia malayi (Fuhramn et al. 1992) e

Leishmania (Shakarian et al. 1998). É interessante ressaltar que as quitinases secretadas



27

por parasitas como Plasmodium falciparum (Vinetz et al. 1999) e Plasmodium

gallinaceum (Vinetz et al. 2000) estão associadas com a ruptura da matriz peritrófica.

Em vertebrados, as quitinases são encontradas no trato digestivo, participando

do metabolismo de carboidratos (Boot et al. 2001). Em mamíferos, a quitinase

quitotriosidase está presente no soro humano e em macrófagos (Boot et al. 1995), sendo

secretada em mamíferos conforme mostrado em trabalhos de Suzuki et al. (2001). A

enzima quitotriosidase é um membro da família de quitinases capaz de hidrolizar

quitina. Embora apresente homologia com outras quitinases de plantas, bactérias,

fungos, nematodas e insetos; esta enzima é utilizada como marcador de ativação em

doenças como Gaucher e é particularmente aumentada em indivíduos com

arteriosclerose (Hollak et al. 1994).

I.5.2. CLASSIFICAÇÃO

As quitinases são enzimas que pertencem à superfamília das glicosilhidrolases

e apresentam-se distribuídas em duas famílias, 18 e 19, de acordo com a homologia

existente entre as seqüências de aminoácidos e diferenças nos mecanismos de reação

(Brameld & Goddard III 1998) (Tabela 1).



28



29

A família 18 tem sido encontrada em bactérias, fungos, animais e plantas das

classes III e V. O domínio catalítico da família 18 apresenta uma estrutura em forma de

α/β 8- TIM BARRIL (Hollis et al. 2002), hidroliza as pontes glicosídicas, produzindo

fragmentos ß-anoméricos (Iseli et al. 1996), apresenta sensibilidade a alosamidina e

possui a seqüência consenso característica DXDXE, com exceção da glicoproteína

humana GP39 (Hakal et al. 1993), da lecitina concanavalina B isolada da planta

Canavalia ensiforms (Henning et al. 1995) e de fatores de crescimento dependentes de

insulina em Drosophila melanogaster (Kawamura et al. 1999).

A catálise assistida pelo substrato é o modelo aceito para o mecanismo

catalítico da família 18 (Brameld et al. 1998) o qual envolve resíduos conservados no

motivo DXDXE e o grupo N-acetil do açúcar. Vaaje-Kolstad (2004) propõe o seguinte

modelo descrito na Figura 1: A- ligação do substrato, B- distorção do substrato e

protonação da ligação glicosídica e ataque nucleofílico do grupo N-acetil no carbono

anomérico, C- formação do intermediário íon oxazolinium e partida do grupo, sendo

que uma molécula de água é destinada a hidrólise do íon; é dirigida ao carbono

anomérico. A catálise é denominada ácida porque depende de um grupo ácido que

posiciona-se próximo ao domínio catalítico. Em C. immitis experimentos de mutagênese

sítio-dirigida demonstraram que o resíduo Glu171 é o grupo ácido catalítico (Hollis et

al. 2000).



30

Fig. 1 Mecanismo catalítico das glicosil hidrolases da família 18. A figura mostra o seguinte mecanismo A- Ligação ao substrato B- Distorção do substrato e protonação da ligação glicosídica C- Formação do íon intermediário oxazolinium e partida do grupo .



31

Na família 19 estão incluídas as quitinases de plantas das classes I, II e IV

(Neuhaus et al. 1996), bactérias do gênero Streptomyces sp (Saito et al. 1999),

Streptomyces griseus (Ohno et al. 1996) e Aeromonas sp. (Ueda et al. 2003). A presença

dessas quitinases em bactérias tem sido atribuída a uma possível transferência vertical

desses genes provenientes de plantas superiores durante associação em um mesmo

habitat (Watanabe et al. 1999).

A classificação das quitinases de plantas é baseada na estrutura do domínio

catalítico e no mecanismo de ação. Entretanto algumas informações adicionais têm sido

feitas. As classes I e IV de plantas possuem um domínio com core altamente conservado

situado na região N-terminal rico em cisteína envolvido na ligação ao substrato, seguido

de peptídeo sinal (Shinshi et al. 1990). As quitinases da classe IV são menores que as da

classe I. As da classe III homólogas às da classe I e as da classe II são homólogas às da

classe I e IV, mas não apresentam o domínio rico em cisteína.

De acordo com análises da estrutura cristalográfica de enzimas da família 19,

estas apresentam um alto conteúdo α- hélice com core conservado em seu domínio

catalítico (Monzingo et al. 1996). O mecanismo de hidrólise envolve inversão com

produção de fragmentos α-anoméricos (Ohno et al. 1996) utilizando um mecanismo

catalítico ácido-básico (Garcia-Casado et al. 1998). O resíduo Glu 315 tem sido

identificado como o resíduo doador de prótons envolvidos na catálise e apresenta-se

conservado em domínios catalíticos da família 18. Em Clostridium paratrificum, este

resíduo Glu é conservado como Glu 184 (Morimoto et al. 1997).

O sequenciamento do genoma de A. fumigatus, A. nidulans e C. immitis

permitiu identificar um grande número de genes que codificam quitinases. As quitinases

foram classificadas como quitinases fungo/bactéria devido à similaridade encontrada em

relação às quitinases de bactérias, com massa molecular de aproximadamente 46-48

kDa e não apresentando papel morfogenético. As quitinases fungo/planta similares

àquelas descritas em plantas apresentaram massa molecular de 83-97 kDa atuando

durante o crescimento e morfogênese (Jaques et al. 2003).



32

I.5.3. CARACTERÍSTICAS

I.5.3.1 ESTRUTURA

Uma das principais características observadas em quitinases de vários

organismos é a presença de uma arquitetura em multidomínio, que inclui um domínio

catalítico, um domínio rico em serina-treonina glicosilado e um domínio de ligação à

quitina rico em cisteína (Yan et al. 2002).

O domínio catalítico está envolvido na catálise da hidrólise de ligações

glicosídicas e o domínio de ligação ao polissacarídeo é responsável pela mediação da

ligação da enzima ao substrato. Entretanto, alguns organismos, como o inseto Glossina

morsitans morsitans não apresentam o domínio rico em serina-treonina (Yan et al.

2002).

O domínio catalítico das quitinases da família 18 apresenta uma estrutura em

forma de α/β 8- TIM BARRIL em Vibrio carchariae (Suginta et al. 2004) e em C.

immitis (Hollis et al. 2000) além de apresentarem a seqüência característica DXDXE, de

acordo com análises de similaridades estruturais entre eles.

O domínio de ligação à quitina deve definir a localizacão da enzima na parede

celular (Kuranda et al. 1991). Algumas enzimas de fungos possuem este domínio

(Kuranda et al. 1991) enquanto que outras não possuem (Hayes et al. 1994). Em

mamíferos a ausência deste domínio não impede a hidrólise do substrato solúvel, mas

leva à perda da capacidade de hidrólise de quitina insolúvel (Tjoelker et al. 2000). As

quitinases de nematodas e insetos têm como função ancorar a enzima ao substrato

facilitando o processo de hidrólise. Essa capacidade é dada pela presença de um

domínio rico em cisteína na região C-terminal (Barry et al. 1999).

Variações na estrutura usual já descrita para quitinase podem ocorrer, como a

estrutura apresentada por Tenebrio molitor em que a análise da seqüência mostrou cinco

unidades de quitinase de aproximadamente 480 aminoácidos com similaridade às

quitinases da família 18. Estas unidades são separadas por regiões menos conservadas

contendo seqüências ricas em prolina, ácido glutâmico, serina e treonina, domínios de

ligação à quitina e domínios de ligação à mucina (Royer et al. 2002). Essas quitinases

estão envolvidas na lise de mucina, uma glicoproteína de alto peso molecular, sendo

80% deste determinado por carboidratos, com a função de fornecer energia para o



33

metabolismo de parasitas. Este fato também pode ser observado em Trichomonas

vaginalis (Loiseau et al 2002).

A organização estrutural de quitinases (Sasaki et al.2002) define-se por

módulos em função da associação dos domínios e sub-sítios catalíticos de acordo coma

família. Embora sejam bactérias, as Aeromonas sp, pertencentes à família 19, possuem

quitinases com sub-sítios característicos da família 18, definindo um novo tipo de

quitinase da família 19 com propriedades estruturais e funcionais (Ueda et al. 2003).

As quitinases das classes I e IV de plantas apresentam peptídeo sinal de

direcionamento extracelular (Shinshi et al. 1990). Em Vibrio cholerae este peptídeo

sinal está localizado entre os aminoácidos 75 e 555 (Folster et al. 2002). Em

Onchocerca volvulus (Wu et al. 2003), Entamoeba histolytica (Ghosh et al. 1999), Bufo

japonicus (Oshima et al. 2002), Glossina morsitans morsitans, Heterodera glycines

(Gao et al. 2002) e Sacccharomyces cerevisae (Kuranda &Robbins 1991) o número de

aminoácidos que compõem este domínio varia de 12 a 24. A quitinase de Trichoderma

harzianum possui dois peptídeos, sendo um com 22 aminoácidos e outro com 12

aminoácidos (Garcia et al. 1994).

I.5.3.2 REGULAÇÃO

A síntese e a hidrólise de quitina são processos regulados durante a transição

levedura-micélio em fungos dimórficos. Em Candida albicans a enzima mostra-se ativa

na fase leveduriforme (Selvaggini et al. 2004).

