Manganese import protects Salmonella enterica serovar Typhimurium against nitrosative stress.
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ABSTRACT:
SUBMITTER: Yousuf S
PROVIDER: S-EPMC7677218 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The divalent transition metal cation manganese is important for protein function, particularly under conditions of iron limitation, nitrosative stress, and oxidative stress, but can mediate substantial toxicity in excess. Salmonella enterica serovar Typhimurium possesses multiple manganese importers, but the pathways for manganese efflux remain poorly defined. The S. Typhimurium ATCC 14028s genome was analyzed for putative manganese export pathways, which identified a previously uncharacterized homologue of the Escherichia coli manganese exporter mntP, stm1834, and two cation diffusion facilitator family transporters, zitB (stm0758) and yiiP (stm4061). Manganese acquisition by S. Typhimurium has been shown to occur in response to nitric oxide, an important chemical mediator of the mammalian innate immune response. However, cellular manganese can rapidly return to prechallenge levels, strongly suggesting that one or more S. Typhimurium exporters may contribute to this process. Here, we report that mntP and yiiP contribute to manganese resistance and export in S. Typhimurium. YiiP, also known as FieF, has previously been associated with zinc and iron transport, although its physiological role remains ambiguous due to a lack of zinc-sensitive phenotypes in yiiP mutant strains of S. Typhimurium and E. coli. We report that S. Typhimurium ΔmntP ΔyiiP mutants are exquisitely sensitive to manganese and show that both YiiP and MntP contribute to manganese efflux following nitric oxide exposure. IMPORTANCE Transition metal cations are required for the function of many proteins but can mediate toxicity when present in excess. Identifying transporters that facilitate metal ion export, the conditions under which they are expressed, and the role they play in bacterial physiology is an evolving area of interest for environmental and pathogenic organisms. Determining the native targets of metal transporters has proved challenging since bioinformatic predictions, in vitro transport data, and mutant phenotypes do not always agree. This work identifies two transporters that mediate manganese efflux from the Gram-negative pathogen Salmonella enterica serovar Typhimurium in response to manganese overload and nitric oxide stress. While homologues of MntP have been characterized previously, this is the first observation of YiiP contributing to manganese export.
| S-EPMC8754126 | biostudies-literature
Project description:Nitric oxide (NO·) is an important mediator of innate immunity. The facultative intracellular pathogen Salmonella has evolved mechanisms to detoxify and evade the antimicrobial actions of host-derived NO· produced during infection. Expression of the NO·-detoxifying flavohaemoglobin Hmp is controlled by the NO·-sensing transcriptional repressor NsrR and is required for Salmonella virulence. In this study we show that NsrR responds to very low NO· concentrations, suggesting that it plays a primary role in the nitrosative stress response. Additionally, we have defined the NsrR regulon in Salmonella enterica sv. Typhimurium 14028s using transcriptional microarray, qRT-PCR and in silico methods. A novel NsrR-regulated gene designated STM1808 has been identified, along with hmp, hcp-hcr, yeaR-yoaG, ygbA and ytfE. STM1808 and ygbA are important for S. Typhimurium growth during nitrosative stress, and the hcp-hcr locus plays a supportive role in NO· detoxification. ICP-MS analysis of purified STM1808 suggests that it is a zinc metalloprotein, with histidine residues H32 and H82 required for NO· resistance and zinc binding. Moreover, STM1808 and ytfE promote Salmonella growth during systemic infection of mice. Collectively, these findings demonstrate that NsrR-regulated genes in addition to hmp are important for NO· detoxification, nitrosative stress resistance and Salmonella virulence.
| S-EPMC3438343 | biostudies-literature
Project description:UnlabelledMultidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump in Salmonella, has previously been linked to virulence in mice. The molecular mechanism of this link between macAB and infection is unclear. We demonstrate that macAB plays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection. macAB is induced upon exposure to H2O2 and is critical for survival of Salmonella enterica serovar Typhimurium in the presence of peroxide. Furthermore, we determined that macAB is required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages. macAB mutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay, macAB mutants appear to be unable to induce protection against exogenous H2O2 in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue the macAB mutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection of Salmonella against oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2 compound secreted by Salmonella cells through a MacAB-dependent mechanism.ImportanceThe ABC-type multidrug efflux pump MacAB is known to be required for Salmonella enterica serovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killing in vitro and for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect of macAB mutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump.
