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Staphylococcus aureus (S. aureus) is an opportunistic pathogen causing diseases ranging from mild skin infections to life threatening conditions, including endocarditis, pneumonia, and sepsis. To identify host genes modulating this host-pathogen interaction, we infected 25 Collaborative Cross (CC) mouse strains with methicillin-resistant S. aureus (MRSA) and monitored disease progression for seven days using a surgically implanted telemetry system. CC strains varied widely in their response to intravenous MRSA infection. We identified eight 'susceptible' CC strains with high bacterial load, tissue damage, and reduced survival. Among the surviving strains, six with minimal colonization were classified as 'resistant', while the remaining six tolerated higher organ colonization ('tolerant'). The kidney was the most heavily colonized organ, but liver, spleen and lung colonization were better correlated with reduced survival. Resistant strains had higher pre-infection circulating neutrophils and lower post-infection tissue damage compared to susceptible and tolerant strains. We identified four CC strains with sexual dimorphism: all females survived the study period while all males met our euthanasia criteria earlier. In these CC strains, males had more baseline circulating monocytes and red blood cells. We identified several CC strains that may be useful as new models for endocarditis, myocarditis, pneumonia, and resistance to MRSA infection. Quantitative Trait Locus (QTL) analysis identified two significant loci, on Chromosomes 18 and 3, involved in early susceptibility and late survival after infection. We prioritized Npc1 and Ifi44l genes as the strongest candidates influencing survival using variant analysis and mRNA expression data from kidneys within these intervals.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection.

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of "nutritional immunity", where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

Rift Valley fever Virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease, Rift Valley fever (RVF). In both humans and livestock, protective immunity can be achieved through vaccination. Earlier and more recent vaccine trials in cattle and sheep demonstrated a strong neutralizing antibody and total IgG response induced by the RVF vaccine, authentic recombinant MP-12 (arMP-12). From previous work, protective immunity in sheep and cattle vaccinates normally occurs from 7 to 21 days after inoculation with arMP-12. While the serology and protective response induced by arMP-12 has been studied, little attention has been paid to the underlying molecular and genetic events occurring prior to the serologic immune response. To address this, we isolated RNA from whole blood of vaccinated calves over a time course of 21 days before and after vaccination with arMP-12. The time course RNAs were sequenced by RNASeq and bioinformatically analyzed. Our results revealed time-dependent activation or repression of numerous gene ontologies and pathways related to the vaccine induced immune response and its regulation. Additional bioinformatic analyses identified a correlative relationship between specific host immune response genes and protective immunity prior to the detection of protective serum neutralizing antibody responses. These results contribute an important proof of concept for identifying molecular and genetic components underlying the immune response to RVF vaccination and protection prior to serologic detection.

[This corrects the article DOI: 10.1371/journal.pone.0147027.].

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

Chicks are ideal to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Taxonomic/metagenomic analyses captured the development of the chick microbiota in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm) during development. Taxonomic analysis suggests that colonization by the chicken microbiota takes place in several waves. The cecal microbiota stabilizes at day 12 posthatch with prominent Gammaproteobacteria and Clostridiales. Introduction of S. Typhimurium at day 4 posthatch disrupted the expected waves of intestinal colonization. Taxonomic and metagenomic shotgun sequencing analyses allowed us to identify species present in uninfected chicks. Untargeted metabolomics suggested different metabolic activities in infected chick microbiota. This analysis and gas chromatography-mass spectrometry on ingesta confirmed that lactic acid in cecal content coincides with the stable presence of enterococci in STm-infected chicks. Unique metabolites, including 2-isopropylmalic acid, an intermediate in the biosynthesis of leucine, were present only in the cecal content of STm-infected chicks. The metagenomic data suggested that the microbiota in STm-infected chicks contained a higher abundance of genes, from STm itself, involved in branched-chain amino acid synthesis. We generated an ilvC deletion mutant (STM3909) encoding ketol-acid-reductoisomerase, a gene required for the production of l-isoleucine and l-valine. ΔilvC mutants are disadvantaged for growth during competitive infection with the wild type. Providing the ilvC gene in trans restored the growth of the ΔilvC mutant. Our integrative approach identified biochemical pathways used by STm to establish a colonization niche in the chick intestine during development. IMPORTANCE Chicks are an ideal model to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Using taxonomic and metagenomic analyses, we captured the development of chick microbiota to 19 days posthatch in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm). We show that normal development of the microbiota takes place in waves and is altered in the presence of a pathogen. Metagenomics and metabolomics suggested that branched-chain amino acid biosynthesis is especially important for Salmonella growth in the infected chick intestine. Salmonella mutants unable to make l-isoleucine and l-valine colonize the chick intestine poorly. Restoration of the pathway for biosynthesis of these amino acids restored the colonizing ability of Salmonella. Integration of multiple analyses allowed us to correctly identify biochemical pathways used by Salmonella to establish a niche for colonization in the chick intestine during development.

