RESUMO
Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.
Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Porinas/metabolismo , Salmonelose Animal/metabolismo , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Regulação para Baixo , Escherichia coli/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Porinas/genética , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidadeRESUMO
Here we demonstrate that OmpD, the most abundant porin in Salmonella enterica serovar Typhimurium, facilitates uptake of hydrogen peroxide (H2O2) and that its expression is negatively regulated by ArcA upon peroxide exposure. When exposed to sublethal concentrations of H2O2, a S. Typhimurium ompD mutant showed decreased peroxide levels compared to those observed in the wild type strain, suggesting that H2O2 could be channeled inside the cell through OmpD. Further evidence came from in vitro studies using OmpD-containing reconstituted proteoliposomes, which showed enhanced H2O2 uptake compared to control liposomes with no porins. RT-PCR and western blot analyses were consistent with a negative regulation mechanism of ompD expression in wild type S. Typhimurium exposed to H2O2. In silico analysis aimed at detecting putative transcriptional regulator binding regions led to identification of an ArcA global regulator motif in the ompD promoter region. The interaction of ArcA with its putative binding site was confirmed in vitro by electrophoretic mobility shift assays. In addition, RT-PCR and western blot experiments demonstrated that the ompD downregulation, observed when the wild type strain was grown in the presence of H2O2, was not retained in arcA mutants, suggesting that ArcA could act as an ompD transcriptional repressor.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Regulação para Baixo , Peróxido de Hidrogênio/farmacologia , Porinas/metabolismo , Proteínas Repressoras/metabolismo , Salmonella typhimurium/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Porinas/genética , Proteínas Repressoras/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
Porins are channels that enable passive diffusion of hydrophilic solutes, nutrients and toxins through the outer bacterial membrane. This explains in part the ability of Gram-negative microorganisms to grow in several different environments, as well as their drug resistance. OmpD is an outer membrane channel that works with the inner membrane pump YddG to expel methyl viologen (MV) from Salmonella enterica serovar Typhimurium; this occurs independently of SmvA, also involved in MV resistance. On the other hand, DeltatolC strains show increased MV resistance when compared to wild-type cells, suggesting that there may be other porin(s) that could function with SmvA to pump MV out of S. typhimurium. A strong candidate is OmpW. Here we show that DeltaompW strains of S. typhimurium are 2.5-fold more sensitive to MV than the wild-type strain. Transcriptional fusions replacing ompW by lacZ show that ompW is induced at least 2-fold in the presence of MV. This result was observed both at the mRNA and protein levels, suggesting that ompW participates in MV resistance. In addition, DeltasmvADeltaompW strains are not fully complemented by smvA, suggesting that OmpW may function through an independent pathway different from that used by SmvA to move MV outside the cell.