Salmonella Pathogenicity Island 1 Genes Research Articles

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.

Phosphate (Pi) Transporter PIT1 Induces Pi Starvation in Salmonella-Containing Vacuole in HeLa Cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen, causes diarrheal illness and gastrointestinal diseases. S. Typhimurium survives and replicates in phagocytic and non-phagocytic cells for acute or chronic infections. In these cells, S. Typhimurium resides within Salmonella-containing vacuoles (SCVs), in which the phosphate (Pi) concentration is low. S. Typhimurium senses low Pi and expresses virulence factors to modify host cells. However, the mechanism by which host cells reduce the Pi concentration in SCVs is not clear. In this study, we show that through the TLR4-MyD88-NF-κB signaling pathway, S. Typhimurium upregulates PIT1, which in turn transports Pi from SCVs into the cytosol and results in Pi starvation in SCVs. Immunofluorescence and western blotting analysis reveal that after the internalization of S. Typhimurium, PIT1 is located on SCV membranes. Silencing or overexpressing PIT1 inhibits or promotes Pi starvation, Salmonella pathogenicity island-2 (SPI-2) gene expression, and replication in SCVs. The S. Typhimurium ΔmsbB mutant or silenced TLR4-MyD88-NF-κB pathway suppresses the expression of the SPI-2 genes and promotes the fusion of SCVs with lysosomes. Our results illustrate that S. Typhimurium exploits the host innate immune responses as signals to promote intracellular replication, and they provide new insights for the development of broad-spectrum therapeutics to combat bacterial infections.

Salmonella Derby adaptation to swine and simultaneous attenuation for humans go through decay of Salmonella Pathogenicity Island I.

We report the identification, within the global population of Salmonella Derby, of a lineage highly diffused and expanding in swine while being significantly under-represented in humans which carries stop mutations in the Salmonella Pathogenicity Island 1 (SPI-1) genes sipA and hilC. Single-cell analysis of invasion, vacuolar load, and cytosolic hyper-replication show that these mutations attenuate S. Derby virulence in human intestinal cells. Identification of this lineage follows the previous detection of another lineage over-represented in swine compared with humans which carries a loss-of-function mutation in hilD, the major SPI-1 regulator, responsible for reduced infection of human cells. Notably, this hilD-mutated lineage represents a sub-population of the wider sipA and hilC-mutated lineages identified in this study, indicating that SPI-1 is subjected to genetic decay in this part of the Salmonella Derby population adapted to swine. Virulence assessment on swine intestinal cells showed drastically lower levels for both wild-type and mutant S. Derby compared with human cells. Still, S. Derby with impaired sipA and hilC showed further attenuation also in swine cells compared with the wild type. These data suggest that invasion of swine intestinal epithelium is not needed by S. Derby to circulate in pigs, leading to the decay of SPI-1, which in turn determines attenuation for humans. Accordingly, a further S. Derby lineage under-represented in humans compared with pigs was found carrying truncated or missing SPI-1 genes, indicating that the S. Derby population is undergoing converging evolution in its adaptation to swine while attenuating for humans through loss of SPI-1 function.IMPORTANCEThis study integrated population data with in vitro assessment of virulence phenotypes to unveil that a considerable part of the global population of Salmonella Derby is evolving to enhance its host adaptation to the swine host and that this evolution is simultaneously increasing its attenuation for humans. The study shows that the fixation of deleterious mutations in SPI-1 has a role in this process. This evidence indicates that SPI-1 has a key role for S. Derby virulence in humans but not for its circulation in swine. The results show that genes generally considered essential for Salmonella pathogenesis do not play the same key role for all Salmonella serovars or lineages and/or all hosts. The study helps in understanding the molecular mechanisms underlying the ecology and host adaptation of Salmonella showing that the adaptation process can vary for different types of Salmonella and hosts.