To identify the resistance phenotype against Mycobacterium bovis in cattle, we used a bactericidal assay that has been considered a marker of this trait. Three of 24 cows (12.5%) were phenotyped as resistant and 21 as susceptible. Resistance of bovine macrophages (MΦ) to BCG challenge was evaluated for its association with SLC11A1 GT microsatellite polymorphisms within 3'UTR region. Twenty-three cows (95.8%) had a GT13 genotype, reported as resistant, consequently the SLC11A1 polymorphism was not in agreement with our bactericidal assay results. MΦ of cows with resistant or susceptible phenotype were challenged in vitro with virulent M. bovis field strain or BCG, and nitric oxide production, bacterial killing and apoptosis induction were measured in resting and LPS-primed states. M. bovis field strain induced more apoptosis than BCG, although the difference was not significant. Resistant MΦ controlled better the replication of M. bovis (P

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.

Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

LuxR-type transcription factors are typically used by bacteria to determine the population density of their own species by detecting N-acylhomoserine lactones (AHLs). However, while Escherichia and Salmonella encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species.In this report, we tested the hypothesis that SdiA detects the AHL-production of other bacterial species within the animal host. SdiA did not detect AHLs during the transit of Salmonella through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during the transit of Salmonella through turtles. All turtles examined were colonized by the AHL-producing species Aeromonas hydrophila.We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals.

Abscisic acid (ABA) is an ancient stress hormone and is detectable in a wide variety of organisms where it regulates innate immunity and inflammation. Previously, we showed that oral supplementation with ABA decreased parasitemia in a mouse model of malaria, decreased liver and spleen pathology and reduced parasite transmission to mosquitoes. Here, we report that higher circulating ABA levels were associated with a reduced risk of symptomatic malaria in a cohort of Plasmodium falciparum-infected Ugandan children. To understand possible mechanisms of ABA protection in malaria, we returned to our mouse model to show that ABA effects on Plasmodium yoelii 17XNL infection were accompanied by minimal effects on complete blood count and blood chemistry analytes, suggesting a benefit to host health. In addition, orally delivered ABA induced patterns of gene expression in mouse liver and spleen that suggested enhancement of host anti-parasite defenses. To test these inferences, we utilized passive immunization and knockout mice to demonstrate that ABA supplementation increases circulating levels of protective, parasite-specific IgG and requires caspase-1 to reduce parasitemia. Collectively, ABA induces host responses that ameliorate infection and disease in an animal model and suggest that further studies of ABA in the context of human malaria are warranted.

