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 flagellumchemotaxis regulon between these clinically relevant pathogens. Salmonella enterica is one of the most prevalent human and animal pathogens, consisting of more than 2,500 serovars. Some Salmonella serovars, such as Salmonella enterica serovar Typhimurium, are ubiquitous pathogens that can infect a broad range of animal and human hosts. In contrast, other Salmonella serovars are host restricted and highly adapted in their pathogenesis. Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi A, and Salmonella enterica serovar Sendai are humanrestricted serovars and the causative agents of enteric fever (1, 2). This is an invasive, life-threatening systemic disease with a global annual estimation of over 25 million cases, resulting in more than 200,000 deaths (3). In recent years, the occurrence of infection with S. Paratyphi A has been rising, and in some regions of the world (particularly in eastern and southern Asia), it is accountable for up to 50% of all enteric fever cases (4, 5). Increasing rates of infection, the lack of a commercially available vaccine, and steadily increasing resistance of S. Paratyphi A isolates to antimicrobial agents make S. Paratyphi A infections a significant public health concern.Bacterial invasion of nonphagocytic cells is one of the hallmarks of S. enterica and is pivotal for its pathogenicity. Active invasion by Salmonella of eukaryotic ...

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 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. Salmonella enterica is a highly diverse and ubiquitous pathogen containing more than 2,600 different serovars classified by their antigenic presentation (1). Salmonella serovars differ by their host specificity and by the clinical syndromes they cause, ranging from asymptomatic carriage to invasive systemic disease. While many nontyphoidal Salmonella (NTS) serovars, such as Typhimurium and Enteritidis, are generalist pathogens with broad host specificity, a few S. enterica serovars, including Typhi, Sendai, and Paratyphi A, are highly adapted to humans. These specialist pathogens, collectively referred to as typhoidal Salmonella serovars, are the causative agents of enteric fever, posing an estimated global annual burden of over 27 million cases, resulting in more than 200,000 deaths (2). While NTS cause inflammatory gastroenteritis that is confined to the terminal ileum and colon in immunocompetent patients, typhoidal serovars do not induce a strong inflammatory response during the initial invasion of the intestinal mucosa (3-5). This noninflammatory phase associated with typhoidal infections is thought to facilitate its dissemination to systemic sites (6).Regardless of the clinical manifestation, both typhoidal and NTS ...