Orientadores: Tomomasa Yano, Maria Celia S. Lanna / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T10:36:24Z (GMT). No. of bitstreams: 1 Ribeiro_DanielaAlves_M.pdf: 9526817 bytes, checksum: 451baed1219c35350316a70f67d5b336 (MD5) Previous issue date: 2006 / Resumo: Estudos de infecções causadas pela ingestão de águas contaminadas, em especial por Escherichia coli, são importantes para definir o papel dessas bactérias em casos de gastroenterites. Devido à ocorrência freqüente da diarréia infantil na cidade de Ouro Preto-MG, análises microbiológicas da sua água de consumo determinaram a presença de E. coli. No presente trabalho, propriedades fenotípicas e genotípicas de virulência foram estudadas em 97 amostras de E. coli isoladas da água de consumo desse município. Os resultados obtidos para padrões de adesão mostraram que 79 (81,4%) amostras aderiram a células HEp-2 em diferentes padrões de adesão, sendo que 49 (62%) amostras aderiram de forma agregativa, 16 (20,3%) apresentaram o padrão difuso, 12 (15,2%) aderiram de forma não característica e 2 (2,5%) apresentaram o perfil de adesão localizada ¿like¿. Por ensaios de hemaglutinação verificou-se que 70 (72%) amostras hemaglutinaram hemácias de cobaia, 64 (65,9%) aglutinaram hemácias eqüinas, 5 (5,2%) foram positivas com hemácias humanas do grupo A e 10 (10,3%) aglutinaram hemácias bovinas. Todas essas amostras evidenciaram um perfil de hemaglutinação manose sensível, o que está associado à presença da fímbria tipo 1. Outro fator de virulência detectado foi a produção de aerobactina em 11 (11,3%) isolados. Em relação à expressão de hemolisinas em meio sólido, 50 amostras de E. coli foram positivas em placas de ágar sangue contendo hemácias de cavalo, 37 lisaram hemácias de carneiro, 28 lisaram eritrócitos de cobaia, nove lisaram eritrócitos humanos e seis foram positivas com hemácias bovinas. Entretanto, os sobrenadantes de cultura de todas essas bactérias não apresentaram atividade hemolítica ¿in vitro¿. Em relação à atividade citotóxica dos sobrenadantes de cultura das amostras, verificou-se que 89 deles provocaram diferentes alterações morfológicas em células Vero, HeLa e CHO. Deste total, 39 apresentaram efeito citotóxico caracterísitico de desarranjo do citoesqueleto celular, foram estáveis ao aquecimento por 100ºC e demonstraram atividade enterotóxica em camundongos recém nascidos. Estudos genotípicos pela PCR, identificaram nove amostras eltIA+, uma amostra stI+, 14 stII+, seis _ hLy+, três sat+, três astA+, duas pic+, 10 kps+, 11 iucD+, 88 fimH+, seis eae+, três papC+ e uma papG1+. Nenhuma amostra foi positiva para os genes stx1, stx2, stx2v, eltIIA, cnfs, cdt, enhly, pet, bfp, inv, K88 e 987p, aggA, aafA e papGII/GIII. Baseado na aderência em células, na produção de toxinas e na presença de genes de virulência, classificou-se 24 amostras como ETEC, 6 como EPEC atípicas , 36 EAEC e 13 como DAEC. Nenhuma amostra foi classificada como EHEC e EIEC. Estes resultados sugerem um possível envolvimento destas amostras de E. coli como um dos agentes enteropatogênicos envolvidos nas infecções entéricas ocorridas na cidade de Ouro Preto / Abstract: Infections studies caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the pathogenic role of these bacteria in gastroenteritis. Due to frequent infantile diarrhea in the city of Ouro Preto-MG, this city's water microbiologic analysis displayed the presence of E. coli. In the present study, genotypic and phenotypic virulence properties were studied among 97 E. coli strains isolated from the drinking water provided for this city. The obtained results for adhesion patterns showed that 81,4% of the samples adhere to HEp-2 cells in different adhesion patterns, wherein 49 strains adhere in a aggregative pattern, 16 displayed the diffuse pattern, 12 adhered in a non-characteristic pattern and 2 showed the localized adhesion profile ¿like¿. By means of hemagglutination tests we verified that 72 % of the strains hemagglutinated guinea pig erythrocytes, 65,9% agglutinated horse erythrocytes, 5,2% were positives on group A human erythrocytes and 10,3% agglutinated bovine erythrocytes. All these strains made evident a manose sensible