Em fungos, os genes de quitinase são induzidos na presença de quitina e

reprimidos na presença de glicose (Mach et al. 1999). Este comportamento tem sido

descrito para quitinases de Aphanocladium álbum (Blaiseau et al. 1992), Trichoderma

harzianum (Carsolio et al 1999), Streptomyces thermoviolaceus (Tsujibo et al 1998) e

Enterobacter sp. Neste último, uma seqüência localizada na porção acima da região

promotora está envolvida na regulação do gene de quitinase (Park et al. 1997). A

regulação da expressão dos maiores genes de quitinase ech42 e nag1 de T. harzianum

foi avaliada e significante expressão de ech42 foi observada após prolongada limitação

de fontes de carbono (Mach et al. 1999).

Vários genes de quitinase têm sido isolados de linhagens de S. cerevisae,

Trichoderma spp., Metahirzium anisopliae, Histoplasma capsulatum, T. harzianum e os



34

níveis de expressão, bem como condições limitantes in vivo e in vitro (Baek et al. 1999)

têm sido avaliados. A expressão de quitinase e os seus efeitos na morfologia da levedura

de S. pombe foram mostrados por Shimono et al. (2002), indicando que o sistema de

expressão é eficiente pela presença de um gene regulador Ace2p que controla a

produção de pseudohifas.

As bactérias sintetizam uma grande variedade de enzimas quitinolíticas, dentre

elas duas endoquitinases presentes em C. violaceum reguladas por um sistema de

“quórum-sensing” induzido por AHL (N-acyl homoserine lactone), que apresenta-se

como uma molécula sinal capaz de controlar a produção enzimática de quitinase

(Chernin et al. 1998). Em Pseudomonas aeruginosa o mesmo sistema regula a

produção de quitinase no sobrenadante do filtrado celular (Thompson et al. 2001).

I.5.3.3 ATIVIDADE

A atividade enzimática das quitinases tem sido avaliada através de métodos de

detecção que incluem ensaios colorimétricos (Reissig et al. 1955), ensaios radioativos

(Molano et al. 1977), detecção direta utilizando a eletroforese em gel de poliacrilamida

(Pan et al. 1991), HPLC-Cromatografia Líquida de Alta Performance (Koga et al. 1999)

e espectroscopia (Roberts et al. 1994).

Um método típico para avaliar a atividade de quitinases e que permite selecionar

microrganismos quitinolíticos, envolve a incubação com o substrato e a quantificação

colorimétrica de quitooligossacarídeos produzidos pela reação diagnóstica com ácido

dinitrosalicílico (DNAS) e que se baseia na avaliação de açúcares redutores liberados

(Monreal & Reese 1969). Esta técnica apresenta pouca sensibilidade e não é facilmente

aplicável a microrganismos fracamente quitinolíticos.

O uso de quitina coloidal como substrato marcada com Remazol brilhante (CC-

RBB) tem melhorado a detecção e aumentado a sensibilidade por apresentar

solubilidade mais rápida, ausência de toxicidade para microrganismos podendo ser

utilizado como fonte de carbono e indutor de quitinases em meio de cultura (Ramirez

et al. 2004).

A quitinase extracelular produzida hidroliza o substato, liberando

proporcionalmente o substrato corado que não foi utilizado, o qual é dosado em

espectrofotômetro (Ramírez et al. 2004). As quitinases secretadas também podem ser



35

detectadas utilizando um substrato fluorescente 4-Methylumbelliferil sintético (4-MU),

como mostrado para quitinases de G. morsitans morsitans (Yan et al. 2002), Aspergillus

fumigatus (Overdijk et al. 1999) e em Pseudomonas aeruginosa (Thompson et al. 2001).

O método mais recente é baseado na associação de outras substâncias ao

substrato ligado ao calcofluor. Este método foi utilizado para avaliação da atividade

enzimática em plantas (Velasquez et al. 2004). Um outro método, o qual utiliza quitina

azure como substrato apresenta vantagens com relação`a linearidade apresentada pelos

resultados detectados (Guo et al. 2004).

Como já foi descrito, as quitinases podem apresentar atividade de

endoquitinase, exoquitinase e de transglicosilação. Kang et al. (1999) detectaram uma

quitinase de Metahirzium anisopliae com atividade endo e exo, baseado na degradação

de oligômeros de quitina e vários compostos químicos. O polímero de quitina foi

utilizado como substrato para detectar a atividade endo e a enzima purificada exibiu alta

atividade quitinolítica em presença do substrato Pnp-nag2, indicando que esta enzima

tem uma caraterística de quitobiase (exoquitinase). Em Aspergillus fumigatus foi

identificada uma quitinase que possui atividade de endoquitinase, exoquitinase e

transglicosilase com um único modo de ação (Xia et al. 2001).

As quitinases e as quitinas sintases estão associadas com N-

acetilglicosaminidases formando um sistema integrado triplo que parece estar envolvido

na extensão apical e ramificação da levedura (Rast et al. 2000). Um pequeno número

destas hidrolases deve ser necessário para modificações da parede celular em

organismos que apresentam dimorfismo como mostrado em S. cerevisae que contém

somente dois genes de quitinases CTS I e CTS2 (Giaver et al 2002) e em C. albicans

que contém quatro ou cinco genes de quitinases. Além disso, pode haver uma interação

de enzimas hidrolíticas como quitinases e glucanases que podem atuar sinergisticamente

envolvendo diferentes isoformas de quitinases e ß-1,3 glucanases como demonstrado

em Nicotiana tabacum (Buurlage et al. 1993).

As quitinases de microrganismos apresentam termoestabilidade, pois após três

horas a 60°C as mesmas se encontram ativas (Yuli et al. 2004, Wen et al. 2002). Em A.

fumigatus essa condição é explicada pela presença de 10,22% de leucina em sua

composição (Xia et al. 2001).

A quitinase de P. aeruginosa 385 é constitutiva e dosagens da atividade

quitinolítica mostraram uma maior atividade no citoplasma (66%), do que em frações de

membrana (2%). Nenhuma atividade foi verificada no meio de cultura, indicando que a

enzima não é secretada (Loiseau et al.2002). Sabe-se que esta bactéria possui um



36

sistema regulador “quórum-sensing” que controla a secreção mas não a produção

enzimática. Este fato pode ser explicado pela ausência nesta linhagem deste sistema de

regulação (Thompson et al. 2001).

I.5.3.4. INIBIDORES

A enzima quitinase pode ter sua atividade bloqueada por drogas inibidoras.

Embora a alosamidina seja de alto custo e de difícil síntese, é o inibidor mais potente

em estudo. Atualmente a alosamidina, derivado de Streptomyces (Nishhimoto et al.

1991) é capaz de inibir a atividade de quitinase proveniente de Candida (Milewski et al.

1992). Esse glicosídeo é um inibidor competitivo da quitinase do fungo apresentando

afinidade com quitinase de S.cerevisae e similaridade com intermediários da reação

(Bortone et al. 2002). Estudos têm mostrado que a alosamidina é capaz de inibir todas

as quitinases da família 18 atuando através de um mecanismo de inibição competitiva

(Blattner et al. 1996). Em T. vaginalis entre as atividades qutinolíticas, N-

acetilhexosaminidase (NH-ase), quitotriosidase (endo) quitotobiosidase (exo) descritas

(Sanon et al. 1998), apenas a atividade de NHA-se não foi afetada pela alosamidina

(Loiseau et al. 2002).

A demetilalosamidina, um dos derivados da alosamidina, é um potente inibidor

de quitinase de S. cerevisae afetando o crescimento de leveduras (Sakuda et al. 1990).

Atualmente os peptídeos argifina e argadina, os quais apresentam modificações como a

acetilação e ciclização, têm sido estudados como inibidores (Houston et al. 2002). Outro

inibidor sintetizado recentemente, HM508 (N,N'-diacetylchitobionoxime-N-

phenylcarbamate ), foi capaz de inibir a atividade de quitinases em S. marcescens .

Recentemente um peptídeo mais simples CI-4 [cyclo-(L-Arg-D-Pro)],

produzido pela bactéria marinha Pseudomonas, foi identificado como um inibidor

natural de quitinase (Houston et al. 2002). Ensaios in vitro mostraram que CI-4 inibe a

separação de células filhas de S. cerevisae e impede a diferenciação de C. albicans para

a forma leveduriforme (Izumida et al. 1996). Em S. marcescens a estrutura de CI-4

revela que esse dipeptídeo cíclico inibe a atividade da enzima por apresentar

simularidade estrutural a um intermediário da reação (Houston et al. 2002). Os

dipeptídeos cíclicos derivados de CI-4 [(cyclo-l-Arg-Pro),(cyclo-l-Gly-Pro),(cyclo-l-



37

Hys-Pro), (cyclo-l-Tyr-Pro)] testados para quitinases de S. marcescens apresentaram um

valor de inibição quatro vezes menor em relação a alosamidina (Houston et al. 2004).

