A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program.

Transcription is crucial for the expression of genetic information and its efficient and accurate termination is required for all living organisms. Rho-dependent termination could rapidly terminate unwanted premature RNAs and play important roles in bacterial adaptation to changing environments. Although Rho has been discovered for about five decades, the regulation mechanisms of Rho-dependent termination are still not fully elucidated. Here we report that Rof is a conserved antiterminator and determine the cryogenic electron microscopy structure of Rho-Rof antitermination complex. Rof binds to the open-ring Rho hexamer and inhibits the initiation of Rho-dependent termination. Rof's N-terminal α-helix undergoes conformational changes upon binding with Rho, and is key in facilitating Rof-Rho interactions. Rof binds to Rho's primary binding site (PBS) and excludes Rho from binding with PBS ligand RNA at the initiation step. Further in vivo analyses in Salmonella Typhimurium show that Rof is required for virulence gene expression and host cell invasion, unveiling a physiological function of Rof and transcription termination in bacterial pathogenesis.

Salmonella exploits membrane reservoirs for invasion of host cells.

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth.

Salmonella enterica serovar Typhimurium (S. Typhimurium) controls lipopolysaccharide (LPS) biosynthesis by regulating proteolysis of LpxC, the rate-limiting enzyme and target of preclinical antibiotics. PbgA/YejM/LapC regulates LpxC levels and controls outer membrane (OM) LPS composition at the log-to-stationary phase transition. Suppressor substitutions in LPS assembly protein B (LapB/YciM) rescue the LPS and OM integrity defects of pbgA-mutant S. Typhimurium. We hypothesized that PbgA regulates LpxC proteolysis by controlling LapB's ability to bind LpxC as a function of the growth phase. According to existing models, when nutrients are abundant, PbgA binds and restricts LapB from interacting with LpxC and FtsH, which limits LpxC proteolysis. However, when nutrients are limited, there is debate whether LapB dissociates from PbgA to bind LpxC and FtsH to enhance degradation. We sought to examine these models and investigate how the structure of LapB enables salmonellae to control LpxC proteolysis and LPS biosynthesis. Salmonellae increase LapB levels during the stationary phase to promote LpxC degradation, which limits lipid A-core production and increases their survival. The deletion of lapB, resulting in unregulated lipid A-core production and LpxC overabundance, leads to bacterial growth retardation. Tetratricopeptide repeats near the cytosol-inner membrane interface are sufficient for LapB to bind LpxC, and remarkably, LapB and PbgA interact in both growth phases, yet LpxC only associates with LapB in the stationary phase. Our findings support that PbgA-LapB exists as a constitutive complex in S. Typhimurium, which differentially binds LpxC to control LpxC proteolysis and limit lipid A-core biosynthesis in response to changes in the environment.IMPORTANCEAntimicrobial resistance has been a costly setback for human health and agriculture. Continued pursuit of new antibiotics and targets is imperative, and an improved understanding of existing ones is necessary. LpxC is an essential target of preclinical trial antibiotics that can eliminate multidrug-resistant Gram-negative bacterial infections. LapB is a natural LpxC inhibitor that targets LpxC for degradation and limits lipopolysaccharide production in Enterobacteriaceae. Contrary to some studies, findings herein support that LapB remains in complex instead of dissociating from its presumed negative regulator, PbgA/YejM/LapC, under conditions where LpxC proteolysis is enhanced. Advanced comprehension of this critical protein-lipid signaling network will lead to future development and refinement of small molecules that can specifically interfere.

Anaerobic respiration of host-derived methionine sulfoxide protects intracellular Salmonella from the phagocyte NADPH oxidase.

Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars.

Salmonella enterica serovar Typhimurium remodels mitochondrial dynamics of macrophages via the T3SS effector SipA to promote intracellular proliferation.

