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ABSTRACTActive invasion into nonphagocytic host cells is central to Salmonella entericapathogenicity and dependent on multiple genes within Salmonellapathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovar S. Paratyphi A compared to that of the nontyphoidal serovar S. 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 in S. Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization by S. 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 and S. 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.

ABSTRACTSalmonella entericaserovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide. Salmonellapathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that in S.Typhimurium, dksAis induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates the Salmonellapathogenicity island 1 and motility-chemotaxis genes and is necessary for S.Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable for S.Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found that dksAis spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infection in vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulence S.Typhimurium programs and therefore is a key virulence regulator of Salmonella.

ABSTRACTSalmonella entericaserovar Paratyphi A is a human-specific serovar that, together with Salmonella entericaserovar Typhi and Salmonella entericaserovar Sendai, causes enteric fever. Unlike the nontyphoidal Salmonella entericaserovar 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 Salmonellapathogenicity 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.

ABSTRACTStaphylococcus aureusis a pathogen that causes significant morbidity and mortality. Nasal carriage is a major source of transmission and of endogenous infection. Persistent carriage is detected in ∼30% of healthy individuals. While Th17 cells have been shown to play a role in S. aureusinfection and clearance, the immune response to carriage is not well understood. Here, we evaluate the Th17 response and its potential inhibitors during S. aureuscarriage. We recruited 25 volunteers, of whom 11 were persistent carriers. Volunteers' peripheral blood mononuclear cells (PBMCs) were stimulated with either their endogenous strain (a strain isolated from that carrier) or exogenous ones (strains not carried by that volunteer). Changes in Th17 cell frequency and numbers, interleukin-17 (IL-17) mRNA expression, and IL-17 protein abundance were measured by fluorescence-activated cell sorting, real-time PCR, and enzyme-linked immunosorbent assay. Similarly, responses of IL-17 suppressors (regulatory T cells [FOXP3], IL-10, IL-27, and IL-19) were measured. Th17 and IL-17 levels in response to stimulation with endogenous strains were significantly lower than those in response to stimulation with exogenous ones. Of the suppressive cytokines tested, only IL-19 exhibited a stronger response to endogenous than to exogenous strains. Addition of recombinant IL-19 to exogenous-strain-stimulated PBMCs caused decreased IL-17 expression, whereas addition of IL-19 antibodies to endogenous-strain-stimulated cells resulted in an increased IL-17 response. Together, our results suggest that S. aureuscarriage induced a tolerogenic response to a carried strain that could be reproduced through the addition of recombinant IL-19 or prevented by the addition of IL-19 antibodies. This differential immune response may play a role in the determination of S. aureuscarriage patterns.