RESUMO
AIM: Detection ofproteases in outer membranes (OM) of ompT+ and ompT- Vibrio cholerae strains of O1 and O139 serogroups. MATERIALS AND METHODS: Specific sterile preparations of OM were obtained by lysis of live V. cholerae cells by 4.5 M urea solution with subsequent differential centrifugation and treatment by nucleases. Extraction of OM proteins previously treated by sodium sarcosinate was carried out by Triton X-100 in the presence of EDTA. Protease and polypeptide spectra were studied in substrate and SDS electrophoresis. Sensitivity of proteases to inhibitors was determined in diffusion test in agarose gel containing substrate by using soy trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF). The presence of ompT was determined in PCR by using specific primers. RESULTS: According to PCR data 13 Vibrio cholerae O1 strains and 3 V. cholerae O139 strains isolated from clinical material as well as 22 V. cholerae O1 strains isolated from environmental objects contained ompT gene. 2 V. cholerae O1 human isolated strains, 9 V. cholerae O1 strains and 2 V. cholerae O139 strains isolated from the environment did not have ompT gene. By using SDS- and enzyme-electrophoresis in polyacrylamide gel quantitative and qualitative differences in composition of polypeptides and proteases of OM ompT+ and ompT- V. cholerae strains that hydrolyze gelatin, casein and protamine sulfate were detected. Inhibition of OM by STI and PMSF resulted in a decrease of their proteolytic activity. CONCLUSION: In preparations and extracts of ompT+ and ompT- V. cholerae OM up to 3 proteases some of which may be related to ompT-like were detected.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/química , Peptídeo Hidrolases/metabolismo , Microbiologia do Solo , Vibrio cholerae O139/enzimologia , Vibrio cholerae O1/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Caseínas/química , Cólera/microbiologia , Ácido Edético , Eletroforese em Gel de Poliacrilamida , Gelatina/química , Humanos , Octoxinol , Peptídeo Hidrolases/isolamento & purificação , Fluoreto de Fenilmetilsulfonil/química , Reação em Cadeia da Polimerase , Protaminas/química , Inibidores de Proteases/química , Sarcosina , Soluções , Ureia , Vibrio cholerae O1/isolamento & purificação , Vibrio cholerae O139/isolamento & purificaçãoRESUMO
High resistance rates to nalidixic acid (NAL) in Vibrio cholerae serogroup O139 strains have been found, and ciprofloxacin (CIP) resistance is also observed. In this study, mutations within the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV from NAL-resistant O139 strains were analysed. The predominant mutation profile was S83I in GyrA in combination with S85L in ParC. In addition, the combination substitutions of D87N in GyrA and D420N in ParE in combination with S83I in GyrA and S85L in ParC as well as D87N in GyrA and P439S in ParE in combination with S83I in GyrA and S85L in ParC were found in the CIP-resistant strains. A series of site-directed mutants comprising D87 in GyrA, D420 in ParE and P439 in ParE were constructed from a wild-type V. cholerae O139 strain carrying the common mutations S83I in GyrA and S85L in ParC. Introduction of the mutation D87N in GyrA increased the CIP minimum inhibitory concentration (MIC) of the mutant strain by nearly 4-fold compared with the initial strain. The second introduction of D420N in ParE further significantly increased the CIP MIC to ca. 23-fold compared with the initial strain. A second introduction of P439S in ParE also increased the CIP MIC by 17-fold. Therefore, it is concluded that the emergence of D87N in GyrA and D420N or P439S in ParE dramatically induces resistance to fluoroquinolones in V. cholerae O139, and the accumulation of multiple mutations in the QRDRs confers significant resistance to fluoroquinolones in V. cholerae.
Assuntos
DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Mutação de Sentido Incorreto , Vibrio cholerae O139/efeitos dos fármacos , Vibrio cholerae O139/genética , Antibacterianos/farmacologia , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Vibrio cholerae O139/enzimologiaRESUMO
Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.
Assuntos
ADP Ribose Transferases/genética , Fatores de Ribosilação do ADP/genética , Toxinas Bacterianas/genética , Toxina da Cólera/genética , Vibrio cholerae O139/genética , Vibrio cholerae não O1/genética , Vibrio cholerae/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Variação Genética , Hepatócitos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Coelhos , Vibrio cholerae/patogenicidade , Vibrio cholerae O139/enzimologia , Vibrio cholerae O139/patogenicidade , Vibrio cholerae não O1/patogenicidade , Fatores de Virulência/genéticaRESUMO
The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains. A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation. All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model. However, the subsequent thyA mutation did not affect their colonisation properties in the same model. These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera.