Mitochondrial dynamics are critical in cellular energy production, metabolism, apoptosis, and immune responses. Pathogenic bacteria have evolved sophisticated mechanisms to manipulate host cells' mitochondrial functions, facilitating their proliferation and dissemination. Salmonella enterica serovar Typhimurium (S. Tm), an intracellular foodborne pathogen, causes diarrhea and exploits host macrophages for survival and replication. However, S. Tm-associated mitochondrial dynamics during macrophage infection remain poorly understood. In this study, we showed that within macrophages, S. Tm remodeled mitochondrial fragmentation to facilitate intracellular proliferation mediated by Salmonella invasion protein A (SipA), a type III secretion system effector encoded by Salmonella pathogenicity island 1. SipA directly targeted mitochondria via its N-terminal mitochondrial targeting sequence, preventing excessive fragmentation and the associated increase in mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and release of mitochondrial DNA and cytochrome c into the cytosol. Macrophage replication assays and animal experiments showed that mitochondria and SipA interact to facilitate intracellular replication and pathogenicity of S. Tm. Furthermore, we showed that SipA delayed mitochondrial fragmentation by indirectly inhibiting the recruitment of cytosolic dynamin-related protein 1, which mediates mitochondrial fragmentation. This study revealed a novel mechanism through which S. Tm manipulates host mitochondrial dynamics, providing insights into the molecular interplay that facilitates S. Tm adaptation within host macrophages.

ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis.

Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen's ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3'UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program. IMPORTANCE: Salmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella.

Salmonella manipulates the host to drive pathogenicity via induction of interleukin 1ß.

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1ß (IL-1ß). However, the role of IL-1ß in intestinal defense against Salmonella remains unclear. Here, we show that IL-1ß production is detrimental during Salmonella infection. Mice lacking IL-1ß (IL-1ß -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1ß -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1ß induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1ß loss prevented mortality in Salmonella-infected IL-1ß -/- mice. Finally, we found that IL-1ß expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1ß signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1ß signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis.

Spermidine constitutes a key determinant of motility and attachment of Salmonella Typhimurium through a novel regulatory mechanism.

Spermidine is a poly-cationic molecule belonging to the family of polyamines and is ubiquitously present in all organisms. Salmonella synthesizes, and harbours specialized transporters to import spermidine. A group of polyamines have been shown to assist in Salmonella Typhimurium's virulence and regulation of Salmonella pathogenicity Inslad 1 (SPI-1) genes and stress resistance; however, the mechanism remains elusive. The virulence trait of Salmonella depends on its ability to employ multiple surface structures to attach and adhere to the surface of the target cells before invasion and colonization of the host niche. Our study discovers the mechanism by which spermidine assists in the early stages of Salmonella pathogenesis. For the first time, we report that Salmonella Typhimurium regulates spermidine transport and biosynthesis processes in a mutually inclusive manner. Using a mouse model, we show that spermidine is critical for invasion into the murine Peyer's patches, which further validated our in vitro cell line observation. We show that spermidine controls the mRNA expression of fimbrial (fimA) and non-fimbrial adhesins (siiE, pagN) in Salmonella and thereby assists in attachment to host cell surfaces. Spermidine also regulated the motility through the expression of flagellin genes by enhancing the translation of sigma-28, which features an unusual start codon and a poor Shine-Dalgarno sequence. Besides regulating the formation of the adhesive structures, spermidine tunes the expression of the two-component system BarA/SirA to regulate SPI-1 encoded genes. Thus, our study unravels a novel regulatory mechanism by which spermidine exerts critical functions during Salmonella Typhimurium pathogenesis.

Interactions between the AraC/XylS-like transcriptional activator InvF of Salmonella Typhimurium, the RNA polymerase alpha subunit and the chaperone SicA.