| S-EPMC3809562 | biostudies-literature
Project description:The antimicrobial action of the curing agent sodium nitrite (NaNO2), which is added as a preservative to raw meat products, depends on its conversion to nitric oxide and other reactive nitrogen species under acidic conditions. In this study, we used RNA sequencing to analyze the acidified-NaNO2 shock and adaptive responses of Salmonella enterica serovar Typhimurium, a frequent contaminant in raw meat, considering parameters relevant for the production of raw-cured sausages. Upon a 10-min exposure to 150 mg/liter NaNO2 in LB (pH 5.5) acidified with lactic acid, genes involved in nitrosative-stress protection, together with several other stress-related genes, were induced. In contrast, genes involved in translation, transcription, replication, and motility were downregulated. The induction of stress tolerance and the reduction of cell proliferation obviously promote survival under harsh acidified-NaNO2 stress. The subsequent adaptive response was characterized by upregulation of NsrR-regulated genes and iron uptake systems and by downregulation of genes involved in anaerobic respiratory pathways. Strikingly, amino acid decarboxylase systems, which contribute to acid tolerance, displayed increased transcript levels in response to acidified NaNO2. The induction of systems known to be involved in acid resistance indicates a nitrite-mediated increase in the level of acid stress. Deletion of cadA, which encodes lysine decarboxylase, resulted in increased sensitivity to acidified NaNO2. Intracellular pH measurements using a pH-sensitive green fluorescent protein (GFP) variant showed that the cytoplasmic pH of S. Typhimurium in LB medium (pH 5.5) is decreased upon the addition of NaNO2. This study provides the first evidence that intracellular acidification is an additional antibacterial mode of action of acidified NaNO2.
| S-EPMC4178661 | biostudies-literature
Project description:Nontyphoidal salmonellae, particularly Salmonella enterica serovar Typhimurium, are a major cause of invasive disease in Africa, affecting mainly young children and HIV-infected individuals. Glycoconjugate vaccines provide a safe and reliable strategy against invasive polysaccharide-encapsulated pathogens, and lipopolysaccharide (LPS) is a target of protective immune responses. With the aim of designing an effective vaccine against S. Typhimurium, we have synthesized different glycoconjugates, by linking O-antigen and core sugars (OAg) of LPS to the nontoxic mutant of diphtheria toxin (CRM(197)). The OAg-CRM(197) conjugates varied in (i) OAg source, with three S. Typhimurium strains used for OAg extraction, producing OAg with differences in structural specificities, (ii) OAg chain length, and (iii) OAg/CRM(197) ratio. All glycoconjugates were compared for immunogenicity and ability to induce serum bactericidal activity in mice. In vivo enhancement of bacterial clearance was assessed for a selected S. Typhimurium glycoconjugate by challenge with live Salmonella. We found that the largest anti-OAg antibody responses were elicited by (i) vaccines synthesized from OAg with the highest glucosylation levels, (ii) OAg composed of mixed- or medium-molecular-weight populations, and (iii) a lower OAg/CRM(197) ratio. In addition, we found that bactericidal activity can be influenced by S. Typhimurium OAg strain, most likely as a result of differences in OAg O-acetylation and glucosylation. Finally, we confirmed that mice immunized with the selected OAg-conjugate were protected against S. Typhimurium colonization of the spleen and liver. In conclusion, our findings indicate that differences in the design of OAg-based glycoconjugate vaccines against invasive African S. Typhimurium can have profound effects on immunogenicity and therefore optimal vaccine design requires careful consideration.
