Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella.

Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.

Improved RNA stability estimation through Bayesian modeling reveals most Salmonella transcripts have subminute half-lives.

RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in posttranscriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 min, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with cross-linking and immunoprecipitation followed by RNA sequencing (CLIP-seq) revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3' untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for ProQ in the oxidative stress response. Our findings provide insights into posttranscriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

Alternate typhoid toxin assembly evolved independently in the two Salmonella species.

AB5-type toxins are a diverse family of protein toxins composed of an enzymatic active (A) subunit and a pentameric delivery (B) subunit. Salmonella enterica serovar Typhi's typhoid toxin features two A subunits, CdtB and PltA, in complex with the B subunit PltB. Recently, it was shown that S. Typhi encodes a horizontally acquired B subunit, PltC, that also assembles with PltA/CdtB to produce a second form of typhoid toxin. S. Typhi therefore produces two AB5 toxins with the same A subunits but distinct B subunits, an evolutionary twist that is unique to typhoid toxin. Here, we show that, remarkably, the Salmonella bongori species independently evolved an analogous capacity to produce two typhoid toxins with distinct B subunits. S. bongori's alternate B subunit, PltD, is evolutionarily distant from both PltB and PltC and outcompetes PltB to form the predominant toxin. We show that, surprisingly, S. bongori elicits similar levels of CdtB-mediated intoxication as S. Typhi during infection of cultured human epithelial cells. This toxicity is exclusively due to the PltB toxin, and strains lacking pltD produce increased amounts of PltB toxin and exhibit increased toxicity compared to the wild type, suggesting that the acquisition of the PltD subunit potentially made S. bongori less virulent toward humans. Collectively, this study unveils a striking example of convergent evolution that highlights the importance of the poorly understood "two-toxin" paradigm for typhoid toxin biology and, more broadly, illustrates how the flexibility of A-B interactions has fueled the evolutionary diversification and expansion of AB5-type toxins. IMPORTANCE: Typhoid toxin is an important Salmonella Typhi virulence factor and an attractive target for therapeutic interventions to combat typhoid fever. The recent discovery of a second version of this toxin has substantial implications for understanding S. Typhi pathogenesis and combating typhoid fever. In this study, we discover that a remarkably similar two-toxin paradigm evolved independently in Salmonella bongori, which strongly suggests that this is a critical aspect of typhoid toxin biology. We observe significant parallels between how the two toxins assemble and their capacity to intoxicate host cells during infection in S. Typhi and S. bongori, which provides clues to the biological significance of this unusual toxin arrangement. More broadly, AB5 toxins with diverse activities and mechanisms are essential virulence factors for numerous important bacterial pathogens. This study illustrates the capacity for novel A-B interactions to evolve and thus provides insight into how such a diverse arsenal of toxins might have emerged.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Scs system links copper and redox homeostasis in bacterial pathogens.

The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.

Outer Membrane Proteins and Efflux Pumps Mediated Multi-Drug Resistance in Salmonella: Rising Threat to Antimicrobial Therapy.

Despite colossal achievements in antibiotic therapy in recent decades, drug-resistant pathogens have remained a leading cause of death and economic loss globally. One such WHO-critical group pathogen is Salmonella. The extensive and inappropriate treatments for Salmonella infections have led from multi-drug resistance (MDR) to extensive drug resistance (XDR). The synergy between efflux-mediated systems and outer membrane proteins (OMPs) may favor MDR in Salmonella. Differential expression of the efflux system and OMPs (influx) and positional mutations are the factors that can be correlated to the development of drug resistance. Insights into the mechanism of influx and efflux of antibiotics can aid in developing a structurally stable molecule that can be proficient at escaping from the resistance loops in Salmonella. Understanding the strategic responsibilities and developing policies to address the surge of drug resistance at the national, regional, and global levels are the needs of the hour. In this Review, we attempt to aggregate all the available research findings and delineate the resistance mechanisms by dissecting the involvement of OMPs and efflux systems. Integrating major OMPs and the efflux system's differential expression and positional mutation in Salmonella may provide insight into developing strategic therapies for one health application.