Salmonella enterica serovar Typhimurium causes gastroenteritis and systemic infections in humans. For this bacterium the expression of a type III secretion system (T3SS) and effector proteins encoded in the Salmonella pathogenicity island-1 (SPI-1), is keystone for the virulence of this bacterium. Expression of these is controlled by a regulatory cascade starting with the transcriptional regulators HilD, HilC and RtsA that induce the expression of HilA, which then activates expression of the regulator InvF, a transcriptional regulator of the AraC/XylS family. InvF needs to interact with the chaperone SicA to activate transcription of SPI-1 genes including sicA, sopB, sptP, sopE, sopE2, and STM1239. InvF very likely acts as a classical activator; however, whether InvF interacts with the RNA polymerase alpha subunit RpoA has not been determined. Results from this study confirm the interaction between InvF with SicA and reveal that both proteins interact with the RNAP alpha subunit. Thus, our study further supports that the InvF/SicA complex acts as a classical activator. Additionally, we showed for the first time an interaction between a chaperone of T3SS effectors (SicA) and the RNAP.

Expression of accessory genes in Salmonella requires the presence of the Gre factors.

Genomic studies with Salmonella enterica serovar Typhimurium reveal a crucial role of horizontal gene transfer (HGT) in the acquisition of accessory cellular functions involved in host-interaction. Many virulence genes are located in genomic islands, plasmids and prophages. GreA and GreB proteins, Gre factors, interact transiently with the RNA polymerase alleviating backtracked complexes during transcription elongation. The overall effect of Gre factors depletion in Salmonella expression profile was studied. Both proteins are functionally redundant since only when both Gre factors were depleted a major effect in gene expression was detected. Remarkably, the accessory gene pool is particularly sensitive to the lack of Gre factors, with 18.6% of accessory genes stimulated by the Gre factors versus 4.4% of core genome genes. Gre factors involvement is particularly relevant for the expression of genes located in genomic islands. Our data reveal that Gre factors are required for the expression of accessory genes.

Val43 residue of NsrR is crucial for the nitric oxide response of Salmonella Typhimurium.

ABSTRACT: In pathogenic bacteria, the flavohemoglobin Hmp is crucial in metabolizing the cytotoxic levels of nitric oxide (NO) produced in phagocytic cells, contributing to bacterial virulence. Hmp expression is predominantly regulated by the Rrf2 family transcription repressor NsrR in an NO-dependent manner; however, the underlying molecular mechanism in enterobacteria remains poorly understood. In this study, we identified Val43 of Salmonella Typhimurium NsrR (StNsrR) as a critical amino acid residue for regulating Hmp expression. The Val43-to-Ala-substituted mutant NsrR isolated through random and site-directed mutagenesis showed high binding affinity to the target DNA irrespective of NO exposure, resulting in a severe reduction in hmp transcription and slow NO metabolism in Salmonella under NO-producing conditions. Conversely, the Val43-to-Glu-substituted NsrR caused effects similar to nsrR null mutation, which directed hmp transcription and NO metabolism in a constitutive way. Comparative analysis of the primary sequences of NsrR and another NO-sensing Rrf2 family regulator, IscR, from diverse bacteria, revealed that Val43 of enterobacterial NsrR corresponds to Ala in Pseudomonas aeruginosa or Streptomyces coelicolor NsrR and Glu in enterobacterial IscR, all of which are located in the DNA recognition helix α3. The predicted structure of StNsrR in complex with the hmp DNA suggests dissimilar spatial stoichiometry in the interactions of Val43 and its substituted residues with the target DNA, consistent with the observed phenotypic changes in StNsrR Val43 mutants. Our findings highlight the discriminative roles of the NsrR recognition helix in regulating species-specific target gene expression, facilitating effective NO detoxification strategies in bacteria across diverse environments. IMPORTANCE: The precise regulation of flavohemoglobin Hmp expression by NsrR is critical for bacterial fitness, as excessive Hmp expression in the absence of NO can disturb bacterial redox homeostasis. While the molecular structure of Streptomyces coelicolor NsrR has been recently identified, the specific molecular structures of NsrR proteins in enterobacteria remain unknown. Our discovery of the crucial role of Val43 in the DNA recognition helix α3 of Salmonella NsrR offers valuable insights into the Hmp modulation under NO stress. Furthermore, the observed amino acid polymorphisms in the α3 helices of NsrR proteins across different bacterial species suggest the diverse evolution of NsrR structure and gene regulation in response to varying levels of NO pressure within their ecological niches.