Proteomic analysis reveals the global effect of the BarA/SirA-Csr regulatory cascade in Salmonella Typhimurium grown in conditions that favor the expression of invasion genes

In many bacteria, the BarA/SirA and Csr regulatory systems control expression of genes encoding a wide variety of cellular functions. The BarA/SirA two-component system induces the expression of CsrB and CsrC, two small non-coding RNAs that sequester CsrA, a protein that binds to target mRNAs and thus negatively or positively regulates their expression. BarA/SirA and CsrB/C induce expression of the Salmonella Pathogenicity Island 1 (SPI-1) genes required for Salmonella invasion of host cells. To further investigate the regulatory role of the BarA/SirA and Csr systems in Salmonella, we performed LC-MS/MS proteomic analysis using the WT S. Typhimurium strain and its derived ΔsirA and ΔcsrB ΔcsrC mutants grown in SPI-1-inducing conditions. The expression of 164 proteins with a wide diversity, or unknown, functions was significantly affected positively or negatively by the absence of SirA and/or CsrB/C. Interestingly, 19 proteins were identified as new targets for SirA-CsrB/C. Our results support that SirA and CsrB/C act in a cascade fashion to regulate gene expression in S. Typhimurium in the conditions tested. Notably, our results show that SirA-CsrB/C-CsrA controls expression of proteins required for the replication of Salmonella in the intestinal lumen, in an opposite way to its control exerted on the SPI-1 proteins. SignificanceThe BarA/SirA and Csr global regulatory systems control a wide range of cellular processes, including the expression of virulence genes. For instance, in Salmonella, BarA/SirA and CsrB/C positively regulate expression of the SPI-1 genes, which are required for Salmonella invasion to host cells. In this study, by performing a proteomic analysis, we identified 164 proteins whose expression was positively or negatively controlled by SirA and CsrB/C in SPI-1-inducing conditions, including 19 new possible targets of these systems. Our results support the action of SirA and CsrB/C in a cascade fashion to control different cellular processes in Salmonella. Interestingly, our data indicate that SirA-CsrB/C-CsrA controls inversely the expression of proteins required for invasion of the intestinal epithelium and for replication in the intestinal lumen, which suggests a role for this regulatory cascade as a molecular switch for Salmonella virulence. Thus, our study further expands the insight into the regulatory mechanisms governing the virulence and physiology of an important pathogen.

CsrA Positively and Directly Regulates the Expression of the pdu, pocR, and eut Genes Required for the Luminal Replication of Salmonella Typhimurium.

Enteric pathogens, such as Salmonella, have evolved to thrive in the inflamed gut. Genes located within the Salmonella pathogenicity island 1 (SPI-1) mediate the invasion of cells from the intestinal epithelium and the induction of an intestinal inflammatory response. Alternative electron acceptors become available in the inflamed gut and are utilized by Salmonella for luminal replication through the metabolism of propanediol and ethanolamine, using the enzymes encoded by the pdu and eut genes. The RNA-binding protein CsrA inhibits the expression of HilD, which is the central transcriptional regulator of the SPI-1 genes. Previous studies suggest that CsrA also regulates the expression of the pdu and eut genes, but the mechanism for this regulation is unknown. In this work, we show that CsrA positively regulates the pdu genes by binding to the pocR and pduA transcripts as well as the eut genes by binding to the eutS transcript. Furthermore, our results show that the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes mediated by PocR or EutR, which are the positive AraC-like transcriptional regulators for the pdu and eut genes, respectively. By oppositely regulating the expression of genes for invasion and for luminal replication, the SirA-CsrB/CsrC-CsrA regulatory cascade could be involved in the generation of two Salmonella populations that cooperate for intestinal colonization and transmission. Our study provides new insight into the regulatory mechanisms that govern Salmonella virulence. IMPORTANCE The regulatory mechanisms that control the expression of virulence genes are essential for bacteria to infect hosts. Salmonella has developed diverse regulatory mechanisms to colonize the host gut. For instance, the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the SPI-1 genes, which are required for this bacterium to invade intestinal epithelium cells and for the induction of an intestinal inflammatory response. In this study, we determine the mechanisms by which the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes, which are necessary for the replication of Salmonella in the intestinal lumen. Thus, our data, together with the results of previous reports, indicate that the SirA-CsrB/CsrC-CsrA regulatory cascade has an important role in the intestinal colonization by Salmonella.