Diverse gut pathogens exploit the host engulfment pathway via a conserved mechanism.

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

Physiological stress drives the emergence of a Salmonella subpopulation through ribosomal RNA regulation.

Bacteria undergo cycles of growth and starvation to which they must adapt swiftly. One important strategy for adjusting growth rates relies on ribosomal levels. Although high ribosomal levels are required for fast growth, their dynamics during starvation remain unclear. Here, we analyzed ribosomal RNA (rRNA) content of individual Salmonella cells by using fluorescence in situ hybridization (rRNA-FISH) and measured a dramatic decrease in rRNA numbers only in a subpopulation during nutrient limitation, resulting in a bimodal distribution of cells with high and low rRNA content. During nutritional upshifts, the two subpopulations were associated with distinct phenotypes. Using a transposon screen coupled with rRNA-FISH, we identified two mutants, DksA and RNase I, acting on rRNA transcription shutdown and degradation, which abolished the formation of the subpopulation with low rRNA content. Our work identifies a bacterial mechanism for regulation of ribosomal bimodality that may be beneficial for population survival during starvation.

Acetylation of K188 and K192 inhibits the DNA-binding ability of NarL to regulate Salmonella virulence.

Salmonella infection significantly increases nitrate levels in the intestine, immune cells, and immune organs of the host, and it can exploit nitrate as an electron acceptor to enhance its growth. In the presence of nitrate or nitrite, NarL, a regulatory protein of the Nar two-component system, is activated and regulates a number of genes involved in nitrate metabolism. However, research on NarL at the post-translational level is limited. In this study, we demonstrate that the DNA-binding sites K188 and 192 of NarL can be acetylated by bacterial metabolite acetyl phosphate and that the degree of acetylation has a considerable influence on the regulatory function of NarL. Specifically, acetylation of NarL negatively regulates the transcription of narG, narK, and napF, which affects the utilization of nitrate in Salmonella. Besides, both cell and mouse models show that acetylated K188 and K192 result in attenuated replication in RAW 264.7 cells, as well as impaired virulence in mouse model. Together, this research identifies a novel NarL acetylation mechanism that regulates Salmonella virulence, providing a new insight and target for salmonellosis treatment.IMPORTANCESalmonella is an important intracellular pathogen that can cause limited gastroenteritis and self-limiting gastroenteritis in immunocompetent humans. Nitrate, the highest oxidation state form of nitrogen, is critical in the formation of systemic infection in Salmonella. It functions as a signaling molecule that influences Salmonella chemotaxis, in addition to acting as a reduced external electron acceptor for Salmonella anaerobic respiration. NarL is an essential regulatory protein involved in nitrate metabolism in Salmonella, and comprehending its regulatory mechanism is necessary. Previous research has linked NarL phosphorylation to the formation of its dimer, which is required for NarL to perform its regulatory functions. Our research demonstrated that acetylation also affects the regulatory function of NarL. We found that acetylation affects Salmonella pathogenicity by weakening the ability of NarL to bind to the target sequence, further refining the mechanism of the anaerobic nitrate respiration pathway.

TamAB is regulated by PhoPQ and functions in outer membrane homeostasis during Salmonella pathogenesis.