Apoptotic signaling clears engineered Salmonella in an organ-specific manner.

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. When a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we engineered S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. To clear an infection, cells may have specific tasks that they must complete before they die; different modes of cell death could initiate these 'bucket lists' in either convergent or divergent ways.

Although alive and healthy cells are essential for survival, in certain circumstances ­ such as when a cell becomes infected ­ it is beneficial for cells to deliberately die through a process known as regulated cell death. There are several types of regulated cell death, each with distinct pathways and mechanisms. However, if the initial pathway is blocked, cells can use an alternative one, suggesting that they can compensate for one other. Two forms of regulated cell death ­ named pyroptosis and apoptosis ­ can be used by infected cells to limit the spread of pathogens. However, it was not clear if these two forms or additional 'back-up' apoptosis pathways ­ which are induced when pyroptosis fails ­ are equally efficient at clearing infections and how they might vary in different cell types. To address this, Abele et al. investigated cell death in live mice infected with the bacterium Salmonella. Different organs in which the bacterium infects distinct cell types were examined. Experiments showed that pyroptosis could eliminate bacteria from both intestinal cells as well as immune cells found throughout the body, called macrophages. In contrast, apoptosis was only able to clear infection from intestinal cells. The findings can be explained by prior studies showing both apoptosis and pyroptosis lead to the same outcome in intestinal cells ­ dead cells are expelled from the body through a process called extrusion to maintain the barrier function of the intestine. However, in macrophages, the different pathways lead to different outcomes, indicating they are not entirely interchangeable. Overall, the findings of Abele et al. underscore the complexity of cellular responses to infection and the nuanced roles of different cell death pathways. This provides further evidence that cells might have specific tasks they need to complete before death in order to effectively clear an infection. These tasks may differ depending on cell type and the form of regulated cell death, and may not be equally efficient at clearing an infection.

Microbiome diversity protects against pathogens by nutrient blocking.

The human gut microbiome plays an important role in resisting colonization of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Furthermore, we applied our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.

The Salmonella Typhimurium Effector SpvB Subverts Host Membrane Trafficking by Targeting Clathrin and AP-1.

Salmonella enterica, the etiological agent of gastrointestinal and systemic diseases, translocates a plethora of virulence factors through its type III secretion systems to host cells during infection. Among them, SpvB has been reported to harbor an ADP-ribosyltransferase domain in its C terminus, which destabilizes host cytoskeleton by modifying actin. However, whether this effector targets other host factors as well as the function of its N terminus still remains to be determined. Here, we found that SpvB targets clathrin and its adaptor AP-1 (adaptor protein 1) via interactions with its N-terminal domain. Notably, our data suggest that SpvB-clathrin/AP-1 associations disrupt clathrin-mediated endocytosis and protein secretion pathway as well. In addition, knocking down of AP-1 promotes Salmonella intracellular survival and proliferation in host cells.

Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of Salmonella typhimurium in mice.

IMPORTANCE: The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

c-di-GMP inhibits the DNA binding activity of H-NS in Salmonella.

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria.

Benzalkonium chloride forces selective evolution of resistance towards antibiotics in Salmonella enterica serovar Typhimurium.

