RESUMO
Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen that is commonly found in the human upper respiratory tract, has only four identified two-component signal transduction systems. One of these, an ortholog to the QseBC (quorum-sensing Escherichia coli) system, was characterized. This system, designated firRS, was found to be transcribed in an operon with a gene encoding a small, predicted periplasmic protein with an unknown function, ygiW. The ygiW-firRS operon exhibited a unique feature with an attenuator present between ygiW and firR that caused the ygiW transcript level to be 6-fold higher than the ygiW-firRS transcript level. FirRS induced expression of ygiW and firR, demonstrating that FirR is an autoactivator. Unlike the QseBC system of E. coli, FirRS does not respond to epinephrine or norepinephrine. FirRS signal transduction was stimulated when NTHI cultures were exposed to ferrous iron or zinc but was unresponsive to ferric iron. Notably, the ferrous iron-responsive activation only occurred when a putative iron-binding site in FirS and the key phosphorylation aspartate in FirR were intact. FirRS was also activated when cultures were exposed to cold shock. Mutants in ygiW, firR, and firS were attenuated during pulmonary infection, but not otitis media. These data demonstrate that the H. influenzae strain 2019 FirRS is a two-component regulatory system that senses ferrous iron and autoregulates its own operon.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Haemophilus influenzae/metabolismo , Ferro/metabolismo , Animais , Proteínas de Bactérias/genética , Chinchila , Temperatura Baixa , Compostos Ferrosos/farmacologia , Deleção de Genes , Proteínas de Fluorescência Verde , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/genética , Haemophilus influenzae/patogenicidade , Ferro/farmacologia , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Óperon/fisiologia , Otite Média/microbiologia , Mutação Puntual , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VirulênciaRESUMO
Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His(6)-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind beta-lactams, indicating these putative PBPs have penicillin-binding activity.