Salmonella survive and replicate in macrophages, which normally kill bacteria by exposing them to a variety of harsh conditions and antimicrobial effectors, many of which target the bacterial cell envelope. The PhoPQ two-component system responds to the phagosome environment and induces factors that protect the outer membrane, allowing adaptation and growth in the macrophage. We show that PhoPQ induces the transcription of the tamAB operon both in vitro and in macrophages. The TamA protein is structurally similar to BamA, an essential protein in the Bam complex that assembles ß-barrel proteins in the outer membrane, while TamB is an AsmA-family protein implicated in lipid transport between the inner and outer membranes. We show that the Bam machinery is stressed in vitro under low Mg2+, low pH conditions that mimic the phagosome. Not surprisingly, mutations affecting Bam function confer significant virulence defects. Although loss of TamAB alone confers no virulence defect, a tamAB deletion confers a synthetic phenotype in bam mutant backgrounds in animals and macrophages, and in vitro upon treatment with vancomycin or sodium dodecyl sulfate. Mutations affecting YhdP, which functions in partial redundancy with TamB, also confer synthetic phenotypes with bam mutations in the animal, but this interaction is not evident in vitro. Thus, in the harsh phagocytic environment of the macrophage, the outer membrane Bam machinery is compromised, and the TamAB system, and perhaps other PhoPQ-regulated factors, is induced to compensate. It is most likely that TamAB and other systems assist the Bam complex indirectly by affecting outer membrane properties. IMPORTANCE The TamAB system has been implicated in both outer membrane protein localization and phospholipid transport between the inner and outer membranes. We show that the ß-barrel protein assembly complex, Bam, is stressed under conditions thought to mimic the macrophage phagosome. TamAB expression is controlled by the PhoPQ two-component system and induced in macrophages. This system somehow compensates for the Bam complex as evidenced by the fact that mutations affecting the two systems confer synthetic phenotypes in animals, macrophages, and in vitro in the presence of vancomycin or SDS. This study has implications concerning the role of TamAB in outer membrane homeostasis. It also contributes to our understanding of the systems necessary for Salmonella to adapt and reproduce within the macrophage phagosome.

Phages on filaments: A genetic screen elucidates the complex interactions between Salmonella enterica flagellin and bacteriophage Chi.

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia.

Parkinson's disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella.

Cell-intrinsic defences constitute the first line of defence against intracellular pathogens. The guanosine triphosphatase RAB32 orchestrates one such defence response against the bacterial pathogen Salmonella, through delivery of antimicrobial itaconate. Here we show that the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) orchestrates this defence response by scaffolding a complex between RAB32 and aconitate decarboxylase 1, which synthesizes itaconate from mitochondrial precursors. Itaconate delivery to Salmonella-containing vacuoles was impaired and Salmonella replication increased in LRRK2-deficient cells. Loss of LRRK2 also restored virulence of a Salmonella mutant defective in neutralizing this RAB32-dependent host defence pathway in mice. Cryo-electron tomography revealed tether formation between Salmonella-containing vacuoles and host mitochondria upon Salmonella infection, which was significantly impaired in LRRK2-deficient cells. This positions LRRK2 centrally within a host defence mechanism, which may have favoured selection of a common familial Parkinson's disease mutant allele in the human population.

Speaking the host language: how Salmonella effector proteins manipulate the host.

Salmonella injects over 40 virulence factors, termed effectors, into host cells to subvert diverse host cellular processes. Of these 40 Salmonella effectors, at least 25 have been described as mediating eukaryotic-like, biochemical post-translational modifications (PTMs) of host proteins, altering the outcome of infection. The downstream changes mediated by an effector's enzymatic activity range from highly specific to multifunctional, and altogether their combined action impacts the function of an impressive array of host cellular processes, including signal transduction, membrane trafficking, and both innate and adaptive immune responses. Salmonella and related Gram-negative pathogens have been a rich resource for the discovery of unique enzymatic activities, expanding our understanding of host signalling networks, bacterial pathogenesis as well as basic biochemistry. In this review, we provide an up-to-date assessment of host manipulation mediated by the Salmonella type III secretion system injectosome, exploring the cellular effects of diverse effector activities with a particular focus on PTMs and the implications for infection outcomes. We also highlight activities and functions of numerous effectors that remain poorly characterized.

Salmonella T3SS effector SseK1 arginine-glycosylates the two-component response regulator OmpR to alter bile salt resistance.

Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.

Engineered Salmonella inhibits GPX4 expression and induces ferroptosis to suppress glioma growth in vitro and in vivo.