I.5.4. APLICAÇÕES

A principal aplicação das quitinases é a produção industrial de quitosana a

partir de quitinas. Esse processo envolve uma reação de deacetilação que consome 40%

de solução de hidróxido de sódio, liberando um resíduo tóxico, e o uso do processo

catalítico eliminaria este inconveniente. A quitosana obtida por catálise pode ser

utilizada para os mais diversos fins. Como exemplos podemos citar o desenvolvimento

de membranas sintéticas para imobilização de poluentes orgânicos em ambientes

aquáticos em áreas industriais, conservante de alimentos, obtenção de películas e

membranas artificiais e até manufatura de cosméticos. É necessário considerar que

existem limitações no uso de enzimas industriais com relação ao custo do isolamento

das mesmas provenientes dos recursos naturais e a dificuldade da manutenção da

atividade que deve ser mantida em condições de temperatura e pH ideais.

Ainda recentemente, as quitinases de Brugyia malayi, Wuchereria bancrofti e

O. volvulus têm sido apontadas como candidatas a vacinas, esta estratégia tem

apresentado complicações pós-imunização (Harrison et al. 2000).

As quitinases têm sido aplicadas em processos de bioconversão. Após o

consumo de camarões, principalmente em países costeiros, o exoesqueleto é descartado.

Em alguns países são produzidos mais de 2,5 milhões de toneladas de lixo dessa

natureza. Assim o desenvolvimento de métodos de conversão desse lixo em produtos

derivados de quitina é de grande importância (Deshpande et al. 1986).

Na agricultura, as quitinases têm sido utilizadas como agentes antifúngicos no

controle biológico (Chernin et al. 1995).



38



39

II. JUSTIFICATIVA

P. brasiliensis apresenta um teor de quitina duas vezes maior na fase

leveduriforme parasítica quando comparado à fase miceliana saprofítica. O estudo de

enzimas que degradam um dos principais componentes estruturais da parede celular, a

quitina, possibilita um maior entendimento sobre o ciclo biológico do fungo e a sua

importância em processos como o crescimento e divisão celular.

Nosso grupo tem trabalhado na caracterização e expressão de proteínas/genes

envolvidos no metabolismo da parede celular de P. brasiliensis. Neste trabalho

realizamos a clonagem, caracterização, expressão e atividade de quitinase de P.

brasiliensis, visando, em uma próxima etapa, a obtenção da proteína recombinante.

Nosso objetivo final é estudar a importância da quitinase de P. brasiliensis e selecionar

inibidores da mesma. Esperamos que inibidores de moléculas envolvidas no

metabolismo de parede celular do fungo apresentem baixa ou nenhuma toxicidade ao

homem.



40



41

III. OBJETIVOS

Este trabalho teve como objetivos:

1- Clonagem e caracterização do cDNA completo codificante para a enzima

quitinase de P. brasiliensis (PbCTS I) através do rastreamento da biblioteca de cDNA.

2- Análises filogenéticas das quitinases de vários organismos.

3- Determinação do número de cópias do gene PbctsI .

4- Avaliação da expressão do gene Pbcts1 através de Northern blot e RT-PCR,

nas formas de levedura e micélio, bem como durante os processos de diferenciação

celular e infecção em camundongo.

5- Avaliação da atividade de N-acetylglucosaminidase (NAGase), quitobiase e

quitinase em frações celulares de P. brasiliensis.



42



43

Chitinase from Paracoccidioides brasiliensis: Molecular

cloning, structural, phylogenetic, expression and

activity analysis

Artigo submetido à publicação na revista FEMS Immunology

and Medical Microbiology



44



45

15-Jul-2005 Dear Dr. Maristela Pereira, The manuscript you submitted to our journal, Chitinase from Paracoccidioides brasiliensis: Molecular cloning, structural, phylogenetic, expression and activity analysis, by Bonfim, Sheyla; Cruz, Aline; Jesuino, Rosalia; Ulhoa, Cirano; Molinari-Madlum, Eugenia; Soares, Celia; Pereira, Maristela, has been uploaded to Manuscript Central. As the submitting author, you will receive future communications via e-mail. Your manuscript number is FEMSIM-05-07-0156. We thank you for submitting your work for publication in one of the FEMS Microbiology journals. Please be sure to save the word processing and graphics files from your manuscript. You may need them later again for production purposes if your manuscript is accepted. You can keep track of your manuscript by logging on periodically to our site (http://mc.manuscriptcentral.com/femsim ), where the status will be displayed in your Submitting Author Center. Again, thank you for the submission of your manuscript. Kindest regards, Dr Montserrat Blázquez-Domingo FEMS Editorial Administrator On behalf of Chief Editor, Dr Alex van Belkum FEMS Immunology & Medical Microbiology Note: It is the responsibility of the corresponding author (or submitting author if different) that all authors of this submitted manuscript are informed about the submission and its progress.



46

Chitinase from Paracoccidioides brasiliensis: Molecular cloning,

structural, phylogenetic, expression and activity analysis

Sheyla M.R.C.Bonfim a, Aline H.S.Cruz a, Rosália S.A. Jesuíno a, Cirano

J.Ulhoa b,

Eugênia E.W.I. Molinari-Madlum c, Célia M.A. Soares a , Maristela Pereira a

aLaboratório de Biologia Molecular, Instituto de Ciências Biológicas,

Universidade Federal de Goiás, 74001-970, Goiânia-Goiás, Brazil

bLaboratório de Enzimologia, Instituto de Ciências Biológicas, Universidade

Federal de Goiás, 74001-970, Goiânia-Goiás, Brazil

cLaboratório de Imunopatologia, Instituto de Patologia Tropical e Saúde

Pública, Universidade Federal de Goiás, 74001-970, Goiânia-Goiás, Brazil

Corresponding author: Dr. M. Pereira, Laboratório de Biologia Molecular, ICB II,

Campus II - Universidade Federal de Goiás, 74001-970, Goiânia-Goiás, Brazil

Tel/fax: 55-62-521-1110

E-mail address: [email protected]



47

Abstract

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening

a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA

consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein

of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of

two signature family 18 catalytic domains and seems to belong to

fungal/bacterial class. Southern blot analysis indicated that Pbcts1 is a single

copy. Phylogenetic analysis with PbCTS1 and other chitinases point to the

possibility of paralogous of several chitinases to be grouped based in

specialized functions, which may reflect the multiple and diverse roles played by

fungi chitinases. Computer-based sequence analysis revealed that PbCTS1

presents a complex structure of domains that could imply in multi functionality.

Although PbCTS1 does not seem to present chitin-binding domains (CBD) and

serine/threonine/proline-rich regions (STP), the immunoglobulin C-Type domain

could propitiate interaction with the chitin chain during catalysis. Glycosyl

hydrolase activity was evaluated and the results demonstrated that P.

brasiliensis is able to produce and secrete these enzymes mainly during

transition from yeast to mycelium. In this way, P. brasiliensis should be able to

use chitin as a carbon source. In addition, PbCTS1 presents an Aamy domain,

indicating the possibility of alpha amylase activity. The presence of an endocytic

signal in the deduced protein suggests that it could be secreted by a vesicular

nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during

differentiation from mycelium to yeast and in yeast cells obtained from infected

mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1

as an attractive drug target.



48

Key words: Paracoccidioides brasiliensis, chitinase, enzymatic activity, cellular

differentiation.

1. Introduction

Fungi are the causative agents of a wide range of human pathogenesis. Indeed, the

last two decades has witnessed a remarkable increase in the incidence of deep-seated,

disseminated mycoses [1]. Immune-compromised patients and the development of

fungal resistance to conventional treatments have contributed for this scenario [2,3,4,

5,6]. In this way, the search for new drug targets has been necessary. One of the most

prevalent fungi in Latin America is Paracoccidioides brasiliensis. Over ten million

people are estimated to be infected, but only up to 2% develop the disease [7].

Enzymes involved on the cell wall metabolism have been approached as

interesting targets to be explored to the design of specific antifungal agents, since this

structure is absent in human [8]. Chitin, a β-1,4-linked polymer of N-acetylglucosamine

(GlcNAc), is a major component of fungal cell walls [9]. Chitin synthases, chitinases

and N-acetyl-β-D-glucosaminidases are hydrolytic enzymes involved in the cell wall

metabolism and required for cell growth [9,10]. In dimorphic fungi, the transition of

phases involves changes in the cell wall composition. An increase in chitin levels in

parasitic phases is detected in Candida albicans [11] and P. brasiliensis [12], hyphae

and yeast-like, respectively, defining a cell wall thickness to the fungus inside the host.

O-Glycosyl hydrolases (EC 3.2.1.-) are a widespread group of enzymes that

hydrolyze the glycosidic bond between two or more carbohydrates, or between a

carbohydrate and a non-carbohydrate moiety. A classification system for glycosyl

hydrolases, based on sequence similarity, has led to the definition of 85 different



49

families. This classification is available on the CAZy (Carbohydrate-Active EnZymes)

web site. Because the fold of proteins is better conserved than their sequences, some of

the families can be grouped in “clans” [13].

Chitinases (EC 3.2.1.14) cleave the β-1,4-linkage between GlcNAc residues in the

chitin homopolymer. Genes encoding chitinases have been cloned from a wide variety

of organisms, for instance bacteria, fungi, insects, plants and animals. Henrissat and

Bairoch [14] classified chitinases into two families 18 and 19, based on amino acid

sequence similarities. Family 18 includes chitinases found in bacteria, fungi, viruses,

animals and class III and V of plant chitinases. Family 19 include class I, II and IV

chitinases of plant origin only, except chitinase C from Streptomyces griseus HUT 6037

[15], and chitinases F and G from Streptomyces coelicolor [16]. Within family 18 two

distinct classes of chitinases may be identified based on the similarity of the enzymes to

family 18 chitinases from plants or bacteria [17,18]. Chitinases of the plant class have

been detected in dimorphic, yeast and filamentous fungi and play roles during growth

and morphogenesis in fungi. Chitinases of the bacterial class have been found in

filamentous fungi, but not in yeasts. The role of these chitinases has not yet been

demonstrated.