hemagglutination profile, which is associated with type 1 fimbriae. Another virulence factor detected was the production of aerobactin in 11,3% of the isolates. Regarding the hemolysin expression in solid medium, 50 E. coli strains were positive in blood agar plates containing horse erythrocytes, 37 lysated sheep erythrocytes, 28 lysated guinea pig erythrocytes, 9 lysated human erythrocytes and 6 were positives on bovine erythrocytes. However, the culture supernatants of all these bacterias didn't displayed any hemolytic activity ¿in vitro¿. This could be associated with a possible b-hemolysin production. In respect of the cytotoxic activity produced by the strains culture supernatants, we verified that 89 of them provoked different morphological alterations in Vero, HeLa and CHO cells. From these, 39 showed a characteristic cytotoxic effect of disarrangement of the cellular cytoskeleton, remained stable when heated by 100ºC and also showed enterotoxic activity in suckling mice. Genotipic studies performed by PCR identified nine eltIA+ strains, one stI+ strain, 14 stII+, six _ hLy+, three sat+, three astA+, two pic+, 10 kps+, 11 iucD+, 88 fimH+, six eae+, three papC+ and one papG1+. None of these results were positive for the following genes stx1, stx2, stx2v, eltIIA, cnfs, cdt, enhly, pet, bfp, inv, K88 e 987p, aggA, aafA e papGII/GIII. Based on the adherence to cells, toxin production and the presence of the virulence genes, 24 strains were classified as ETEC, 6 as atypical EPEC, 36 as EAEC and 13 as DAEC. Anyone strain were classified as EHEC and EIEC. These results suggest a possible involvement of these E.coli strains as one of the enterophatogenic agents related with the enteric infections occurred in the city of Ouro Preto / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Dermatophytes are a group of fungi, some of which can cause skin diseases in humans and animals due to their ability to degrade keratinized tissue. Representatives of this group also include strains from the Trichophyton benhamiae complex, known to cause dermatophytosis especially of small rodents and rabbits. In the last decade, one of four populations of this complex has spread epidemically across Europe among guinea pigs and their breeders. To answer the question what stands behind the successful spread of this population, the gene expression and production of volatile organic compounds of epidemic and non-epidemic populations of T. benhamiae was investigated. Gene expression of three strains from each population was studied during growth in liquid medium and on ex vivo mouse skin models prepared according to a newly optimized protocol. RNAseq and RT-qPCR methods were chosen for the gene expression analysis. Based on the literature and the results of RNAseq preliminary analysis, several genes were selected for which specific primers were designed. The spectra of the produced volatile organic compounds of the same strains growing on sheep wool in vials were analyzed by GC-MS. While non-epidemic populations did not differ in gene expression and production of volatile organic compounds, the...

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. Complementation of a high-passage non-adherent B. burgdorferi strain reveals that the expression of DbpA, DbpB, or BBK32, is sufficient to confer efficient spirochete attachment to 293 epithelial cells. Epithelial cell attachment by DbpA and B was mediated by dermatan sulfate, while BBK32 recognized dermatan and heparan sulfate. The GAG binding properties of bacteria expressing DbpB or DbpA were distinguishable in that DbpB, but not DbpA, promoted spirochetal attachment to C6 glial cells. Furthermore, DbpA alleles from diverse Lyme disease spirochetes exhibit allelic variation with respect to binding decorin, dermatan sulfate, and epithelial cells. Targeted disruption of bbk32 resulted in decreased spirochete binding to fibronectin, GAGs, and mammalian cells. Thus, DbpA, DbpB, and BBK32 may play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains and targeted gene disruption provide a comprehensive genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.

Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.

