Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-stressed Salmonella. Further, a prophage gene, STM2699, induced in cold-stressed Salmonella and a spectrin gene, SPTAN1, induced in Salmonella-infected intestinal epithelial cells were found to have a significant contribution in increased adhesion and invasion of cold-stressed Salmonella in epithelial cells.

Active invasion into nonphagocytic host cells is central to Salmonella enterica pathogenicity and dependent on multiple genes within Salmonella pathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovarS Paratyphi A compared to that of the nontyphoidal serovarS Typhimurium. We demonstrate that while S. Typhimurium is equally invasive under both aerobic and microaerobic conditions, S. Paratyphi A invades only following growth under microaerobic conditions. Transcriptome sequencing (RNA-Seq), reverse transcription-PCR (RT-PCR), Western blot, and secretome analyses established that S. Paratyphi A expresses much lower levels of SPI-1 genes and secretes lesser amounts of SPI-1 effector proteins than S. Typhimurium, especially under aerobic growth. Bypassing the native SPI-1 regulation by inducible expression of the SPI-1 activator, HilA, considerably elevated SPI-1 gene expression, host cell invasion, disruption of epithelial integrity, and induction of proinflammatory cytokine secretion by S. Paratyphi A but not by S. Typhimurium, suggesting that SPI-1 expression is naturally downregulated inS Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization byS Paratyphi A and showed moderately higher pathology and intestinal inflammation in mice infected with S. Paratyphi A overexpressing hilA Collectively, our results reveal unexpected differences in SPI-1 expression between S. Paratyphi A andS Typhimurium, indicate that S. Paratyphi A host cell invasion is suppressed under aerobic conditions, and suggest that lower invasion in aerobic sites and suppressed expression of immunogenic SPI-1 components contributes to the restrained inflammatory infection elicited by S. Paratyphi A.

Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies.

Bacterial surface ligands mediate interactions with the host cell during association that determines the specific outcome for the host-microbe association. The association begins with receptors on the host cell binding ligands on the microbial cell to form a partnership that initiates responses in both cells. Methods to determine the specific cognate partnerships are lacking. Determining these molecular interactions between the host and microbial surfaces are difficult, yet crucial in defining biologically important events that are triggered during association of the microbiome, and critical in defining the initiating signal from the host membrane that results in pathology or commensal association. In this study, we designed an approach to discover cognate host-microbe receptor/ligand pairs using a covalent cross-linking strategy with whole cells. Protein/protein cross-linking occurred when the interacting molecules were within 9-12 Å, allowing for identification of specific pairs of proteins from the host and microbe that define the molecular interaction during association. To validate the method three different bacteria with three previously known protein/protein partnerships were examined. The exact interactions were confirmed and led to discovery of additional partnerships that were not recognized as cognate partners, but were previously reported to be involved in bacterial interactions. Additionally, three unknown receptor/ligand partners were discovered and validated with in vitro infection assays by blocking the putative host receptor and deleting the bacterial ligand. Subsequently, Salmonella enterica sv. Typhimurium was cross-linked to differentiated colonic epithelial cells (caco-2) to discover four previously unknown host receptors bound to three previously undefined host ligands for Salmonella. This approach resulted in a priori discovery of previously unknown and biologically important molecules for host/microbe association that were casually reported to mediate bacterial invasion. The whole cell cross-linking approach promises to enable discovery of possible targets to modulate interaction of the microbiome with the host that are important in infection and commensalism, both of with initiate a host response.

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org).

Salmonella enterica serovar Paratyphi A is a human-specific serovar that, together with Salmonella enterica serovar Typhi and Salmonella enterica serovar Sendai, causes enteric fever. Unlike the nontyphoidal Salmonella enterica serovar Typhimurium, the genomes of S. Typhi and S. Paratyphi A are characterized by inactivation of multiple genes, including in the flagellum-chemotaxis pathway. Here, we explored the motility phenotype of S. Paratyphi A and the role of flagellin in key virulence-associated phenotypes. Motility studies established that the human-adapted typhoidal S. Typhi, S. Paratyphi A, and S. Sendai are all noticeably less motile than S. Typhimurium, and comparative transcriptome sequencing (RNA-Seq) showed that in S. Paratyphi A, the entire motility-chemotaxis regulon is expressed at significantly lowers levels than in S. Typhimurium. Nevertheless, S. Paratyphi A, like S. Typhimurium, requires a functional flagellum for epithelial cell invasion and macrophage uptake, probably in a motility-independent mechanism. In contrast, flagella were found to be dispensable for host cell adhesion. Moreover, we demonstrate that in S. Paratyphi A, but not in S. Typhimurium, the lack of flagellin results in increased transcription of the flagellar and the Salmonella pathogenicity island 1 (SPI-1) regulons in a FliZ-dependent manner and in oversecretion of SPI-1 effectors via type three secretion system 1. Collectively, these results suggest a novel regulatory linkage between flagellin and SPI-1 in S. Paratyphi A that does not occur in S. Typhimurium and demonstrate curious distinctions in motility and the expression of the flagellum-chemotaxis regulon between these clinically relevant pathogens.