Staphylococcus aureus (S. aureus) colonizes humans asymptomatically but can also cause opportunistic infections, ranging from mild skin infections to severe life-threatening conditions. Resistance and tolerance are two ways a host can survive an infection. Resistance is limiting the pathogen burden, while tolerance is limiting the health impact of a given pathogen burden. In previous work, we established that collaborative cross (CC) mouse line CC061 is highly susceptible to Methicillin-resistant S. aureus infection (MRSA, USA300), while CC024 is tolerant. To identify host genes involved in tolerance after S. aureus infection, we crossed CC061 mice and CC024 mice to generate F1 and F2 populations. Survival after MRSA infection in the F1 and F2 generations was 65% and 55% and followed a complex dominant inheritance pattern for the CC024 increased survival phenotype. Colonization in F2 animals was more extreme than in their parents, suggesting successful segregation of genetic factors. We identified a Quantitative Trait Locus (QTL) peak on chromosome 7 for survival and weight change after infection. In this QTL, the WSB/EiJ (WSB) allele was present in CC024 mice and contributed to their MRSA tolerant phenotype. Two genes, C5ar1 and C5ar2, have high-impact variants in this region. C5ar1 and C5ar2 are receptors for the complement factor C5a, an anaphylatoxin that can trigger a massive immune response by binding to these receptors. We hypothesize that C5a may have altered binding to variant receptors in CC024 mice, reducing damage caused by the cytokine storm and resulting in the ability to tolerate a higher pathogen burden and longer survival. Author summary: Staphylococcus aureus causes a wide range of life-threatening diseases in humans. Resistance and tolerance are two ways a host can survive an infection. Resistance is limiting the pathogen burden, while tolerance is limiting the health impact of a given pathogen burden. Tolerance mechanisms are poorly understood in context of host-pathogen interaction. To identify host genes involved in tolerance after S. aureus infection, we crossed CC061 mice and CC024 mice. The genetic factors controlling tolerance were well segregated in the F2 population. Using QTL mapping, we identified a significant peak on chromosome 7 for survival and weight change after infection. Two genes, C5ar1 and C5ar2, have high-impact variants in this region. We hypothesize that C5a may have altered binding to variant receptors in CC024 mice, reducing damage caused by the cytokine storm and resulting in the ability to tolerate a higher pathogen burden and longer survival. [ABSTRACT FROM AUTHOR]