Haemophilus influenzae is a pathogenic Gram-negative bacterium that colonizes the upper respiratory tract of humans and can cause otitis media, upper and lower respiratory infections, and meningitis. Factors important for H. influenzae to colonize humans and cause disease are not fully understood. Different bacterial pathogens are armed with virulence mechanisms unique to their specific strategies for interacting with their hosts. Many of the proteins mediating these interactions are secreted and contain disulfide bonds required for function or stability. I postulated that identifying the set of secreted proteins in H. influenzae that require periplasmic disulfide bonds would provide better understanding of this bacterium's pathogenic mechanisms. In this thesis, the periplasmic disulfide bond oxidoreductase protein, DsbA, was found to be essential for colonization and virulence of H. influenzae. Mutants of dsbA were also found to be sensitive to the bactericidal effects of serum. However, the DsbA-dependent proteins important for pathogenesis of this organism have not been previously identified. To find them, putative targets of the periplasmic disulfide bond pathway were identified and examined for factors which might be important for mediating critical virulence aspects. By doing so, novel virulence factors were discovered including those important for heme and zinc acquisition, as well as resistance to complement. Overall, the work presented here provides insight into requirements for H. influenzae to survive within various host environments.

The bacterium Streptococcus pneumoniae (pneumococcus), is considered to be a leading cause of morbidity and mortality globally, particularly in infants and the elderly. It is one of the most frequent causes of respiratory tract infections, which sporadically have the potential to develop into serious invasive symptoms including sepsis and meningitis. The development of effective vaccination against this pathogen is essential for reducing the morbidity and mortality it causes since the currently available vaccines can protect against only a limited number of the 100 pneumococci serotypes which target the polysaccharidic capsule of the bacterium. The potential use of conserved protein antigens could provide a defense to a wider range of serotypes and clonal types. The immunogenic properties of the proteins MalX and PrsA as well as their role in vital biological functions of S. pneumoniae have made them stand out as potential targets. MalX is a crucial membrane protein involved in the metabolism of maltose, whereas PrsA is a chaperone-like protein that is connected to the cell envelope. To understand these proteins' potential as vaccine candidates, it is essential to understand their immunogenic characteristics and physiological roles. In this project, we tried to characterize the two antigens to determine the functional significance of different regions and domains in antigen recognition and their expression dynamics in bacterial host. A better understanding of the antigenic properties of the PrsA and MalX proteins will drive the construction of improved versions of antigens for vaccine prototypes. Some approaches were used to clarify the structural characteristics and antigenic determinants associated with these proteins including, protein expression, purification, and structural characterization. Additionally, their expression in E. coli was examined using immunological assays including ELISA and Western blot. The identification of antigenic regions of these proteins also provides insight into how to develop epitope-based vaccinations that specifically target S. pneumoniae. This project discusses the possibility of using membrane vesicles (MVs) as a platform for vaccination. Membrane vesicles made from bacterial cells have innate immunogenic qualities that expose the immune system to a wide variety of antigens. Incorporating MalX and PrsA into such vesicles can improve the vaccine candidate's overall immunogenicity and effectiveness and trigger a stronger immune response against S. pneumoniae.

