RESUMO
Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the ß-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in ß galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.
Assuntos
Escherichia coli , Salmonella typhimurium , Salmonella typhimurium/genética , Malato Sintase , Óperon , Estresse Oxidativo/genética , Acetatos , Carbono , NutrientesRESUMO
Host-generated antimicrobial peptides (AMPs) play a pivotal role in defense against bacterial pathogens. AMPs kill invading bacteria majorly by disrupting the bacterial cell walls. AMPs are actively synthesized and released into the lumen of the gastrointestinal tract to limit colonization of enteric pathogens like Salmonella typhimurium (S. typhimurium). However, S. typhimurium has evolved several resistance mechanisms to defend AMPs. The multicomponent SapABCDF uptake transporter is one such system that helps in resisting AMPs. In the current study, we analyzed the role of S. typhimurium SapA against stress survival and virulence of this bacterium. ∆sapA mutant strain showed hypersensitivity to AMPs, like melittin and mastoparan. Further, ∆sapA mutant showed more than 22 folds (p = 0.019) hypersensitivity to neutrophils as compared to the WT strain of S. typhimurium. In addition, ∆sapA strain showed defective survival in mice. In conclusion, the results of the current study suggest that the SapA is essential for survival against AMPs and virulence of S. typhimurium.
Assuntos
Neutrófilos , Salmonella typhimurium , Animais , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/genética , Camundongos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Virulência , Peptídeos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Camundongos Endogâmicos BALB C , Infecções por Salmonella/microbiologia , Peptídeos Catiônicos Antimicrobianos/farmacologiaRESUMO
Sulphur containing amino acids, methionine and cysteine are highly prone to oxidation. Reduction of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) enhances the survival of bacterial pathogens under oxidative stress conditions. S. Typhimurium encodes two types (cytoplasmic and periplasmic) of Msrs. Periplasmic proteins, due to their location are highly vulnerable to host-generated oxidants. Therefore, the periplasmic Msr (MsrP) mediated repair (as compared to the cytoplasmic counterpart) might play a more imperative role in defending host-generated oxidants. Contrary to this, we show that in comparison to the ΔmsrP strain, the mutant strains in the cytoplasmic Msrs (ΔmsrA and ΔmsrAC strains) showed many folds more susceptibility to chloramine-T and neutrophils. Further ΔmsrA and ΔmsrAC strains accumulated higher levels of ROS and showed compromised fitness in mice spleen and liver. Our data suggest the pivotal role of cytoplasmic Msrs in oxidative stress survival of S. Typhimurium.