PURPOSE: Glioma is a life-threatening malignancy where conventional therapies are ineffective. Bacterial cancer therapy has shown potential for glioma treatment, in particular, the facultative anaerobe Salmonella has been extensively studied. Meanwhile, ferroptosis is a newly characterized form of cell death. Nevertheless, the role of ferroptosis in Salmonella-induced tumour cell death remains unclear. Therefore, we aim to elucidate whether Salmonella YB1 exerts therapeutic effects via inducing ferroptosis in glioma. METHODS: Following Salmonella YB1 infection, mRNA sequencing was applied to detect ferroptosis-related gene expression and the levels of reactive oxygen species, malondialdehyde, and glutathione were quantified. Transmission electron microscopy (TEM) was then used to observe the changes in the mitochondrial morphology of glioma cells. The role of ferroptosis in the anti-tumor effect of YB1 was assessed in vivo in mouse tumor xenograft models. RESULTS: Whole-transcriptome analysis revealed that Salmonella YB1 infection alters ferroptosis-related gene expression in the U87 glioma cell line. Moreover, we found that Salmonella-induced ferroptosis is correlated with reduced levels of glutathione and glutathione peroxidase-4 (GPX4) and increased levels of reactive oxygen species and malondialdehyde in vitro. Meanwhile, TEM revealed that mitochondria are shrunken and mitochondrial membrane density increases in infected glioma cells. Experiments in vivo further showed that tumor growth in the Salmonella-treated group was significantly slower compared to the control and Fer-1 groups. However, Salmonella-induced tumor suppression can be reversed in vivo by Fer-1 treatment. CONCLUSION: Salmonella YB1 inhibits GPX4 expression and induces ferroptosis to suppress glioma growth. Hence, ferroptosis regulation might represent a promising strategy to improve the efficacy of bacterial cancer therapy.

Salmonella enterica serovar Typhi uses two type 3 secretion systems to replicate in human macrophages and colonize humanized mice.

Salmonella enterica serovar Typhi (S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S. Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an S. Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflict with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.

Arginine Metabolism Powers Salmonella Resistance to Oxidative Stress.

Salmonella invades host cells and replicates inside acidified, remodeled vacuoles that are exposed to reactive oxygen species (ROS) generated by the innate immune response. Oxidative products of the phagocyte NADPH oxidase mediate antimicrobial activity, in part, by collapsing the ΔpH of intracellular Salmonella. Given the role of arginine in bacterial resistance to acidic pH, we screened a library of 54 single-gene mutants in Salmonella that are each involved in, but do not entirely block, arginine metabolism. We identified several mutants that affected Salmonella virulence in mice. The triple mutant ΔargCBH, which is deficient in arginine biosynthesis, was attenuated in immunocompetent mice, but recovered virulence in phagocyte NADPH oxidase deficient Cybb-/- mice. Furthermore, ΔargCBH Salmonella was profoundly susceptible to the bacteriostatic and bactericidal effects of hydrogen peroxide. Peroxide stress led to a larger collapse of the ΔpH in ΔargCBH mutants than occurred in wild-type Salmonella. The addition of exogenous arginine rescued ΔargCBH Salmonella from peroxide-induced ΔpH collapse and killing. Combined, these observations suggest that arginine metabolism is a hitherto unknown determinant of virulence that contributes to the antioxidant defenses of Salmonella by preserving pH homeostasis. In the absence of phagocyte NADPH oxidase-produced ROS, host cell-derived l-arginine appears to satisfy the needs of intracellular Salmonella. However, under oxidative stress, Salmonella must additionally rely on de novo biosynthesis to maintain full virulence.

Functional flexibility and plasticity in immune control of systemic Salmonella infection.

Immunity to systemic Salmonella infection depends on multiple effector mechanisms. Lymphocyte-derived interferon gamma (IFN-γ) enhances cell-intrinsic bactericidal capabilities to antagonize the hijacking of phagocytes as replicative niches for Salmonella. Programmed cell death (PCD) provides another means through which phagocytes fight against intracellular Salmonella. We describe remarkable levels of flexibility with which the host coordinates and adapts these responses. This involves interchangeable cellular sources of IFN-γ regulated by innate and adaptive cues, and the rewiring of PCD pathways in previously unknown ways. We discuss that such plasticity is likely the consequence of host-pathogen coevolution and raise the possibility of further functional overlap between these seemingly distinct processes.

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole.

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.