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

UnlabelledTo establish a replicative niche during its infectious cycle between the intestinal lumen and tissue, the enteric pathogen Salmonella enterica serovar Typhimurium requires numerous virulence genes, including genes for two type III secretion systems (T3SS) and their cognate effectors. To better understand the host-pathogen relationship, including early infection dynamics and induction kinetics of the bacterial virulence program in the context of a natural host, we monitored the subcellular localization and temporal expression of T3SS-1 and T3SS-2 using fluorescent single-cell reporters in a bovine, ligated ileal loop model of infection. We observed that the majority of bacteria at 2 h postinfection are flagellated, express T3SS-1 but not T3SS-2, and are associated with the epithelium or with extruding enterocytes. In epithelial cells, S. Typhimurium cells were surrounded by intact vacuolar membranes or present within membrane-compromised vacuoles that typically contained numerous vesicular structures. By 8 h postinfection, T3SS-2-expressing bacteria were detected in the lamina propria and in the underlying mucosa, while T3SS-1-expressing bacteria were in the lumen. Our work identifies for the first time the temporal and spatial regulation of T3SS-1 and -2 expression during an enteric infection in a natural host and provides further support for the concept of cytosolic S. Typhimurium in extruding epithelium as a mechanism for reseeding the lumen.ImportanceThe pathogenic bacterium Salmonella enterica serovar Typhimurium invades and persists within host cells using distinct sets of virulence genes. Genes from Salmonella pathogenicity island 1 (SPI-1) are used to initiate contact and facilitate uptake into nonphagocytic host cells, while genes within SPI-2 allow the pathogen to colonize host cells. While many studies have identified bacterial virulence determinants in animal models of infection, very few have focused on virulence gene expression at the single-cell level during an in vivo infection. To better understand when and where bacterial virulence factors are expressed during an acute enteric infection of a natural host, we infected bovine jejunal-ileal loops with S. Typhimurium cells harboring fluorescent transcriptional reporters for SPI-1 and -2 (PinvF and PssaG, respectively). After a prescribed time of infection, tissue and luminal fluid were collected and analyzed by microscopy. During early infection (≤2 h), bacteria within both intact and compromised membrane-bound vacuoles were observed within the epithelium, with the majority expressing SPI-1. As the infection progressed, S. Typhimurium displayed differential expression of the SPI-1 and SPI-2 regulons, with the majority of tissue-associated bacteria expressing SPI-2 and the majority of lumen-associated bacteria expressing SPI-1. This underscores the finding that Salmonella virulence gene expression changes as the pathogen transitions from one anatomical location to the next.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen causing diseases ranging from mild skin infections to life threatening conditions, including endocarditis, pneumonia, and sepsis. To identify host genes modulating this host-pathogen interaction, we infected 25 Collaborative Cross (CC) mouse strains with methicillin-resistant S. aureus (MRSA) and monitored disease progression for seven days using a surgically implanted telemetry system. CC strains varied widely in their response to intravenous MRSA infection. We identified eight 'susceptible' CC strains with high bacterial load, tissue damage, and reduced survival. Among the surviving strains, six with minimal colonization were classified as 'resistant', while the remaining six tolerated higher organ colonization ('tolerant'). The kidney was the most heavily colonized organ, but liver, spleen and lung colonization were better correlated with reduced survival. Resistant strains had higher pre-infection circulating neutrophils and lower post-infection tissue damage compared to susceptible and tolerant strains. We identified four CC strains with sexual dimorphism: all females survived the study period while all males met our euthanasia criteria earlier. In these CC strains, males had more baseline circulating monocytes and red blood cells. We identified several CC strains that may be useful as new models for endocarditis, myocarditis, pneumonia, and resistance to MRSA infection. Quantitative Trait Locus (QTL) analysis identified two significant loci, on Chromosomes 18 and 3, involved in early susceptibility and late survival after infection. We prioritized Npc1 and Ifi44l genes as the strongest candidates influencing survival using variant analysis and mRNA expression data from kidneys within these intervals. Author summary: Methicillin-resistant Staphylococcus aureus is a human opportunistic pathogen that can cause life-threatening diseases. To study the influence of host genetics on the outcome of MRSA infection, we infected a collection of genetically diverse mice. We identified different phenotypes for survival, organ colonization, and tissue damage, and classified CC strains into MRSA susceptible, tolerant, and resistant categories. We identified several parameters that correlated with these phenotypes. Four CC strains exhibited strong sexual dimorphism in infection outcome: females lived longer, and males had higher baseline circulating monocytes and red blood cells. Several of the CC strains we characterize may represent better animal models for diseases caused by MRSA. QTL analysis identified two genes, Npc1 and Ifi44l, as strong candidates for involvement in early susceptibility and late survival after MRSA infection. Our data suggests a strong involvement of host genetics in MRSA infection outcome. [ABSTRACT FROM AUTHOR]

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock. Understanding the mechanisms underlying Salmonella-host interactions are important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.

Salmonella enterica serotype Typhimurium (S. Typhimurium) causes acute gut inflammation by using its virulence factors to invade the intestinal epithelium and survive in mucosal macrophages. The inflammatory response enhances the transmission success of S. Typhimurium by promoting its outgrowth in the gut lumen through unknown mechanisms. Here we show that reactive oxygen species generated during inflammation react with endogenous, luminal sulphur compounds (thiosulphate) to form a new respiratory electron acceptor, tetrathionate. The genes conferring the ability to use tetrathionate as an electron acceptor produce a growth advantage for S. Typhimurium over the competing microbiota in the lumen of the inflamed gut. We conclude that S. Typhimurium virulence factors induce host-driven production of a new electron acceptor that allows the pathogen to use respiration to compete with fermenting gut microbes. Thus the ability to trigger intestinal inflammation is crucial for the biology of this diarrhoeal pathogen.