Human infection with typhoidal Salmonella serovars causes a febrile systemic disease, termed enteric fever. Here we establish that in response to a temperature equivalent to fever (39 °C-42 °C) Salmonella enterica serovars Typhi, Paratyphi A, and Sendai significantly attenuate their motility, epithelial cell invasion, and uptake by macrophages. Under these feverlike conditions, the residual epithelial cell invasion of S. Paratyphi A occurs in a type III secretion system (T3SS) 1-independent manner and results in restrained disruption of epithelium integrity. The impaired motility and invasion are associated with down-regulation of T3SS-1 genes and class II and III (but not I) of the flagella-chemotaxis regulon. In contrast, we demonstrate up-regulation of particular Salmonella pathogenicity island 2 genes (especially spiC) and increased intraepithelial growth in a T3SS-2-dependent manner. These results indicate that elevated physiological temperature is a novel cue controlling virulence phenotypes in typhoidal serovars, which is likely to play a role in the distinct clinical manifestations elicited by typhoidal and nontyphoidal salmonellae.

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.

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.

Background

Although chronic infections by typhoidal Salmonella are well-known, prolonged human infections by nontyphoidal Salmonella (NTS) are poorly characterized.

Methods

We retrospectively analyzed 48 345 culture-confirmed NTS infections that occurred in Israel 1995-2012. A case-control study was performed to identify risk factors associated with persistent infections. Whole-genome-sequencing, pulsed-field gel electrophoresis (PFGE), and a mouse infection model were used to study genetic and phenotypic differences between same-patient persistent, recurring isolates.

Results

In total, 1047 cases of persistent NTS infections, comprising 2.2% of all reported cases of salmonellosis, were identified. The persistence periods ranged between 30 days to 8.3 years. The majority (93%) of the persistently infected patients were immunocompetent, and 65% were symptomatic with relapsing diarrhea, indicating a distinct clinical manifestation from the asymptomatic carriage of typhoidal Salmonella. Four NTS serovars (Mbandaka, Bredeney, Infantis and Virchow) were found to be significantly more frequently associated with persistence than others. Comparative genomics between early and later isolates obtained from the same patients confirmed clonal infection and showed 0 to 10 SNPs between persistent isolates. A different composition of mobile genetic elements (plasmids and phages) or amino acid substitutions in global regulators was identified in multiple cases. These changes resulted in differences in phenotype and virulence between early and later same-patient isolates.

Conclusions

These results illuminate the overlooked clinical manifestation of persistent salmonellosis that can serve as a human reservoir for NTS infections. Additionally, we demonstrate mechanisms of in-host microevolution and exhibit their potential to shape Salmonella pathogenicity, antimicrobial resistance and host-pathogen interactions.

DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the ubiquitous Salmonella serovars worldwide and a major cause of food-born outbreaks, which are often associated with poultry and poultry derivatives. Here we report a nation-wide S. Enteritidis clonal outbreak that occurred in Israel during the last third of 2015. Pulsed field gel electrophoresis and whole genome sequencing identified genetically related strains that were circulating in Israel as early as 2008. Global comparison linked this outbreak strain to several clinical and marine environmental isolates that were previously isolated in California and Canada, indicating that similar strains are prevalent outside of Israel. Phenotypic comparison between the 2015 outbreak strain and other clinical and reference S. Enteritidis strains showed only limited intra-serovar phenotypic variation in growth in rich medium, invasion into Caco-2 cells, uptake by J774.1A macrophages, and host cell cytotoxicity. In contrast, significant phenotypic variation was shown among different S. Enteritidis isolates when biofilm-formation, motility, invasion into HeLa cells and uptake by THP-1 human macrophages were studied. Interestingly, the 2015 outbreak clone was found to possess superior intra-macrophage replication ability within both murine and human macrophages in comparison to the other S. Enteritidis strains studied. This phenotype is likely to play a role in the virulence and host-pathogen interactions of this emerging clone.

Salmonella enterica is the leading etiologic agent of bacterial food-borne outbreaks worldwide. This ubiquitous species contains more than 2,600 serovars that may differ in their host specificity, clinical manifestations, and epidemiology. To characterize salmonellosis epidemiology in Israel and to study the association of nontyphoidal Salmonella (NTS) serovars with invasive infections, 48,345 Salmonella cases reported and serotyped at the National Salmonella Reference Center between 1995 and 2012 were analyzed. A quasi-Poisson regression was used to identify irregular clusters of illness, and pulsed-field gel electrophoresis in conjunction with whole-genome sequencing was applied to molecularly characterize strains of interest. Three hundred twenty-nine human salmonellosis clusters were identified, representing an annual average of 23 (95% confidence interval [CI], 20 to 26) potential outbreaks. We show that the previously unsequenced S. enterica serovar 9,12:l,v:- belongs to the B clade of Salmonella enterica subspecies enterica, and we show its frequent association with extraintestinal infections, compared to other NTS serovars. Furthermore, we identified the dissemination of two prevalent Salmonella enterica serovar Typhimurium DT104 clones in Israel, which are genetically distinct from other global DT104 isolates. Accumulatively, these findings indicate a severe underreporting of Salmonella outbreaks in Israel and provide insights into the epidemiology and genomics of prevalent serovars, responsible for recurring illness.