BACKGROUND: Although food-grade disinfectants are extensively used worldwide, it has been reported that the long-term exposure of bacteria to these compounds may represent a selective force inducing evolution including the emergence of antibiotic resistance. However, the mechanism underlying this correlation has not been elucidated. This study aims to investigate the genomic evolution caused by long-term disinfectant exposure in terms of antibiotic resistance in Salmonella enterica Typhimurium. METHODS: S. Typhimurium isolates were exposed to increasing concentrations of benzalkonium chloride (BAC) and variations of their antibiotic susceptibilities were monitored. Strains that survived BAC exposure were analyzed at whole genome perspective using comparative genomics, and Sanger sequencing-confirmed mutations in ramR gene were identified. Next, the efflux activity in ramR-mutated strains shown as bisbenzimide accumulation and expression of genes involved in AcrAB-TolC efflux pump using quantitative reverse transcriptase PCR were determined. RESULTS: Mutation rates of evolved strains varied from 5.82 × 10-9 to 5.56 × 10-8, with fold increase from 18.55 to 1.20 when compared with strains evolved without BAC. Mutations in ramR gene were found in evolved strains. Upregulated expression and increased activity of AcrAB-TolC was observed in evolved strains, which may contribute to their increased resistance to clinically relevant antibiotics. In addition, several indels and point mutations in ramR were identified, including L158P, A37V, G42E, F45L, and R46H which have not yet been linked to antimicrobial resistance. Resistance and mutations were stable after seven consecutive cultivations without BAC exposure. These results suggest that strains with sequence type (ST) ST34 were the most prone to mutations in ramR among the three STs tested (ST34, ST19, ST36). CONCLUSIONS: This work demonstrated that disinfectants, specifically BAC forces S. Typhimurium to enter a specific evolutionary trajectory towards antibiotic resistance illustrating the side effects of long-term exposure to BAC and probably also to other disinfectants. Most significantly, this study provides new insights in understanding the emergence of antibiotic resistance in modern society.

Development of recombinant subunit vaccine targeting InvH protein of Salmonella Typhimurium and evaluation of its immunoprotective efficacy against salmonellosis.

Salmonella Typhimurium is the most prevalent non-host specific Salmonella serovars and a major concern for both human and animal health systems worldwide contributing to significant economic loss. Type 3 secretion system (T3SS) of Salmonella plays an important role in bacterial adherence and entry into the host epithelial cells. The product of invH gene of Salmonella is an important component of the needle complex of the type 3 secretion system. Hence, the present study was undertaken to clone and express the 15 kDa InvH surface protein of Salmonella Typhimurium in an E. coli host and to evaluate its immune potency in mice. The purified recombinant InvH (r-InvH) protein provoked a significant (p < 0.01) rise in IgG in the inoculated mice. The immunized mice were completely (100%) protected against the challenge dose of 107.5 LD50, while protection against challenge with the same dose of heterologous serovars was 90%. The bacterin-vaccinated group showed homologous protection of 60% against all three serovars. Findings in this study suggest the potential of the r-InvH protein of S. Typhimurium as an effective vaccine candidate against Salmonella infections.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Strategies adopted by Salmonella to survive in host: a review.

Salmonella, a Gram-negative bacterium that infects humans and animals, causes diseases ranging from gastroenteritis to severe systemic infections. Here, we discuss various strategies used by Salmonella against host cell defenses. Epithelial cell invasion largely depends on a Salmonella pathogenicity island (SPI)-1-encoded type 3 secretion system, a molecular syringe for injecting effector proteins directly into host cells. The internalization of Salmonella into macrophages is primarily driven by phagocytosis. After entering the host cell cytoplasm, Salmonella releases many effectors to achieve intracellular survival and replication using several secretion systems, primarily an SPI-2-encoded type 3 secretion system. Salmonella-containing vacuoles protect Salmonella from contacting bactericidal substances in epithelial cells and macrophages. Salmonella modulates the immunity, metabolism, cell cycle, and viability of host cells to expand its survival in the host, and the intracellular environment of Salmonella-infected cells promotes its virulence. This review provides insights into how Salmonella subverts host cell defenses for survival.