SPI-1 virulence gene expression modulates motility of Salmonella Typhimurium in a proton motive force- and adhesins-dependent manner.

Both the bacterial flagellum and the evolutionary related injectisome encoded on the Salmonella pathogenicity island 1 (SPI-1) play crucial roles during the infection cycle of Salmonella species. The interplay of both is highlighted by the complex cross-regulation that includes transcriptional control of the flagellar master regulatory operon flhDC by HilD, the master regulator of SPI-1 gene expression. Contrary to the HilD-dependent activation of flagellar gene expression, we report here that activation of HilD resulted in a dramatic loss of motility, which was dependent on the presence of SPI-1. Single cell analyses revealed that HilD-activation triggers a SPI-1-dependent induction of the stringent response and a substantial decrease in proton motive force (PMF), while flagellation remains unaffected. We further found that HilD activation enhances the adhesion of Salmonella to epithelial cells. A transcriptome analysis revealed a simultaneous upregulation of several adhesin systems, which, when overproduced, phenocopied the HilD-induced motility defect. We propose a model where the SPI-1-dependent depletion of the PMF and the upregulation of adhesins upon HilD-activation enable flagellated Salmonella to rapidly modulate their motility during infection, thereby enabling efficient adhesion to host cells and delivery of effector proteins.

Small RNAs Activate Salmonella Pathogenicity Island 1 by Modulating mRNA Stability through the hilD mRNA 3' Untranslated Region.

Salmonella enterica serovar Typhimurium is an enteric pathogen associated with foodborne disease. Salmonella invades the intestinal epithelium using a type three secretion system encoded on Salmonella pathogenicity island 1 (SPI-1). SPI-1 genes are tightly regulated by a complex feed-forward loop to ensure proper spatial and temporal expression. Most regulatory input is integrated at HilD, through control of hilD mRNA translation or HilD protein activity. The hilD mRNA possesses a 310-nucleotide 3' untranslated region (UTR) that influences HilD and SPI-1 expression, and this regulation is dependent on Hfq and RNase E, cofactors known to mediate small RNA (sRNA) activities. Thus, we hypothesized that the hilD mRNA 3' UTR is a target for sRNAs. Here, we show that two sRNAs, SdsR and Spot 42, regulate SPI-1 by targeting different regions of the hilD mRNA 3' UTR. Regulatory activities of these sRNAs depended on Hfq and RNase E, in agreement with previous roles found for both at the hilD 3' UTR. Salmonella mutants lacking SdsR and Spot 42 had decreased virulence in a mouse model of infection. Collectively, this work suggests that these sRNAs targeting the hilD mRNA 3' UTR increase hilD mRNA levels by interfering with RNase E-dependent mRNA degradation and that this regulatory effect is required for Salmonella invasiveness. Our work provides novel insights into mechanisms of sRNA regulation at bacterial mRNA 3' UTRs and adds to our knowledge of post-transcriptional regulation of the SPI-1 complex feed-forward loop. IMPORTANCE Salmonella enterica serovar Typhimurium is a prominent foodborne pathogen, infecting millions of people a year. To express virulence genes at the correct time and place in the host, Salmonella uses a complex regulatory network that senses environmental conditions. Known for their role in allowing quick responses to stress and virulence conditions, we investigated the role of small RNAs in facilitating precise expression of virulence genes. We found that the 3' untranslated region of the hilD mRNA, encoding a key virulence regulator, is a target for small RNAs and RNase E. The small RNAs stabilize hilD mRNA to allow proper expression of Salmonella virulence genes in the host.