Our group has finished the transcriptome of P. brasiliensis in mycelium and yeast

cells (http://www.biomol.unb.br/Pb). By using bioinformatics tools we have identified

new genes of P. brasiliensis that could be used as targets for new antibiotics discoveries

[19,20]. Open reading frames (ORFs) homologous to genes involved in the construction

and maintenance of the polymers of the cell wall and cell-wall-associated molecules of

P. brasiliensis were detected [21]. Here we report the molecular cloning, structural and

phylogenetic analysis, as well as the expression and activity analysis of a chitinase P.

brasiliensis, PbCTS1, which is expressed in mycelium, yeast, during differentiation

from mycelium to yeast and in yeast cells obtained from infected mice. In addition



50

PbCTS1 presents motifs that indicate its probable bifunctionality and secretion to into

the medium.

2. Materials and methods

2.1. Growth conditions and phase transition

P. brasiliensis strain Pb01 (ATCC MYA 826) was used throughout this study.

Yeast cells were grown at 36°C in semi-solid Fava-Netto medium (1% w/v peptone;

0.5% w/v yeast extract; 0.3% w/v proteose peptone; 0.5% beef extract; 0.5% w/v NaCl;

4% w/v glucose, and 1.4% w/v agar, pH 7.2) [22] and subcultured every 7 days.

Mycelium was grown at 22°C in this same medium, by sub culturing every 15 days.

The differentiation was performed in liquid medium (Fava-Netto medium) by

changing the temperature from 22°C to 36°C, to the mycelium to yeast and from 36°C

to 22°C, to the yeast to mycelium transition [23]. The differentiation was visualized by

optic microscopy and evaluated daily by counting the yeast cells remaining in the

culture at 22°C, or the yeast cells in the culture transferred to the 36 °C. After cells

growth at 0, 12, 24 and 120 h the cellular and medium fractions were harvested to the

processing of proteins extracts.

2.2. Generation of a cDNA probe by PCR

cDNA library from yeast was used as a template for the PCR amplification of a

partial fragment encoding the chitinase. Oligonucleotide primers were designed based on

the ORF encoding the Pbcts1 cDNA from the transcriptome of P. brasiliensis. The

CHIT1 (5’- GTCAACTGGGCGATCTAC -3’) and the CHIT2 (5’-



51

CCGATGGAGAGGAGCACA -3’) primers were used in a PCR reaction that was

conducted in a total volume of 50 µL. The resulting 257 bp product was subcloned into

pGEM-T-Easy (Promega. Madison, USA). The sequence was determined on both strands

by automated DNA sequencing, applying the Sanger DNA sequencing method [24].

2.3. Cloning of the cDNA encoding Pbcts1

A cDNA library from yeast cells was constructed in EcoRI and XhoI sites of

Lambda ZAP II (Stratagene Inc., LaJolla, CA, USA). Approximately 5x106 recombinant

phage plaques from the cDNA library were plated, according to standard procedures

[25]. Duplicate filters of the plates were hybridized overnight with the 257 bp probe

labeled with [α-32P]-dCTP at 45°C, in solution containing 50% formamide. Eight

positive clones were obtained and phage particles were plaque purified. The in vivo

excision of pBluescript SK+ phagemids in Escherichia coli XL1-Blue MRF’ cells was

performed by using the exassist/SOLR protocol (Stratagene).

2.4. Sequencing and Computer-based sequence analysis

Nucleotide sequence of the cDNAs were determined on both strands, by using

primer walking by the double-strand dideoxy-chain terminator method using a

MegaBace 1000 sequencer (Amersham Biosciences, Little Chalfont, UK) for automated

sequence analysis.

cDNA sequence analysis was carried out by using the following programs

available at various sites at the World Wide Web: predicted protein sequence analysis

were performed with the ExPaSy programs: ScanProsite

(http://us.expasy.org/tools/scanprosite/) [26], PSORT (http://www.psort.org/) [27], DAS



52

(http://www.sbc.su.se/~miklos/DAS/tmdas.cgi) [28], SMART (http://smart.embl-

heidelberg.de/) [29], Pfam (http://pfam.wustl.edu/hmmsearch.shtml) [30], Signal P

(http://www.cbs.dtu.dk/services/SignalP/) [31], ELM (http://elm.eu.org/) [32], InterPro

Scan (http://www.ebi.ac.uk/InterProScan/) [33], PSIPRED

(http://bioinf.cs.ucl.ac.uk/psipred/) [34], PROSCAN (http://npsa-pbil.ibcp.fr/cgi-

bin/pattern_prosite.pl) [35], YinOYang (http://www.cbs.dtu.dk/services/YinOYang/)

and Compute pI/Mw (http://us.expasy.org/tools/pi_tool.html) [36]. Blast database

searches were proceed using the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/)

[37]. The antigenic determinants of the deduced protein were predicted by using the

algorithm described by Kolaskar and Tongaonkar [38]

(http://www.bioweb.pasteur.fr/seganal/interfaces/antigenic.html). Multiple sequence

alignments were performed using Clustal X [39].

2.5. Comparison of sequences and inferred phylogeny

The deduced amino acid sequence was aligned with putative homologues from the

GenBank database. The phylogenetic relationships of PbCTS1 were generated with 79

complete deduced fungal chitinases available at databases. A phylogenetic tree was

constructed by multiple sequence alignments using the CLUSTAL X program [39].

Tree was generated by the neighbor-joining method [40] and visualized using the Tree

View software. Robustness of branches was estimated using 100 bootstraped replicates

and indicates the percentage of times all species appear as a monophyletic cluster.

2.6. DNA extraction



53

P. brasiliensis yeast cells were harvested, washed and frozen in liquid nitrogen.

Grinding disrupted cells to extract genomic DNA by the cationic hexadecyl trimethyl

ammonium bromide (CTAB) method according to Del Sal et al. [41]. The cell powered

was added of 10 mL of extraction buffer [2% (w/v) polyvinylpolypyrrolidone (PVP),

1.4 M NaCl, 0.1 M tris-HCl pH 8.0, 0.02 M ethylenediaminetetraacetic acid (EDTA),

2% (w/v) CTAB]. The mixture was incubated at 65°C for 1 h, extracted with 50%

chloroform/50% isoamyl alcohol (v/v) and precipitated with 100% ethanol. After

treatment with RNase I and ethanol precipitation, the DNA was resuspended in water.

2.7. Southern blot analysis

Southern blot analysis was performed according to standard procedures [25]. The

DNA of P. brasiliensis was digested with selected restriction endonucleases PstI,

HaeIII and DraI. Digestion products were fractioned on a 1.0% (w/v) agarose gel and

blotted to a nylon membrane, after denaturation for 15 min in 0.5 M NaOH. The

homologous 257 bp probe was labeled with [α-32P]-dCTP by using a random primer

DNA labeling Kit RPN 1604 (Amersham Biosciences). Pre- hibridization and

hibridization reactions were performed at 42°C in a blocking reagent containing 50 %

(v/v) formamide. The blots were washed at 45°C with 0.1 x SSC (sodium saline citrate

0.015 M, NaCl 0.015 M), 0.1 % (w/v) SDS.

2.8. Preparation of inocula and infection of mice

Male mice, 8 to 12 weeks old, from the susceptible strain (B10.A), pathogen free,

were used for infection. Pb01 yeast cells were maintained by weekly subcultivation in

semi-solid Fava-Netto medium [22] at 36°C and used on the 7th day of culture. Yeast



54

cells suspensions were washed in phosphate-buffered saline (PBS) and adjusted to 10 x

106 cells/mL based on haemocytometer counts. Viability was determined by the Janus

Green B vital stain (Merck, Germany) [42] and was always higher than 90%. Mice were

intraperitoneally inoculated with 5x106 yeast cells contained in 0.5 mL of PBS. Matched

groups of animals were injected with sterile PBS and used as normal controls.

2.9. Assay for colonies-forming units

The number of viable microorganisms in the liver of infected mice was

determined by counting the number of colonies forming units (CFU). Briefly, the

animals were sacrificed at 7th day after infection, and livers were removed asseptically,

weighed and homogenized in 5 mL of sterile PBS. The cellular suspensions were

washed three times in PBS, and the final pellets were resuspended in 1 mL of PBS.

Aliquots of 100 µL of the homogenate were plated on selective agar for pathogenic

fungi (Merck & Co., Inc.) supplemented with 10% fetal calf serum. Plates were

incubated at 36°C, and fungal growth and colonies counting were evaluated daily. After

14 days of plating the yeast cells were used to obtain the RNA.

2.10. RNA isolation

Total RNAs from yeast, from mycelium, from cells in differentiation and from

cells obtained of infected mice were obtained using the Trizol reagent (10 mL for 0.5-

1.0 g cells) according to the supplier’s instructions (Gibco, Invitrogen, Carlsbad, CA).

To remove genomic DNA contamination, the RNA samples were treated with RNase

free DNase-I at room temperature for 15 min, followed by standard procedures.



