RESUMO
Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.
Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções Urinárias/microbiologia , Animais , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Enterococcus faecalis/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Fibronectinas/metabolismo , Imunofluorescência , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Immunoblotting , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Imunoeletrônica , Ligação Proteica , Proteínas Recombinantes , VirulênciaRESUMO
We evaluated the usefulness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.
Assuntos
Cryptococcus gattii/química , Cryptococcus gattii/classificação , Cryptococcus neoformans/química , Cryptococcus neoformans/classificação , Técnicas Microbiológicas/métodos , Micologia/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Criptococose/diagnóstico , Criptococose/microbiologia , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Stenotrophomonas maltophilia is a microorganism of environmental and clinical importance as well as a frequent airway colonizer of cystic fibrosis (CF) individuals. We combined 2-DE and MALDI-TOF MS to profile the protein expression in S. maltophilia K279a, a completely sequenced clinical isolate, grown at 37 °C with respect to the strain grown at 26 °C. Among the proteins up-regulated at 37 °C, we identified GroEL, a molecular chaperone that mainly assist the folding and unfolding of proteins under both normal and stress conditions. A 2.4-kb groESL mRNA was detected independently by Northern blot analyses with a groES- and a groEL-specific probe, indicating that S. maltophilia groES and groEL form an operon. Primer extension analysis of S. maltophilia groESL done in Escherichia coli showed that 2 promoters, Pσ(32) and Pσ(70), were utilized under the heat-shock and normal condition, respectively, whereas S. maltophilia groEL was shown to act as a heat-shock gene at 37 °C, 42 °C, and, to a lesser extent, at 50 °C by real-time RT-PCR analyses. Finally, immunoblot analyses revealed that S. maltophilia GroEL strongly reacted with sera from CF patients chronically infected by the microorganism, but did not with sera from CF patients with sporadic infection or uninfected.
Assuntos
Proteínas de Bactérias/biossíntese , Chaperoninas/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Stenotrophomonas maltophilia/efeitos da radiação , Northern Blotting , Eletroforese em Gel Bidimensional , Escherichia coli/genética , Perfilação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
BACKGROUND: Fluconazole (FLC), a triazole antifungal drug, is widely used for the maintenance therapy of cryptococcal meningoencephalitis, the most common opportunistic infection in AIDS patients. In this study, we examined changes in the gene expression profile of the C. neoformans reference strain H99 (serotype A) following FLC treatment in order to investigate the adaptive cellular responses to drug stress. RESULTS: Simultaneous analysis of over 6823 transcripts revealed that 476 genes were responsive to FLC. As expected up-regulation of genes involved in ergosterol biosynthesis was observed, including the azole target gene ERG11 and ERG13, ERG1, ERG7, ERG25, ERG2, ERG3 and ERG5. In addition, SRE1 which is a gene encoding a well-known regulator of sterol homeostasis in C. neoformans was up-regulated. Several other genes such as those involved in a variety of important cellular processes (i.e. lipid and fatty acid metabolism, cell wall maintenance, stress and virulence) were found to be up-regulated in response to FLC treatment. Conversely, expression of AFR1, the major transporter of azoles in C. neoformans, was not regulated by FLC. CONCLUSIONS: Short-term exposure of C. neoformans to FLC resulted in a complex altered gene expression profile. Some of the observed changes could represent specific adaptive responses to the antifungal agent in this pathogenic yeast.
Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Fluconazol/farmacologia , Regulação Fúngica da Expressão Gênica , Cryptococcus neoformans/fisiologia , Perfilação da Expressão Gênica , Humanos , Estresse FisiológicoRESUMO
The Vitek 2 yeast susceptibility test was evaluated by testing 122 Candida isolates against fluconazole and voriconazole. Excellent categorical agreement with the CLSI broth microdilution method was observed (97.5% for both the azoles). Moreover, the Vitek 2 system was able to identify all but 2 of 59 investigated fluconazole-resistant organisms.