Transcriptional insight into the effect of benzalkonium chloride on resistance and virulence potential in Salmonella Typhimurium

Benzalkonium chloride (BC), a class of quaternary ammonium compounds (QACs), is widely used as a surface disinfectant in food industries and hospitals. To date, little is known about the underlying mechanisms of how bacterial pathogens respond to and develop resistance against QACs. We investigated the genome-wide transcriptional responses of Salmonella enterica serovar Typhimurium to treatment with BC and identified the genetic determinants required for bacterial resistance to BC, including ramRA, phoU-pstBACS, cpxARP, and ugpQCEAB. Remarkably, RamA, a member of the AraC/XylS family transcription regulators, increased its transcription upon treatment with BC and its absence rendered Salmonella susceptible to BC treatment, indicating the positive role of RamA in BC tolerance. The attenuated BC resistance of the ΔramA mutant strain was complemented by the introduction of AcrA in trans, indicating that the AcrAB-TolC efflux system activated by RamA is required for the resistance of Salmonella to BC. Meanwhile, sublethal concentrations of BC downregulated the mRNA expression of genes associated with Salmonella pathogenicity island 1 (SPI-1) and SPI-2, which are critical determinants of Salmonella virulence. In accordance with the downregulation of SPI-1, HilD, the master regulator of SPI-1, also decreased upon treatment with BC; however, the absence of Lon protease nearly nullified the BC-mediated repression of SPI-1 genes. Intriguingly, overexpression of RamA repressed the transcription of SPI-1 genes; however, its negative regulation of SPI-1 expression is likely to be independent of the Lon-mediated regulation of SPI-1. These results demonstrated that treatment with sublethal concentrations of BC not only stimulates Salmonella to develop resistance mechanisms against BC, but also influences Salmonella virulence.

The Two-Component System CpxRA Represses Salmonella Pathogenicity Island 2 by Directly Acting on the ssrAB Regulatory Operon.

ABSTRACTThe acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The ssrAB bicistronic operon, located in SPI-2, encodes the SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the ssrAB regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of ssrA. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes.IMPORTANCE SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the ssrAB regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.

Pervasive transcription enhances the accessibility of H-NS–silenced promoters and generates bistability in Salmonella virulence gene expression

In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS-silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5' rapid amplification of complementary DNA ends sequencing (5' RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD's ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.

Oxidative stress activates transcription of Salmonella pathogenicity island-2 genes in macrophages

The type III secretion system encoded in the Salmonella pathogenicity island-2 (SPI-2) gene cluster facilitates intracellular growth of nontyphoidal Salmonella by interfering with the maturation of Salmonella-containing vacuoles along the degradative pathway. SPI-2 gene products also protect Salmonella against the antimicrobial activity of reactive oxygen species (ROS) synthesized by the phagocyte NADPH oxidase 2 (NOX2). However, a potential relationship between inflammatory ROS and the activation of transcription of SPI-2 genes by intracellular Salmonella is unclear. Here, we show that ROS engendered in the innate host response stimulate SPI-2 gene transcription. We found that the expression of SPI-2 genes in Salmonella-sustaining oxidative stress conditions involves DksA, a protein otherwise known to regulate the stringent response of bacteria to nutritional stress. We also demonstrate that the J and zinc-2-oxidoreductase domains of DnaJ as well as the ATPase activity of the DnaK chaperone facilitate loading of DksA onto RNA polymerase complexed with SPI-2 promoters. Furthermore, the DksA-driven transcription of SPI-2 genes in Salmonella experiencing oxidative stress is contingent on upstream OmpR, PhoP, and SsrB signaling events that participate in the removal of nucleoid proteins while simultaneously recruiting RNA polymerase to SPI-2 promoter regions. Taken together, our results suggest the activation of SPI-2 gene transcription in Salmonella subjected to ROS produced by the respiratory burst of macrophages protects this intracellular pathogen against NOX2-mediated killing. We propose that Salmonella have co-opted inflammatory ROS to induce SPI-2-mediated protective responses against NOX2 host defenses.