Xylitol har påvisats förebygga karies som orsakas av Streptococcus mutans, en viktig kariespatogen. Syfte: Syftet med denna litteraturstudie var att beskriva xylitolens påverkan på Streptococcus mutans. Metod: Studiedesignen utgjordes av en allmän litteraturstudie som sammanställer resultatet av tidigare forskning inom området. Sökningen genomfördes i databaserna Dentistry of Oral Sciences Source, Medline och Cinahl. Denna litteraturstudie grundas på vetenskapliga invitrostudier med kvantitativ ansats som valdes utifrån inklusions- och exklusionskriterier samt relevanta sökordskombinationer. En urvalsprocess av artiklar utfördes för att säkerställa en systematisk, noggrann samt reliabel presentation av urvalet. Endast artiklar av högkvalitet inkluderades i denna litteraturstudie. Resultat: Resultatet i denna litteraturstudie baserades på en sammanställning av femton vetenskapliga artiklar, som indikerade att xylitol har en inhiberande inverkan på Streptococcus mutans livsduglighet, tillväxt och biofilmbildning, acidogenitet, uppbyggnad, polysackariders kvantitet samt genuttryck. Slutsats: Sammantaget tyder resultatet på att xylitol medför antibakteriell effekt på Streptococcus mutans, genom att hämma dess metaboliska aktiviteter samt virulensfaktorer. / Xylitol has been shown to prevent dental caries caused by Streptococcus mutans, a major caries pathogen. Aim: To describe the impact of xylitol on Streptococcus mutans. Method: The study design included a general literature study summarizing the results of previous research in the chosen field. The research was conducted in the databases Dentistry of Oral Sciences Source, Medline and Cinahl. This literature study is based on scientific invitro studies with a quantitative approach that were selected based on inclusion and exclusion criteria and relevant keyword combination. An article selection process was carried out to ensure a systematic, accurate and reliable presentation of the sample. High-quality articles were included in this study. Results: The result of this literature study were based on fifteen scientific articles, which indicated that xylitol has an inhibitory effect on viability, growth and biofilm formation, acidogenicity, structure, polysaccharide amount and gene expression of Streptococcus mutans. Conclusion: The result of this literature study suggest that xylitol has an antibacterial effect on Streptococcus mutans by inhibiting its metabolic activities and virulence factors.

No description available.

A gama-proteobactéria Pseudomonas aeruginosa é um patógeno oportunista humano frequentemente associado a pacientes com queimadura grave e aos portadores de fibrose cística. O estabelecimento de infecção depende de uma série de fatores que contribuem para a virulência deste patógeno, dentre eles a produção de sideróforos e outros sistemas de captação de ferro. Pioverdina é o principal sideróforo sintetizado por bactérias do gênero Pseudomonas e linhagens deficientes na sua produção são incapazes de estabelecer infecção em modelos animais. A regulação da biossíntese deste sideróforo envolve a agregação entre as células, indicando a dependência de contato para completa indução da sua produção. O contato com uma superfície altera o comportamento das células e diversos fenótipos são dependentes deste sinal mecânico. PrlC é uma oligopeptidase A putativamente envolvida na degradação de peptídeo-sinais e PA14_00800, uma pequena proteína com domínio de função desconhecida, codificada por um gene imediatamente à jusante de prlC. Existem poucos trabalhos na literatura sobre PrlC e seus homólogos e nenhuma informação sobre PA14_00800. Este trabalho teve como objetivo elucidar o envolvimento de PrlC e PA14_00800 na regulação da produção de pioverdina por células em contato com uma superfície. Para estabelecer uma correlação na expressão destes genes, um estudo da organização gênica foi realizado por RT-PCR, confirmando que eles fazem parte do mesmo operon e, portanto, que a expressão destes genes é regulada pelos mesmos fatores. Ensaios classicamente modulados pelo segundo mensageiro c-di-GMP, como formação de biofilme e motilidade, não apresentaram variações nas linhagens mutantes ΔprlC, ΔPA14_00800 ou Δoperon, indicando que a deleção destes genes não altera significativamente os níveis de c-di-GMP nas células. A motilidade do tipo swarming é, no entanto, severamente afetada na linhagem ΔPA14_00800 quando o meio de cultura não contém cloreto de cálcio e glicose, indicando um defeito na sinalização celular ou requerimento energértico desta linhagem nestas condições. PA14_00800 regula a fluorescência de P. aeruginosa em meio sólido e semissólido, mas não em meio líquido. Esta fluorescência depende tanto de pioverdina quanto de PQS, umamolécula de comunicação celular fluorescente, e a possibilidade de outros fatores