55

2.11. Northern blot analysis

10 µg of total RNA from mycelium and yeasts cells were separated by

electrophoresis on 1.2% agarose gel containing formaldehyde and blotted to a nylon

membrane (Amersham Biosciences). The RNAs were fixed on the membrane by UV-

cross-linking. Hybridizations with the radiolabeled cDNA probe (iten 2.2) were

performed under high-stringency conditions [25]. The filters were washed for 30 min at

55°C with 0.1 SSC (sodium saline citrate 0.015 M, NaCl 0.015 M), 0.1 % (w/v) SDS

and exposed to x-ray film.

2.12. RT-PCR analysis

First-strand cDNA synthesis was performed with 1 µg total RNA by using

SuperScript II reverse transcriptase (Gibco, Invitrogen, Carlsbad, CA) in the presence of

the synthetic oligonucleotide antisense primer CHIT2. Reverse transcription (RT)-PCR

reactions were amplified by PCR using the sense primer CHIT1 and antisense primer

CHIT2. The cDNA synthesis reaction was performed at 37°C for 60 min. The PCR was

subjected to an initial denaturation at 94°C for 3 min followed by 40 cycles at 94°C (1

min and 30 s), 55°C (1 min and 30 s), 72°C (1 min and 30 s) and a final extension at

72°C (10 min). The RT-PCR product of 257 bp was gel-purified, subcloned into

plasmid pGEM-T-Easy (Promega, Madison, WI, USA) and sequenced as described

above.

2.13. Obtaining cellular and secreted fractions of P. brasiliensis cells



56

During transition from mycelium to yeast and vice-versa, medium containing cells

grown for 0, 12, 24 and 120 h were processed to obtain supernatants and cellular

fractions, using the method described by Flores-Carreón et al. [43] with modifications.

The secreted fractions were obtained by centrifugation at 5,000g for 15 min of the

cultures and the supernatant was dialyzed against 0.05 M acetate-sodium buffer pH 5.5,

lyophilized and kept at -80°C. The cellular fractions were obtained from cells collected

by centrifugation at 5,000g for 15 min. The cells were washed in cold homogenization

buffer (20 mM Tris-HCl pH 8.8, 2 mM CaCl2), containing protease inhibitors [50

µg/mL N-α-ρ-tosyl-L-lysine chloromethylketone (TLCK); 1 mM 4-

chloromercuribenzoic acid (PCMB); 20 mM leupeptin; 20 mM phenylmethylsulfonyl

fluoride (PMSF) and 5 mM iodoacetamide]. The mixture was frozen in liquid nitrogen

and the cells were disrupted until obtaining a fine powder. Finally, the mixture was

centrifuged at 12,000g for 10 min and the supernatant, corresponding to cellular

fraction, was kept at -80°C and used for the analysis. The secreted fraction of the

transition from yeast to mycelium, performed in Fava-Netto’s medium added of 0.5%

chitin (inducible medium) was also obtained at 24 h of incubation and the chitinase

activity was assayed.

2.14. Enzyme assays

N-acetyl-β-D-glucosaminidase (NAGase) (EC 3.2.1.30) was assayed by

monitoring the rate of formation of p-nitrophenol from p-nitrophenyl-β-N-

acetylglucosamine, as previously described by Yabuki et al. [44]. One unit of activity

was defined as the amount of the enzyme that produced 1 µmol of p-nitrophenol . min-1

at 37°C. Chitinase activity was assayed with the swollen chitin substrate, as previously

described by Ulhoa and Peberdy [45]. One unit of activity was defined as the amount of



57

the enzyme that produced 1 µmol of reducing sugar . h-1 at 37°C. Protein concentration

was determined by method of Bradford [46], using bovine serum albumin as standard

protein.

2.15. Nucleotide sequence accession number

The Pbcts1 nucleotide sequence and the deduced protein reported in this paper

have been submitted to GenBank database under accession number AAQ75798.

3. Results

3.1. Nucleotide and predicted amino acid sequence encoding of Pbcts1

In order to isolate the complete cDNA encoding Pbcts1, we obtained a 257 bp

PCR product using the primers CHIT1 and CHIT2 designed from an expressed

sequence tag (EST) arising from the P. brasiliensis database transcriptome. Homology

search analysis suggested that the 257 bp cDNA encoded a homologue of chitinase of P.

brasiliensis (data not shown). Screening of a cDNA library of the yeast phase of P.

brasiliensis yielded several potentially positives clones. One clone was selected for

further analysis. The cDNA sequence of 1888 bp contained an ORF of 1218 bp, and

presented 377 and 292 bases at the 5’ and 3’ untranslated regions (UTR), respectively.

A polyadenylation signal ATTAA was identified 99 nucleotides downstream of the

TAG termination codon. The deduced amino acid sequence presented 406 residues with

a predicted molecular mass of 45 kDa and pI of 6.0 (Fig. 1).



58

3.2. Characterization of the deduced amino acid sequence

A BLASTP search of the GenBank database revealed that the deduced amino acid

sequence of the Pbcts1 displayed identity to chitinases. Alignment of the P. brasiliensis

predicted protein with reported sequences of chitinases was performed by using the

Clustal X program [39] and is shown in Fig. 2A. This analysis revealed the existence of

several conserved amino acid residues and a variety of domains (Fig. 2B). PbCTS1

presents domains within the query sequence (6-355), which characterize the Glyco 18

family (Gly 18). The two signatures sequences present on proteins belonging to the

family 18 class of chitinases were found in PbCTS1 (94-99; 132-140). The first

signature (LLSIGG) is inside a predictable barrel strand 2 (β2) and the second one

(LDGLDVDWE) is between α3 and α4 putative domains. In particular, the Glu residue

has been described as the key proton donor during hydrolysis of the glycosidic bond is

at position 140. In this region of the gene is present the DxxDxDxE motif described as

essential to the catalytic activity of the chitinases family 18 [47]. Although PbCTS1

seems not to have chitin-binding domains (CBD) found in class I chitinases, and

serine/threonine/proline-rich (STP), an enhancer to enzyme activity, it has an

immunoglobulin C-Type domain (Igc1) (6-54). It has been proposed that

immunoglobulin-like folds in chitinases are involved in interactions of the protein with

the chitin chain during catalysis [48].

PbCTS1 presents a variety of motifs. The predicted protein contains potentially

one tyrosine kinase and two protein kinase C phosphorylation sites (189-196 and 304-

306, 379-381), respectively, seven casein kinase II phosphorylation sites (54-57, 56-59,

66-69, 158-161, 371-374, 390-393, 394-397), one N-glycosylation site (365-368), nine

O-GlcNAc sites (111, 112, 219, 224, 226, 230, 234, 235 and 360), ten N-myristoylation

sites (209-214, 246-251, 247-252, 276-281, 278-283, 281-286, 311-316, 343-348, 361-



59

366, 366-371) and one amidation site (168-171). The modular domains forkhead-

associated (FHA) (T..[ILA]) (379-382) and WW (...[ST]P) (109-114) found in PbCTS1

could mediate protein-protein interaction.

By using the PSORT program to analyze the deduced protein, PbCTS1 seems to

have no N-terminal signal peptide to endoplasmic reticulum targeting. The protein is

predicted as cytoplasmic (39% of probability). On the other hand, PbCTS1 presents the

Yxxphi sorting signal (SS) (Y..[LMVIF]) (326-329, 396-399) responsible in proteins for

the interaction with the mu subunit of AP (Adaptor Protein) complex. This motif is

present in the cytosolic tail of various membrane proteins, as an endocytic motif that

can direct traffic within the endossomal and the secretory pathways.

In addition, PbCTS1 presents an Endosome-Lysosome-Basolateral (ELB) sorting

signal ([DER]…L[LVI]) (150-155) which could function in sorting and internalization

signals directing type I transmembrane proteins from the cell surface or TGN (Trans

Golgi Network) to the lysosomal-endosomal-melanosomal compartments. This signal

Interact with adaptor protein complexes targeting to the endocytic pathway.

A Src homology 3 (SH3) (…[PV]..P) (267-273) domain was detected in PbCTS1.

This domain is found in proteins with enzymatic activity. The function of the SH3

domain is not well understood but they may mediate many diverse processes such as

increasing local concentration of protein, altering their subcellular location [49].

Although low threshold, GRAM (glucosyltransferases domain) (20-89) and Aamy

(Alpha-amylase domain) (41-400) domains were identified in PbCTS1, indicating that

the protein could present glucosyltransferase and alpha amylase activity. Alpha amylase

enzyme is classified as family 13 of the glycosyl hydrolases. The presence of a Src

Homology 2 (SH2) domain (Y[QDEVAIL][DENPYHI]) (205-208) in PbCTS1

indicates that it can have its enzymatic activity modified by interaction to other protein.

SH2 domains are able to bind specific motifs containing a phosphorylated tyrosine



60

residue, modifying enzymatic activities [50]. PbCTS1 presents N-arginine dibasic

convertase (NDR) (.RK/RR[^KR]) (169-171) and subtilisin/kexin-like proprotein

convertase (PCSK) ([RK].[AILMFV][LTKF].) (79-83) cleavage sites. The convertases

are endopeptidases localized in the cytoplasm and at the cell surface, and process latent

precursor proteins into their biologically active products [51].

Glycosaminoglycan attachment (Glyc) site ([ED]{0,3}.(S)[GA].) (351-355) and

Laminin G domain (LamG) (74-279) were found in PbCTS1; this last domain is known

to bind to the heparin. Seventeen putative antigenic residues were found in PbCTS1.