Coordinated interaction between Lon protease and catalase-peroxidase regulates virulence and oxidative stress management during Salmonellosis

ABSTRACT This study investigates the interplay between Lon protease and catalase-peroxidase (KatG) in relation to virulence modulation and the response to oxidative stress in Salmonella Typhimurium (ST). Proteomic comparison of ST wild-type and lon deletion mutant led to the recognition of a highly expressed KatG protein product among five other protein candidates that were significantly affected by lon deletion. By employing a bacterium two-hybrid assay (B2H), we demonstrated that the catalytic domain of Lon protease potentially interacts with the KatG protein that leads to proteolytic cleavage. Assessment of virulence gene expression in single and double lon and katG mutants revealed katG to be a potential positive modulator of both Salmonella pathogenicity Island-1 (SPI-1) and −2, while lon significantly affected SPI-1 genes. ST double deletion mutant, ∆lon∆katG was more susceptible to survival defects within macrophage-like cells and exhibited meager colonization of the mouse spleen compared to the single deletion mutants. The findings reveal a previously unknown function of Lon and KatG interaction in Salmonella virulence. Taken together, our experiments demonstrate the importance of Lon and KatG to cope with oxidative stress, for intracellular survival and in vivo virulence of Salmonella.

σS-Mediated Stress Response Induced by Outer Membrane Perturbation Dampens Virulence in Salmonella enterica serovar Typhimurium.

Salmonella alters cellular processes as a strategy to improve its intracellular fitness during host infection. Alternative σ factors are known to rewire cellular transcriptional regulation in response to environmental stressors. σs factor encoded by the rpoS gene is a key regulator required for eliciting the general stress response in many proteobacteria. In this study, Salmonella Typhimurium deprived of an outer membrane protein YcfR was attenuated in intracellular survival and exhibited downregulation in Salmonella pathogenicity island-2 (SPI-2) genes. This decreased SPI-2 expression caused by the outer membrane perturbation was abolished in the absence of rpoS. Interestingly, regardless of the defects in the outer membrane integrity, RpoS overproduction decreased transcription from the common promoter of ssrA and ssrB, which encode a two-component regulatory system for SPI-2. RpoS was found to compete with RpoD for binding to the PssrA region, and its binding activity with RNA polymerase (RNAP) to form Eσs holoenzyme was stimulated by the small regulatory protein Crl. This study demonstrates that Salmonella undergoing RpoS-associated stress responses due to impaired envelope integrity may reciprocally downregulate the expression of SPI-2 genes to reduce its virulence.

The Small RNA RyhB Homologs from Salmonella Typhimurium Restrain the Intracellular Growth and Modulate the SPI-1 Gene Expression within RAW264.7 Macrophages.

Growing evidence indicates that small noncoding RNAs (sRNAs) play important regulatory roles during bacterial infection. In Salmonella Typhimurium, several sRNAs are strongly up-regulated within macrophages, but little is known about their role during the infection process. Among these sRNAs, the well-characterized paralogs RyhB-1 and RyhB-2 are two regulators of gene expression mainly related with the response to iron availability. To investigate the role of the sRNAs RyhB-1 and RyhB-2 from S. Typhimurium in the infection of RAW264.7 macrophages, we analyzed several phenotypic traits from intracellular mutant strains lacking one and both sRNAs. Deletion of RyhB-1 and/or RyhB-2 resulted in increased intracellular survival and faster replication within macrophages. The bacterial metabolic status inside macrophages was also analyzed, revealing that all the mutant strains exhibited higher intracellular levels of ATP and lower NAD+/NADH ratios than the wild type. Expression analyses from bacteria infecting macrophages showed that RyhB-1 and RyhB-2 affect the intra-macrophage expression of bacterial genes associated with the Salmonella pathogenicity island 1 (SPI-1) and the type III secretion system (T3SS). With a two-plasmid system and compensatory mutations, we confirmed that RyhB-1 and RyhB-2 directly interact with the mRNAs of the invasion chaperone SicA and the regulatory protein RtsB. Altogether, these results indicate that the RyhB homologs contribute to the S. Typhimurium virulence modulation inside macrophages by reducing the intracellular growth and down-regulating the SPI-1 gene expression.