estarem envolvidos neste fenótipo ainda está sob investigação. Análise do transcritoma por RNASeq com a linhagem ΔPA14_00800 comparada à linhagem parental foi realizada a partir de colônias destas linhagens crescidas em M9 modificado. Genes envolvidos no sistema de secreção do tipo III e do tipo VI e na biossíntese de PQS apareceram dentre os genes diferencialmente expressos, bem como genes para o catabolismo de glicose. Este trabalho foi o primeiro a investigar o papel de PA14_00800 na fisiologia de P. aeruginosa, e os conhecimentos adquiridos aqui podem ser transpostos, com cautela, para compreensão da função dos homólogos de PA14_00800 em outras bactérias. / The gamma-proteobacterium Pseudomonas aeruginosa is a human opportunistic pathogen frequently associated with patients with severe burns and those with cystic fibrosis. The establishment of infection depends on several factors that contribute to the virulence of this pathogen, among them siderophore production and other iron uptake systems. Pyoverdine is the main siderophore synthesized by the bacteria of the genus Pseudômonas and pyoverdinedeficient strains are unable to establish infection in animal models. The regulation of biosynthesis of this siderophore involves cell aggregation, indicating contact dependency for complete induction of pyoverdine production. Surface contact alters cell behavior and several phenotypes are dependent on this mechanical cue. PrlC is an oligopeptidase A putatively involved in peptide-signals degradation and PA14_00800, a small protein with a domain of unknown function, encoded by a gene immediately downstream of prlC. There are few papers in the literature on PrlC and its homologues and no information on PA14_00800. This work aimed to elucidate the role of PrlC and PA14_00800 in surface-dependent regulation of pyoverdine production. To establish a correlation in the expression of these genes, a study of the gene organization was performed by RT-PCR, confirming that they are part of an operon and therefore the expression of these genes is regulated by the same factors. Traits classically modulated by the second messenger c-di-GMP, such as biofilm formation and motility, did not show variations in the ΔprlC, ΔPA14_00800 or Δoperon, indicating that the deletion of these genes does not significantly alter the levels of c-di-GMP within the cells. Swarming motility is, however, severely affected in the strain ΔPA14_00800 when the culture medium does not contain calcium chloride and glucose, indicating a cell signaling defect or energetic requirement under these conditions. PA14_00800 regulates surface-dependent fluorescence of P. aeruginosa, in solid and semi-solid medium. This fluorescence depends on both pyoverdine and PQS, a fluorescent cell-to-cell communication molecule, and the investigation of other putative factors involved in this phenotype is still under study. Transcriptomic analysis by RNASeq with the strain ΔPA14_00800 compared to PA14 was performed from colonies ofthese strains grown in modified M9 1% agar. Genes involved in the type III and type VI secretion systems, in PQS biosynthesis and glucose catabolism were differentially expressed. This work was the first to investigate the role of PA14_00800 in the physiology of P. aeruginosa, and the knowledge obtained here can be cautiously transposed to understanding the role of PA14_00800 homologues in other bactéria.

O complexo C. neoformans (no qual as espécies C. neoformans e C. gattii estão inseridas) é constituído por leveduras encapsuladas que causam infecção em humanos e animais, com distribuição mundial. Diante do reconhecimento da importância dessas leveduras, seja no meio ambiente ou na área médica, há um crescente interesse no desenvolvimento de pesquisas que possam levar a uma melhor compreensão de sua epidemiologia e estrutura. Cepas clínicas e ambientais pertencentes ao acervo de amostras do Departamento de Microbiologia do HCFMUSP e ao Laboratório de Leveduras Patogênicas do ICB-USP foram reidentificadas, estudadas quanto aos fatores relacionados à virulência (por pesquisa de exoenzimas), quanto ao perfil de suscetibilidade in vitro frente aos antifúngicos: anfotericina B, fluconazol e voriconazol pela utilização da técnica E-test®, e quanto aos aspectos epidemiológicos (estudo de prontuários dos pacientes). As cepas de origem clínica (60) e ambiental (42) foram reidentificadas e condiziam com o estabelecido pela literatura. Nota-se que quanto às cepas clínicas, 90% delas pertenciam à espécie C. neoformans e 10% à espécie C. gattii e das cepas ambientais, 95,2% eram C. neoformans e 4,8% eram C. gattii. Com relação à produção de exoenzimas, cepas de origem clínica (45%) apresentaram índice 2 (positiva) e cepas de origem ambiental (45,2%) apresentaram índice 3 (fortemente positiva) quanto à proteinase; quanto