The profile defined on the DAS program yielded an overall hydropathy index indicative

of a strongly hydrophilic protein where 91% of amino acids of PbCTS1 are hydrophilic.

3.3. Sequence similarity

The secondary structural elements for PbCTS1 were mapped by using the

PSIPRED program. It shows that PbCTS1 has an eight stranded conformation (β/α)8,

while the N-terminal domain is a discrete unit with a β-sandwich configuration (Fig. 2A).

A search of the public databases of the deduced amino acid sequence of the

PbCTS1 revealed that it displayed strong homology to chitinases of fungal and some

bacteria origin. Alignment of the predicted PbCTS1 with reported sequences of fungal

chitinases by Clustal X analysis revealed high levels of homology. The higher values of

similarity shifted between 79 to 69% and identity between 68 to 55%, in relation to

PbCTS1 (Fig. 2A). Among the compared chitinases, the highest identity was with

Ajellomyces capsulatus.

3.4 Phylogenetic analysis



61

In order to approach phylogenetic analysis among glycosyl hydrolases family 18

fungal chitinases we searched for complete protein sequences in the Pfam, EMBL and

NCBI databases. A total of 79 chitinases sequences (all hits with an e value lower than

6e-18) were downloaded and aligned using the multiple alignments Clustal X program.

From this alignment a neighbor-joining tree was constructed to examine the distances

among these sequences. A tree was then inferred and the tree topology was analyzed

using bootstrapping (100 replicates) (Fig. 3). Several groups were formed.

P. brasiliensis enclosed with others eurotiomycetes pathogenic fungi (E. nidulans

1, C. posadasii 2, A. capsulata 1, A. capsulata 2 and A. dermatitidis). The others

eurotiomycetes clustered inside of the several ascomycota clades (sordariomycetes,

saccharomycetes classes).

3.5. Genomic organization

In order to estimate the number of Pbcts1 copies the genomic DNA was digested

with the restriction enzymes PstI, HaeIII and DraI, which do have or not recognition

sites within the Pbcts1 cDNA sequence. The digested DNA was fractionated on a 0.8%

agarose gel and hybridized to the probe encoding Pbcts1 as shown in Fig. 4A. Hae III

digestion produced fragments consistent in number and sizes with, at least, three

restriction sites presumed to be in Pbcts1. The restriction patterns suggest that, Pbcts1 is

a single copy gene.

3.6. In vitro expression analysis of Pbcts1

The presence of the mRNA transcript encoding the PbCTS1chitinase protein was

determined by Northern blot analysis. Hybridization of total RNA with the 32P-labeled



62

chitinase cDNA probe revealed a single band with a molecular mass of 1.9 kb (Fig. 4B).

This size of the transcript is in agreement with that of the cloned cDNA (Fig. 1). The

transcript was detected only in the yeast phase (Fig. 4B).

In order to investigate the expression profile of the Pbcts1 RT-PCR analysis was

performed on total RNAs from mycelium, yeast cells, from cells in differentiation from

mycelium to yeast, and from yeast cells obtained in infection conditions (Fig. 4C).

Products of 257 bp were obtained and sequenced, showing 100% identity to Pbcts1. The

results indicated that Pbcts1 is a gene expressed in mycelium, yeast, during

differentiation from mycelium to yeast and in yeast cells obtained from infected mice.

3.7. Detection of enzymatic activity in P. Brasiliensis

In this study, the specific activity of NAGase was measured in mycelium, yeast

and during the differentiation of P. brasilensis. The time course of NAGase activity

when grown in Fava-Netto medium is shown in Fig. 5. According to our results, the

morphological change from yeast to mycelium is accompanied by an increase in the

amount of NAGase (Fig. 5A) until 24 hours of cellular differentiation. During the

transition from mycelium to yeast the NAGase activity was maintained constant (Fig.

5B). Cellular and secreted specific activity was found in all time tested, with maximal

activity at 24 hours incubation during the yeast to mycelium transition. After this peak

there was a significant reduction activity in the enzyme activity, suggesting that most of

the chitin was degraded (Fig. 5A).

The secreted chitinase activity at 24 hours of incubation during the yeast to

mycelium transition also was determined after growth of P. brasiliensis in Fava-Netto

medium containing 0.5 % chitin, as carbon and nitrogen sources (inducible medium).

Activity of 0.089 U/min and 0.043 U/min was found in the inducible and non-inducible



63

medium, respectively. However, the specific activity in non-inducible medium (13.6

U/mg) is higher than in the inducible medium (7.4 U/mg) due the high amount of

protein secreted at last.

4. Discussion

By searching in the transcriptome of P. brasiliensis, we identified an ORF related

to a chitinase homologue of this fungus. Using a homologue probe we have screened a

P. brasiliensis yeast cDNA library. In this present study, we report the molecular

cloning, structural and phylogenetic analysis of the deduced protein. The expression of

the Pbcts1 gene was investigated in mycelium, yeast and in cells during the fungus

differentiation. The transcript was also analyzed in yeast cells obtained from infected

mice. In addition we performed enzymatic analysis of chitinase in cells of P.

brasiliensis during the transition of phases.

Pbcts1 encodes a protein with a predicted molecular mass of 45 kDa and

calculated pI of 6.0. The deduced protein PbCTS1 contains 406 amino acid residues,

enclosing in its sequence regions identified as the catalytic residues of the chitinases

[52]. Chitinases are classified into two families, named, families 18 and 19, on the basis

of the amino acid sequence similarity of their catalytic domain [53]. Family 18

chitinases present motif DxxDxDxE at catalytic domain. Among these chitinases, two

catalytic amino acid residues (Glu-315 and Asp-391) are considered to be essential for

chitinase activity [54,55] and are conserved in P. brasiliensis. PbCTS1 seems to belong

to the family 18 chitinases; this and the other signature of family 18 (LLSIGG) were

found in PbCTS1. In the chitinase hevamine from Hevea brasiliensis, both signatures

help to form the attractive site cleft [56]. Also, the sequence analysis of the deduced



64

protein showed very conserved amino acid residues that may form the (β/α)8 barrel

structure characteristic of Glyco 18 family. On the basis of those structural similarities,

and based on the homology with other sequences we were able to suggest that the

cDNA encoding the chitinase of P. brasiliensis has been cloned.

The alignment of the deduced amino acid of PbCTS1 with sequences of the

chitinases of various fungi reveals a high degree of homology. The highest identity

values of 68%, 67% and 60%, were with chitinases from A. dermatitidis, A. capsulatus

and C. posadasii, respectively. High identities of amino acids residues at the catalytic

site were noted.

A large number of chitinase-encoding sequences have been published on database.

Here we approached phylogenetic analysis among 79 glycosyl hydrolases family 18

complete chitinase of fungi. Identity and similarity values ranging between 68-31% and

79-46%, respectively, were found when comparing PbCTS1 with all those fungal

chitinases. Several groups were formed. P. brasiliensis enclosed with others

eurotiomycetes pathogenic fungi (E. nidulans 1, C. posadasii 2, A. capsulata 1, A.

capsulata 2 and A. dermatitidis). Although the eurotiomycetes, the sordariomycetes and

the saccharomycetes classes have been grouped inside several sub-clades, other

chitinases from these families were mixed themselves. The majority of fungi encode

chitinases as multigene families. Some of these paralogous were grouped in the same

clade, indicating possible gene duplication. Others were distributed in different groups,

pointing to a closer relationship among the chitinases from different fungal species and

suggesting interphylum horizontal transfer. Possibly, these paralogous were grouped

based in specialized function, which may reflect the multiple and diverse roles played

by fungal chitinases.

Southern blot analysis performed at high stringency indicated that Pbcts1 is

probable present as a single copy in the P. brasiliensis genome. Northern blot analysis



65

showed a single transcript migrating as a mRNA species of 1.9 kb, reinforcing the

presence of only one gene in the fungal genome. The Pbcts1 expression was analyzed

by RT-PCR in mycelium and yeast phases, during transition from mycelium to yeast

and in cells obtained from infected mice. Although the Northern blot analysis showed

the presence of the Pbcts1 transcript only in the yeast phase, RT-PCR assay shows the

presence of transcript in yeast ( Fig. 3C) and also in the mycelium phase.

In order to analyze if Pbcts1 transcripts were present in yeast cells obtained from

infected mice, RNA processed from yeast cells of P. brasiliensis removed from liver of

infected mice were used in RT-PCR reaction. The presence of amplification shows that

Pbcts1 is present during infection of host tissues and could be important for the process.

Chitinases have been reported to be involved in the infectious process as virulence

factors [57] and have been studied as an attractive target to the development of vaccines

and drugs [58, 59]. In parasites, such as Acanthocheilonema viteae and Onchocerca

volvulus chitinases expressed in the larvae stage, which is not known to contain chitin,

has been suggested playing a role in the infection of host tissues [60,61]. The expression

of chitinase by the parasite in conditions without the presence of chitin [62], associated

to cases that chitinase is identified as an immunodominant antigen [60], suggest that

chitinases may be able to act on substrates other than chitin [63,62].