The Hha-TomB toxin-antitoxin module in Salmonella enterica serovar Typhimurium limits its intracellular survival profile and regulates host immune response.

The key to bacterial virulence relies on an exquisite balance of signals between microbe and hosts. Bacterial toxin-antitoxin (TA) system is known to play a vital role in response to stress adaptation, drug resistance, biofilm formation, intracellular survival, persistence as well as pathogenesis. In the present study, we investigated the role of Hha-TomB TA system in regulating virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium) in a host model system, where we showed that deletion of hha and tomB genes displayed impaired cell adhesion, invasion, and uptake. The isogenic hha and tomB mutant strain was also found to be deficient in intracellular replication in vitro, with a highly repressed Salmonella Pathogenicity Island-2 (SPI-2) genes and downregulation of Salmonella Pathogenicity Island-1 (SPI-1) genes. In addition, the Δhha and ΔtomB did not show acute colitis in C57BL/6 mice and displayed less dissemination to systemic organs followed by their cecal pathology. The TA mutants also showed reduction in serum cytokine and nitric oxide levels both in vitro and in vivo. However, the inflammation phenotype was restored on complementing strain of TA gene to its mutant strain. In silico studies depicted firm interaction of Hha-TomB complex and the regulatory proteins, namely, SsrA, SsrB, PhoP, and PhoQ. Overall, we demonstrate that this study of Hha-TomB TA system is one of the prime regulating networks essential for S. Typhimurium pathogenesis. 1. Role of Hha-TomB toxin-antitoxin (TA) system in Salmonella pathogenesis was examined. 2. The TA mutants resulted in impaired invasion and intracellular replication in vitro. 3. The TA mutants displayed alteration in SPI-1 and SPI-2 regulatory genes inside host cells. 4. Mutation in TA genes also limited systemic colonization and inflammatory response in vivo.

HilE is required for synergistic activation of SPI-1 gene expression in Salmonella enterica serovar Typhimurium

BackgroundSalmonella enterica serovar Typhimurium is an intestinal pathogen capable of infecting a wide range of animals. It initiates infection by invading intestinal epithelial cells using a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). The SPI-1 genes are regulated by multiple interacting transcription factors. The master regulator is HilD. HilE represses SPI-1 gene expression by binding HilD and preventing it from activating its target promoters. Previous work found that acetate and nutrients synergistically induce SPI-1 gene expression. In the present study, we investigated the role of HilE, nominally a repressor of SPI-1 gene expression, in mediating this response to acetate and nutrients.ResultsHilE is necessary for activation of SPI-1 gene expression by acetate and nutrients. In mutants lacking hilE, acetate and nutrients no longer increase SPI-1 gene expression but rather repress it. This puzzling response is not due to the BarA/SirA two component system, which governs the response to acetate. To identify the mechanism, we profiled gene expression using RNAseq in the wild type and a ΔhilE mutant under different growth conditions. Analysis of these data suggested that the Rcs system, which regulates gene expression in response to envelope stress, is involved. Consistent with this hypothesis, acetate and nutrients were able to induce SPI-1 gene expression in mutants lacking hilE and the Rcs system.ConclusionsWhile the exact mechanism is unknown, these results demonstrate the HilE, nominally a repressor of SPI-1 gene expression, can also function as an activator under the growth conditions investigated. Collectively, these results provide new insights regarding SPI-1 gene regulation and demonstrate that HilE is more complex than initially envisioned.

PagR mediates the precise regulation of Salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in Salmonella Typhimurium.