| S-EPMC4333450 | biostudies-literature
Project description:The ability to acetylate lysine residues is conserved across organisms, and acetylation of lysine residues plays important roles in various cellular functions. Maintaining intracellular pH homeostasis is crucial for the survival of enteric bacteria in the acidic gastric tract. It has been shown that eukaryotes can stabilize the intracellular pH by histone deacetylation. However, it remains unknown whether bacteria can utilize a reversible protein acetylation system to adapt to an acidic environment. Here we demonstrate that protein acetylation/deacetylation is critical for Salmonella enterica serovar Typhimurium to survive in an acidic environment. We used RNA sequencing to analyze the transcriptome patterns under acid stress and found that the transcriptional levels of genes involved in NAD(+)/NADH metabolism were significantly changed, leading to an increase in the intracellular NAD(+)/NADH ratio. Moreover, acid stress downregulated the transcriptional level of pat, encoding acetyltransferase, and genes cyaA and crp, encoding adenylate cyclase and cyclic AMP receptor protein, respectively, which are positive regulators of pat. It was found that the acid signal alerts the tricarboxylic acid cycle to promote the consumption of acetyl coenzyme A (Ac-CoA), an acetyl group donor for the acetylation reaction. A lowered acetylation level not only was the bacterial response to acid stress but also increased the survival rate of S. Typhimurium under acid stress. The pat deletion mutant had a more stable intracellular pH, which paralleled the higher survival rate after acid treatment compared with that of both the wild-type strain and the cobB (encoding deacetylase) deletion mutant. Our data indicate that bacteria can downregulate the protein acetylation level to prevent the intracellular pH from further falling under acid stress, and this work may provide a new perspective to understand the bacterial acid resistance mechanism.
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Project description:Serovars within the species Salmonella enterica are some of the most common food and water-borne pathogens worldwide. Some S. enterica serovars have shown a remarkable ability to persist both inside and outside the human body. Salmonella enterica serovar Typhi can cause chronic, asymptomatic infection of the human gallbladder. This organism's ability to survive inside the gallbladder centers around its ability to form biofilms on gallstone surfaces. Currently, chronic carriage of S. Typhi is treated by invasive methods, which are not well suited to areas where Salmonella carriage is prevalent. Herein, we report 2-aminobenzimidazoles that inhibit S. enterica serovar Typhimurium (a surrogate for S. Typhi) biofilm formation in low micromolar concentrations. Modifications to the head, tail, and linker regions of the original hit compound elucidated new, more effective analogues that inhibit S. Typhimurium biofilm formation while being non-toxic to planktonic bacterial growth.
| S-EPMC6151450 | biostudies-literature
Project description:The alternative sigma factor sigma(E) is activated by unfolded outer membrane proteins (OMPs) and plays an essential role in Salmonella pathogenesis. The canonical pathway of sigma(E) activation in response to envelope stress involves sequential proteolysis of the anti-sigma factor RseA by the PDZ proteases DegS and RseP. Here we show that sigma(E) in Salmonella enterica sv. Typhimurium can also be activated by acid stress. A sigma(E)-deficient mutant exhibits increased susceptibility to acid pH and reduced survival in an acidified phagosomal vacuole. Acid activation of sigma(E)-dependent gene expression is independent of the unfolded OMP signal or the DegS protease but requires processing of RseA by RseP. The RseP PDZ domain is indispensable for acid induction, suggesting that acid stress may disrupt an inhibitory interaction between RseA and the RseP PDZ domain to allow RseA proteolysis in the absence of antecedent action of DegS. These observations demonstrate a novel environmental stimulus and activation pathway for the sigma(E) regulon that appear to be critically important during Salmonella-host cell interactions.
| S-EPMC4157319 | biostudies-other
Project description:Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.
| S-EPMC321585 | biostudies-literature
Project description:Two well-characterized enzymes in Salmonella enterica serovar Typhimurium and Escherichia coli are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of pepB pepE mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from E. coli, the product of the iadA gene. The other is the product of the serovar Typhimurium homolog of E. coli ybiK, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated iaaA (isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.
| S-EPMC95209 | biostudies-literature