Although PbCTS1 seems not to have N-terminal signal peptide, and has been

predicted as a cytoplasmic molecule, it presents an endocytic signal that can also direct

traffic within the endossomal and the secretory pathways. The presence of this motif

suggests that PbCTS1 could use nonclassical protein secretion pathways. In this sense,

chitinases have been found inside secretory vesicules in Entamoeba invadens and

Entamoeba histolytica [64]. Many proteins have been demonstrated to be secreted by

unconventional secretion pathways [65]. IL-1β is secreted by a vesicular nonclassical

export pathway [66]. HMG (high mobility group) chromatin-binding protein, HMGB1,



66

localizes to an endolysosomal compartment from which secretion can be triggered by

stimuli known to promote lysosomal exocytosis [67]. Strong evidences in tobacco cells

suggest that chitinase can be secreted into the medium by a novel secretion pathway by

passing through the vacuolar compartment. In this system, although pro-chitinase has

reached the vacuole, only mature chitinase are exported to the medium by a mechanism

time and hormonal-dependent [68].

PbCTS1 presents an Endosome-Lysosome-Basolateral sorting signal, which sort

and internalize signals of proteins to the lysosomal-endosomal-melanosomal

compartments. This signal Interact with adaptor protein complexes targeting to the

endocytic pathway. The presence of this signal could suggest that the enzyme could be

endocytosed from the basolateral surface of the plasma membrane and delivered to the

apical recycling compartment, a central sorting station, underneath the apical surface in

a process regulated by kinases. In this way, PbCTS1 could reach periplasmic space, cell

wall or the external medium. Although PbCTS1 does not seem to have CBD and STP

binding sites it has an immunoglobulin C-Type domain, which could propitiate

interactions of chitinase with the chitin chain during catalysis [48]. It has to be pointed

that truncated chitinases lacking CBD retained or enhanced the rate of chitin hydrolysis

[69,70], indicating different functions of the CBD in individual chitinases.

PbCTS1 has Aamy domain, indicating the possibility of presenting alpha amylase

activity. PbCTS1 could be used by the saprophytic form of P. brasiliensis for

acquisition of energy from polysaccharides found in the nature. PbCTS1 presents

homology to endochitinases although the Transglycosidase domain has been detected.

Chitinase of A. fumigatus have shown endo-, exo- and trans-hydrolytic activities [71].

Bifunctional Glycosyl hydrolase has been described at literature. The secreted

endoglycosidase from Enterococcus faecalis consists of a α-domain with a family 18

glycosyl hydrolase motif and a β-domain similar to family 20 glycosyl hydrolases. This



67

enzyme by two distinct activities hydrolyzes the glycans on RNase B and IgG. Both

activities could be important for the molecular pathogenesis and persistence of E.

faecalis during human infections [72]. The presence of the SH2 domain in PbCTS1

indicates that the enzyme could have modified enzymatic activity by interaction to other

proteins [50].

Glycosaminoglycan (GAG) attachment site and Laminin G, a domain of binding

to heparin, were found in PbCTS1. GAGs are ubiquitous molecules expressed on both

in cell surfaces and in extracellular matrix (ECM). GAGs have emerged as critical

regulators of most events involving cell response to its external environment. Heparin is

a class of GAG. Laminin G domain has been shown to mediate mycobacterial adhesion

to lung epithelial cells and macrophages [73, 74]. In this way, PbCTS1 could mediate

adhesion of P. brasiliensis to different targets tissues by binding to ECM components

rolling a play during the natural course of infection. Moreover, the supposed capacity of

PbCTS1 to bind GAGs extends its potential role in P. brasiliensis virulence beyond a

function as a mere adhesion. Indeed it has lately been described that the interaction

between GAG and bacteria plays a role in multiple aspects of microbial pathogenesis

participating not only in adherence and tissue colonization and cell invasion, but also

contributing to systemic dissemination and potentially affecting functions of the host

cell itself [75].

The presence of several putative antigenic residues and high hydrophilic index of

PbCTS1 suggest its antigenic nature. Chitinases from Bombyx mori [76] and

Acanthocheilonema viteae [60] has been described as antigens. Moonlighting proteins

have been described at literature. The function of these proteins can vary as a

consequence of changes in cellular localization, cell type, oligomeric state, or the

cellular concentration of a ligand, substrate, cofactor or product. These different

mechanisms are not mutually exclusive and, in many cases, a protein uses a



68

combination of methods to switch between functions [77]. SH3 domain was detected in

PbCTS1 that could have its subcellular location and consequently function altered.

Chitinases of glycosyl hydrolase family 18 may be divided into fungal/bacterial

chitinases, similar to chitinases found in bacteria, and fungal/plant chitinases, which are

similar to chitinases from plants. Fungal/bacterial chitinases have deduced molecular

masses of approximately 46-48 kDa, have no effect on growth or morphogenesis, are

secreted and may contribute to the digestion and utilization of exogenous chitin as a

source of organic nutrients for energy and biosynthesis. The fungal/plant chitinases are

bigger, contain serine/threonine-rich domain, GPI anchor and cleavage sites and may

have roles during growth and morphogenesis (for review see [78]). PbCTS1 have a

small-deduced molecular mass, is present in mycelium, yeast, during differentiation

from mycelium to yeast and in yeast cells obtained from infected mice. In addition it

presents endocytic motifs, signals that sort, internalization and direct traffic within the

endossomal and the secretory pathways, suggesting nonclassical secretion. Also, the

deduced protein presents an immunoglobulin C-Type domain, which could propitiate

interactions of chitinase with the chitin chain during catalysis. Altogether those findings

suggest that PbCTS1 could belong to the fungal/bacterial class. PbCTS1 presents a

complex structure of domains and sites that could convey to several pos transduction

modifications and could be involved in many cellular processes in different subcellular

location. Those suggestions cited here must be confirmed by further experiments.

However the presence during infection process elects PbCTS1 as an attractive drug

target.

According to our results, yeast secretes a high quantity of NAGase compared to

mycelium. This data is in accordance with the higher quantity of chitin present in yeast

when compared to mycelium. Yeasts have to disrupt the chitin of the cell wall to growth

and originate a new bud. Adverse results found to strain IVIC Pb9 [43] can be due to



69

different substrates used at enzymatic activity assay. The morphological change from

yeast to mycelium is accompanied by an increase in the amount of NAGase, which is

highest 24 h after transition. After this peak there was a significant reduction in the

enzyme activity, suggesting that most of the chitin was degraded. Addition of chitin

increased the activity of chitinase found in the medium (item 3.7). In this way, P.

brasiliensis is able to use chitin to its maintenance. Small values in the transition from

mycelium to yeast suggest housekeeping NAGase activity.

Acknowledgments

This work was supported by grants from IFS (International Foundation for Science),

CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and

FUNAPE/UFG (Fundação de Apoio a Pesquisa/Universidade Federal de Goiás). Sheyla

M.R.C.Bonfim and Aline H.S.Cruz were recipients of fellowships from CAPES

(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Our thanks also go to

Maria Sueli Soares Felipe from Universidade de Brasília (UnB) to make available the

transcriptome database of P. brasiliensis.



70

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80

Figure Legends

Fig.1. Nucleotide and deduced amino acid sequences of P. brasiliensis cDNA encoding

the PbCTS1 homologue. Arrows mark CHIT1 and CHIT2 oligonucleotides. Numbering

of the amino acid sequence begins at the methionine encoded by the first initiation

codon and end at the first termination codon, both underlined. The deduced amino acid

sequence is shown above the nucleotide sequence. Amino acids inside gray boxes

indicate the two signatures present on protein belonging to the class family 18. The

polyadenylation signal AATAA is double underlined preceding the 3’ end poly (A)

stretch. Brackets indicate predicted antigenic determinants. The sites putatively formed

by amino acid residues are represented by following symbols: ● N-myristoylation sites,

■ tyrosine kinase phosphorylation site, ▼ N-glicosylation site, ^ casein kinase II

phosphorylation site, * protein kinase C phosphorylation site, × amidation site.

Fig. 2. (A) Alignment of the deduced amino acid sequence of PbCTS1 with those of

chitinases from fungi. Conserved regions are shaded in the dark and asterisks indicate

conserved amino acid residues. The symbols (:, .) denote a decreasing order of matching

similarity between each corresponding amino acid pair. Positions in which a gap was

introduced to optimize homology are shown as dashes. The boxed region shows the

consensus sequence in family 18 Glycosyl hydrolases. Accession numbers were as

follows: A. dermatitidis (AAF80371); C. posadasii (AAR18252); C. immitis

(AAB48567). The secondary structure elements are those observed in the PSIPRED

program. ─: α-helix, →: β-strand. (B) Schematic diagram of the deduced PbCTS1

protein. The deduced α-helixes are represented in dark boxes with amino acid positions

indicated above. β-strands are represented in gray box with amino acid positions

indicated below. The domains Gly 18 (Glyco 18 family), Aamy (Alpha-amylase

domain), GRAM (Glucosyltransferases domain), LamG (Laminin G domain), Igc1



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(Immunoglobulin C-Type domain), PCSK (Subtilisin/kexin-like proprotein convertase),

WW, ELB (Endosome-Lysosome-Basolateral), NDR (N-arginine dibasic convertase),

SH2 (Src Homology 2), SH3 (Src Homology 3), Glyc (Glycosaminoglycan attachment

site), FHA (Forkhead-associated) and SS (Yxxphi sorting signal) are represented in the

boxes. The amino acid positions are indicated above.

Fig. 3. Phylogenetic tree illustrating the relationship of PbCTS1 to other fungi

chitinases. Sequences were aligned and subjected to phylogenetic analysis using

maximum parsimony and minimum evolution (neighbour-joining). The numbers on

branches indicate bootstrap values obtained for 100 replications. The species (named

with binomial name) and the respective EMBL accession numbers are shown.