à fosfolipase, 71,7% das cepas de origem clínica apresentaram índice 2 e, 52,4% das cepas ambientais apresentaram índice 3. Todas as cepas foram sensíveis aos antifúngicos testados (com exceção de uma que era sensível de forma dose dependente ao fluconazol). Em estudo epidemiológico, a maioria dos pacientes acometidos de criptococose foi do sexo masculino (70%), com média de acometimento aos 47 anos; 25 pacientes (41,7%) foram ao óbito; a criptococose mais frequente (50%) foi a neurocriptococose; 50% dos pacientes tinham sorologia positiva para o HIV e 26,7% dos pacientes utilizaram anfotericina B associada ao fluconazol como tratamento. Ao final do trabalho chegamos a importantes conclusões: o meio CGB não se apresentou como método capaz de identificar eficazmente cepas C. gattii, sendo útil apenas como uma primeira triagem, devendo ser acoplado à técnica de PCR-RFLP; ensaios de produção da atividade fosfolipásica são extremamente relevantes, podendo-se afirmar que a fosfolipase pode ser a mais importante enzima na patogênese da criptococose (servindo como indicador das espécies do gênero Cryptococcus spp.); homens são mais acometidos de criptococose do que mulheres, sendo estas acometidas mais cedo. A leucemia de células linfoides granulares grandes (LGL), com expressão anômala de CD16+CD56+CD19+, encontrada em 1 paciente HIV negativo, infectado por C. gattii pode sugerir que a substituição de células imunocompetentes por células aberrantes com ineficiência funcional poderia ser a responsável pela imunossupressão do paciente. Esse fato sugere a importância de uma investigação detalhada em pacientes com meningite causada por Cryptococcus spp. com um sistema imune aparentemente competente / C. neoformans complex (the species C. neoformans and C. gattii are inserted) consists of encapsulated yeasts that cause infection in humans and animals, with worldwide distribution. With the recognition of the importance of these yeasts, either in the environment or in the medical field, there is a growing interest in developing research that may lead to a better understanding of its epidemiology and structure. Clinical and environmental strains of the collection of samples from the Department of Microbiology, HC-USP and the Laboratory of Pathogenic Yeasts of ICB- USP were reidentified, studied concerning the factors related to virulence (by exoenzymes research), as the susceptibility profile in vitro front of antifungal: amphotericin B, fluconazole and voriconazole by use of the E-test® technique, and as to the epidemiological (study of medical handbooks of patients). The strains of clinical (60) and environmental origin (42) were reidentified and established in the literature. We notice that as the clinical strains, 90% of them belonged to the species C. neoformans and (10%) to the species C. gattii and of theenvironmental strains, 95,2% were C. neoformans and 4,8 % were C. gattii. With respect to exoenzyme production, strains of clinical origin (4 %) had an index 2 and strains of environmental origin (45,2%) had an index 3, as the phospholipase, 71,7 % of the strains of clinical origin showed index 2 (positive) and 52,4 % of the environmental strains exhibited index 3 (strongly positive). All strains were susceptible to antifungal agents tested (except one that was sensitive dose dependente to fluconazole). In an epidemiological study, the majority of patients with cryptococcosis were male (70%), with age of 47 years, 25 patients (41,7%) died; the cryptococcosis more frequent (50%) was the neurocryptococcosis, 50 % of patients were seropositive for HIV and 26,7 % of patients received amphotericin B associated to fluconazole treatment. At the end of the study we got some important conclusions: the middle CGB is not presented as a method that effectively identify C. gattii strains , being useful only as a first triage, and should be coupled with PCR-RFLP; tests that measuring phospholipase activity are extremely relevant, we can affirm that the phospholipase may be the most important enzyme in the pathogenesis of cryptococcosis (serving as an indicator species of the genus Cryptococcus spp.), men are more affected of cryptococcosis than women, which are affected earlier as men with the age. The NK-cell leukemia found in 1 patient, HIV negative, infected by C. gattii and with a special expression of both their NK cells (CD19+CD16+CD56+ aberrant cells) gave origin to a lymphoid population anomalous CD56+CD19+, and this lymphoid tumor cells lineage may suggest that the substitution of immunocompetent cells by aberrant cells with a functional inefficiency could be responsible for the immunosuppression of the patient, and this suggests the importance of a detailed investigation in patients with meningitis caused by Cryptococcus spp., with an apparently competent immune system