Salmonella is an important cause of bacterial food-borne gastroenteritis. Salmonella encounters multiple abiotic stresses during pathogen elimination methods used in food processing, and these stresses may influence its subsequent survivability within the host or in the environment. Upon ingestion, Salmonella is exposed to gastrointestinal acidity, a first line of the host innate defense system. This study tested the hypothesis that abiotic stresses encountered during food processing alter the metabolic mechanisms in Salmonella that enable survival and persistence during subsequent exposure to the host gastrointestinal acidic environment. Out of the four different abiotic stresses tested, viz., cold, peroxide, osmotic, and acid, preadaptation of the log-phase culture to cold stress (5°C for 5 h) significantly enhanced survival during subsequent acid stress (pH 4.0 for 90 min). The gene expression profile of Salmonella preadapted to cold stress revealed induction of multiple genes associated with amino acid metabolism, oxidative stress, and DNA repair, while only a few of the genes in the above-mentioned stress response and repair pathways were induced upon exposure to acid stress alone. Preadaptation to cold stress decreased the NAD+/NADH ratio and hydroxyl (OH·) radical formation compared with those achieved with the exposure to acid stress alone, indicating alteration of aerobic respiration and the oxidative state of the bacteria. The results from this study suggest that preadaptation to cold stress rescues Salmonella from the deleterious effect of subsequent acid stress exposure by induction of genes involved in stress response and repair pathways, by modification of aerobic respiration, and by redox modulation.

Here, we report a 4.90 Mbp draft genome sequence of Salmonella enterica subsp. enterica serovar Putten strain CRJJGF_00159 isolated from food animal in 2004.

The microbial adaptations to the respiratory burst remain poorly understood, and establishing how the NADPH oxidase (NOX2) kills microbes has proven elusive. Here we demonstrate that NOX2 collapses the ΔpH of intracellular Salmonella Typhimurium. The depolarization experienced by Salmonella undergoing oxidative stress impairs folding of periplasmic proteins. Depolarization in respiring Salmonella mediates intense bactericidal activity of reactive oxygen species (ROS). Salmonella adapts to the challenges oxidative stress imposes on membrane bioenergetics by shifting redox balance to glycolysis and fermentation, thereby diminishing electron flow through the membrane, meeting energetic requirements and anaplerotically generating tricarboxylic acid intermediates. By diverting electrons away from the respiratory chain, glycolysis also enables thiol/disulfide exchange-mediated folding of bacterial cell envelope proteins during periods of oxidative stress. Thus, primordial metabolic pathways, already present in bacteria before aerobic respiration evolved, offer a solution to the stress ROS exert on molecular targets at the bacterial cell envelope.

Outbreaks of salmonellosis linked to the consumption of vegetables have been disproportionately associated with strains of serovar Newport. We tested the hypothesis that strains of sv. Newport have evolved unique adaptations to persistence in plants that are not shared by strains of other Salmonella serovars. We used a genome-wide mutant screen to compare growth in tomato fruit of a sv. Newport strain from an outbreak traced to tomatoes, and a sv. Typhimurium strain from animals. Most genes in the sv. Newport strain that were selected during persistence in tomatoes were shared with, and similarly selected in, the sv. Typhimurium strain. Many of their functions are linked to central metabolism, including amino acid biosynthetic pathways, iron acquisition, and maintenance of cell structure. One exception was a greater need for the core genes involved in purine metabolism in sv. Typhimurium than in sv. Newport. We discovered a gene, papA, that was unique to sv. Newport and contributed to the strain's fitness in tomatoes. The papA gene was present in about 25% of sv. Newport Group III genomes and generally absent from other Salmonella genomes. Homologs of papA were detected in the genomes of Pantoea, Dickeya, and Pectobacterium, members of the Enterobacteriacea family that can colonize both plants and animals.