Fig. 4. Analysis of the P. brasiliensis Pbcts1 gene organization and of the transcript. (A)

Southern blot analysis. Twenty micrograms of total DNA was digested with the

following restriction enzymes: PstI, HaeIII and DraI. The blot was hybridized to the

radiolabeled Pbcts1 cDNA probe. The position of the size markers is indicated. (B)

Northern blot of total RNA yeast (Y) and mycelium (M) cells. Total RNA from yeast

and mycelium was fractionated on a 1,2% formaldehyde agarose gel and hybridized to

the cDNA insert encoding chitinase. The RNA size was calculated by using the 0.24-9,5

kb marker RNA ladder (InvitrogenTM Life Technologies). The amount of loaded RNA

was standardized by ethidium bromide staining of the agarose gels, showing the major

and minor ribosomal RNAs of about 3.2 and 1.6 kb, respectively. (C) RT-PCR analysis

of the Pbcts1 transcripts in P. brasiliensis. Total RNAs were obtained from mycelium,

yeast cells, cells 24 after h the mycelium to yeast differentiation and from yeast cells

harvest from infected mice. The reactions were primed with oligonucleotides antisense,

CHIT2 and sense CHIT1. The RT-PCR products were separated on a 1.0% agarose gel



82

and stained with ethidium bromide. Lanes 1, 3, 5 and 7 control reactions without RT

and lanes 2, 4, 6 and 8 RT-PCR of the mycelium, yeast cells, cells after 24 h from

mycelium to yeast differentiation and from yeast cells harvest from infected mice,

respectively. Lane 9 control reaction without RT and RNA. Size in base pairs is

indicated on left. The amount of loaded RNA was standardized by ethidium bromide

staining of the agarose gels, showing the major and minor ribosomal RNAs of about 3.2

and 1.6 kb, respectively.

Fig. 5. NAGase activity during differentiation of P. brasiliensis. Yeast and mycelium

were grown at 37°C and 22°C, respectively in semi-solid medium and transferred to

liquid medium. The yeast cultures were incubated at 22°C and those of mycelium were

incubated at 37°C for several time intervals, denoted on the bottom of the graphic. The

cellular extracts were obtained and the NAGase activity was measured. Measurements

were also performed in the secreted fraction (A) NAGase activity during yeast to

mycelium and (B) mycelium to yeast transition. White, dotted and black bars

corresponding to secreted, cellular and total NAGase activity, respectively. Errors bars

represent standard errors calculated on three replicates.



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ORGANISMS EMBL Ajellomyces capsulata 1 Q9P4Q1 Ajellomyces capsulata 2 Q9HEW6 Ajellomyces dermatitidis Q9P4Q0 Amanita muscaria Q9C199 Aspergillus fumigatus 1 Q873Y0 Aspergillus fumigatus 2 Q6Q121 Aspergillus fumigatus 3 Q873X9 Aspergillus fumigatus 4 Q870C0 Ashbhya gosssypii Q75CT5 Beauveria bassiana Q8J1Y3 Blumeria graminis Q96VN1 Botrytis cinerea 1 Q8NJP7 Botrytis cinerea 2 Q8NJP5 Candida albicans 1 Q6SYY8 Candida albicans 2 Q9HEQ6 Candida glabrata 1 Q6FJ23 Candida glabrata 2 Q6FY50 Coccidioides immitis 1 Q7ZA41 Coccidioides immitis 2 Q7Z962 Coccidioides posadassi 1 Q5VHW7 Coccidioides posadassi 2 Q5VHW8 Coniothyrium minitans Q9HGU5 Debaryomyces robertisae Q707V9 Debaryomyces hansenni 1 Q6BTF9 Debaryomyces hansenni 2 Q6BJ14 Debaryomyces hansenni 3 Q6BL38 Debaryomyces hansenni 4 Q6BIN9 Debaryomyces hansenni 5 Q6BWH2 Emericella nidulans 1 Q92223 Emericella nidulans 2 Q9HFQ9 Emericella nidulans 3 Q92222 Hypocrea pseudokoningii Q9C1T4 Kluyveromyces lactis 1 Q6CUCH9 Kluyveromyces lactis 2 Q6CPT6 Metarhizium anisopliae 1 O14456 Metarhizium anisopliae 2 O74199 Metarhizium anisopliae 3 Q9HFL9 Neurospora crassa 1 Q7SGZO Neurospora crassa 2 Q871X2 Neurospora crassa 3 Q7SGW0

ORGANISMS EMBL Neurospora crassa 4 Q7S062 Neurospora crassa 5 Q7SC26 Neurospora crassa 6 Q7S8L7 Neurospora crassa 7 Q7RYA7 Neurospora crassa 8 Q7SG57 Neurospora crassa 9 Q7RWD7 Neurospora crassa 10 Q7SE00 Neurospora crassa 11 Q7S4F8 Neurospora crassa 12 Q7S9A1 Nomuraea rileyi Q6WZ15 Paracoccidioides brasiliensis Q5YLCO Pichia acaciae Q707V5 Puccinia triticina Q7ZA28 Rhizopus oligosporus Q92270 Trichoderma atroviride 1 Q7Z8C9 Trichoderma atroviride 2 Q7Z998 Trichoderma hamatum 1 Q99006 Trichoderma hamatum 2 Q9C1T6 Trichoderma hamatum 3 O43111 Trichoderma harzianum 1 Q61VXO Trichoderma harzianum 2 Q9C1T8 Trichoderma harzianum 3 Q8NK36 Trichoderma harzianum 4 Q70B22 Trichoderma harzianum 5 Q9C1T7 Trichoderma harzianum 6 Q6GV13 Trichoderma reesei Q65YQ7 Trichoderma virens 1 O59928 Trichoderma virens 2 Q8TG96 Trichoderma virens 3 Q8TF88 Trichoderma virens 4 Q8TGW8 Trichoderma virens 5 Q9C1T9 Trichoderma viride Q9UV43 Ustilago maydis Q9HEQ7 Verticillium fungicola 1 Q7ZA13 Verticillium lecanii 1 Q5MNU2 Verticillium lecanii 2 Q5MNU1 Yarrowia lipolitica 1 Q6CI44 Yarrowia lipolitica 2 Q6C863 Yarrowia lipolitica 3 Q6C2W7



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V. PERSPECTIVAS As seguintes perspectivas são propostas: 1- Validação dos motivos encontrados na seqüência predita de CTS1 através de ensaios específicos. 2- Continuação do processo de purificação das quitinases de P. brasiliensis. 3- Expressão de CTS1 em E. coli. 4-Produção de anticorpo policlonal para CTS1. 5-Avaliação da citolocalização de CTS1. 6-Avaliação de CTS1 quanto à secreção em P. brasiliensis.



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VII. ANEXOS

ANEXO I. PRODUÇÃO CIENTÍFICA DURANTE O MESTRADO

I- Publicação de artigo:

1)- Tomazett PK, Cruz AHS, Bonfim SMRC, Soares CMA and Pereira M

2004. The cell wall of P. brasiliensis. Genetics and molecular research,

Brasil, 2005.

II- Trabalhos apresentados em eventos:

1)- Bonfim SMRC, Cruz AHS, Tomazett PK, Jesuino RSA, Felipe MMS,

Soares CMA, Pereira M 2004. Molecular cloning and characterization of a

cDNA encoding the chitinase from Paracoccidioides brasiliensis. Anais da

XXIV Reunião Genética de Microorganismos, Gramado.

2)-Cruz AHS, Bonfim SMRC, Tomazett PK, Felipe MSS, Soares CMA,

Pereira M 2004. Molecular cloning of a cDNA encoding the 1,3-ß-

endoglucanase from Paracoccidioides brasiliensis. Anais da XXIV

Reunião Genética de Microorganismos, Gramado 152pp.

3)-Tomazett PK, Faria FP, Bonfim SMRC, Cruz AHS, Soares CMA,

Pereira M 2004. Solubilization of the recombinant catalytic subunit of β-

1.3-glucan synthase gene from Paracoccidioides brasiliensis. Anais da

XXIV Reunião Genética de Microorganismos, Gramado 175pp.

4)- Bonfim SMRC, Cruz AHS, Tomazett PK, Jesuíno RSA, Felipe MSS,

Soares CMA, Pereira M 2003. Clonagem e caracterização do cDNA de



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quitinase do fungo patogênico humano Paracoccidioides brasiliensis.

Anais da XIII Semana Científica Farmacêutica, Goiânia 35 pp.

5)- Tomazett PK, Faria FP, Bonfim SMRC, Cruz AHS, Soares CMA,

Pereira M 2003. Heterologous expression of the 1,3-glucan synthase gene

from Paracoccidioides brasiliensis. Anais do XXII Congresso Brasileiro de

Microbiologia, Florianópolis.

6)- Cruz AHS, Bonfim SMRC, Tomazett PK, Felipe MSS, Soares CMA,

Pereira M 2003. Clonagem do cDNA de 1,3-endoglucanase de

Paracoccidioides brasiliensis. Anais da XIII Semana Farmacêutica,

Goiânia 27 pp.

7)- Zambuzzi PF, Bonfim SMRC, Cruz AHS, Soares CMA, Pereira M

2005. Clonagem, caracterização do cDNA codificante para malato sintase

do fungo patogênico humano Paracoccidioides brasiliensis e produção

heteróloga da proteína recombinante. Anais do I Congresso de pesquisa,

ensino e extensão Conpeex/UFG/Goiânia.

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