To establish systemic infections, Salmonella enterica serovar Typhimurium (S.Typhimurium) requires Salmonella pathogenicity island 2 (SPI-2) to survive and replicate within macrophages. High expression of many SPI-2 genes during the entire intracellular growth period within macrophages is essential, as it contributes to the formation of Salmonella-containing vacuole and bacterial replication. However, the regulatory mechanisms underlying the sustained induction of SPI-2 within macrophages are not fully understood. Here, we revealed a time-dependent regulation of SPI-2 expression mediated by a novel regulator PagR (STM2345) in response to the low Mg2+ and low phosphate (Pi ) signals, which ensured the high induction of SPI-2 during the entire intramacrophage growth period. Deletion of pagR results in reduced bacterial replication in macrophages and attenuation of systemic virulence in mice. The effects of pagR on virulence are dependent on upregulating the expression of slyA, a regulator of SPI-2. At the early (0-4hr) and later (after 4hr) stage post-infection of macrophages, pagR is induced by the low Pi via PhoB/R two-component systems and low Mg2+ via PhoP/Q systems, respectively. Collectively, our findings revealed that the PagR-mediated regulatory mechanism contributes to the precise and sustained activation of SPI-2 genes within macrophages, which is essential for S.Typhimurium systemic virulence.

Site-specific acylation of a bacterial virulence regulator attenuates infection.

Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we discovered that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR-Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells as well as dissemination in vivo. Moreover, a multiple-site HilA lysine-acylation mutant strain of S. Typhimurium was resistant to butyrate-mediated suppression in vivo. Our results suggest prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.

Cinnamaldehyde inhibits type three secretion system in Salmonella enterica serovar Typhimurium by affecting the expression of key effector proteins

The increasing understanding of bacterial pathogenesis has revealed many new targets for the development of non-traditional antibacterial drugs. Interference with bacterial virulence has become a new strategy to treat bacteria-mediated diseases. As an important food-borne pathogen, Salmonella enterica serovar Typhimurium uses type III secretion system (T3SS) to facilitate invasion of host cells. In this study, we identified cinnamaldehyde as a Salmonella pathogenicity island 1 (SPI-1) inhibitor which blocks the secretion of several SPI-1 associated effector proteins and consequently exhibits a strong inhibitory effect on SPI-1-mediated invasion of HeLa cells. Further study revealed that cinnamaldehyde significantly reduced the transcription of some SPI-1 genes, such as sipA and sipB, in S. Typhimurium by affecting multiple SPI-1 regulator genes. In an animal infection model, cinnamaldehyde effectively protected infected mice against S. Typhimurium-induced mortality and pathological damages. In summary, this study presented an effective SPI-1 inhibitor, cinnamaldehyde, which reduces the expression of SPI-1 effector proteins by regulating the transcription of main regulator genes.

Synergistic action of SPI-1 gene expression in Salmonella enterica serovar typhimurium through transcriptional crosstalk with the flagellar system

BackgroundSalmonella enterica serovar Typhimurium is a common food-borne pathogen. S. enterica uses a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1) to invade intestinal epithelial cells. A complex network of interacting transcription factors regulates SPI-1 gene expression. In addition, SPI-1 gene expression is coupled to flagellar gene expression. Both SPI-1 and flagellar gene expression are bistable, with co-existing populations of cells expressing and not expressing these genes. Previous work demonstrated that nutrients could be used to tune the fraction of cells expressing the flagellar genes. In the present study, we tested whether nutrients could also tune the fraction of cells expressing the SPI-1 genes through transcriptional crosstalk with the flagellar genes.ResultsNutrients alone were not found to induce SPI-1 gene expression. However, when the cells were also grown in the presence of acetate, the concentration of nutrients in the growth medium was able to tune the fraction of cells expressing the SPI-1 genes. During growth in nutrient-poor medium, acetate alone was unable to induce SPI-1 gene expression. These results demonstrate that acetate and nutrients synergistically activate SPI-1 gene expression. The response to acetate was governed by the BarA/SirA two-component system and the response to nutrients was governed by transcriptional crosstalk with the flagella system, specifically through the action of the flagellar regulator FliZ.ConclusionsAcetate and nutrients are capable of synergistically activating SPI-1 gene expression. In addition, these signals were found to tune the fraction of cells expressing the SPI-1 genes. The governing mechanism involves transcriptional crosstalk with the flagellar gene network. Collectively, these results further our understanding of SPI-1 gene regulation and provide the basis for future studies investigating this complex regulatory mechanism.