RESUMO
Clostridium difficile is an intestinal pathogen, which produces two main virulence factors, the exotoxins A and B. Other bacterial structures have been implicated in the colonization of the gastrointestinal tract, which is the first step of the pathogenic process. C. difficile expresses adherence factors and also, displays some surface-associated proteolytic activity, which could play a role in the physiopathology of this bacterium. The aim of this work was to study the protein named Cwp84 which displays significant homologies with many cysteine proteases. The coding catalytic domain of this protein has been cloned in the expression system pGEX-6P-1, as an in-frame fusion with the gluthatione S-transferase, and subsequently purified. The purified fraction showed proteolytic activity on gelatine and BAPNA, but not on azocoll, suggesting a highly selective substrate specificity. The results obtained from inhibition experiments confirmed that Cwp84 belongs to the cysteine protease family. Cwp84 could play a role in degrading some specific host proteins or in the maturation of surface-associated bacterial proteins.
Assuntos
Clostridioides difficile/enzimologia , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Clostridioides difficile/patogenicidade , Sequência Conservada , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/enzimologia , Espaço Extracelular/enzimologia , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , VirulênciaRESUMO
For an in vitro mutant of Streptococcus pneumoniae selected on moxifloxacin four- to eightfold-increased MICs of new fluoroquinolones, only a twofold-increased MIC of ciprofloxacin, and a twofold-decreased MIC of novobiocin were observed. This phenotype was conferred by two mutations: Ser81Phe in GyrA and a novel undescribed His103Tyr mutation in ParE, outside the quinolone resistance-determining region, in the putative ATP-binding site of topoisomerase IV.
Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Streptococcus pneumoniae/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , DNA Topoisomerase IV , DNA Topoisomerases Tipo II/metabolismo , Resistência Microbiana a Medicamentos/genética , Fluoroquinolonas , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Fenótipo , Infecções Pneumocócicas/microbiologia , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/genéticaRESUMO
The accumulation of fluoroquinolones (FQs) was studied in a FQ-susceptible laboratory strain of Streptococcus pneumoniae (strain R6). Uptake of FQs was not saturable, was rapidly reversible, and appeared to occur by passive diffusion. In the presence of glucose, which energizes bacteria, the uptake of FQs decreased. Inhibitors of the proton motive force and ATP synthesis increased the uptake of FQs in previously energized bacteria. Similar results were observed with the various FQs tested and may be explained to be a consequence simply of the pH gradient that exists across the cytoplasmic membrane. From a clinical susceptible strain (strain SPn5907) we isolated in vitro on ciprofloxacin an FQ-resistant mutant (strain SPn5929) for which the MICs of hydrophilic molecules were greater than those of hydrophobic molecules, and the mutant was resistant to acriflavine, cetrimide, and ethidium bromide. Strain SPn5929 showed a significantly decreased uptake of ciprofloxacin, and its determinant of resistance to ciprofloxacin was transferred by transformation to susceptible laboratory strain R6 (strain R6tr5929). No mutations in the quinolone resistance-determining regions of the gyrA and parC genes were found. In the presence of arsenate or carbonyl cyanide m-chlorophenylhydrazone, the levels of uptake of ciprofloxacin by the two resistant strains, SPn5929 and R6tr5929, reached the levels of uptake of their susceptible parents. These results suggest an active efflux of ciprofloxacin in strain SPn5929.