Flagellin Is Required for Host Cell Invasion and Normal Salmonella Pathogenicity Island 1 Expression by Salmonella enterica Serovar Paratyphi A

Abstract

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 ...

The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization

“…For in vivo imaging of dksA expression, the dksA promoter region (290 bp upstream of the first methionine) was PCR amplified using the primers dksA promoter Fw and dksA promoter Rev, digested with BamHI and XhoI, and cloned into pCS26. C-terminal two-hemagglutinin (2HA)-tagged versions of SopB, SopE2, and FliC from S. Typhimurium were constructed within pWSK29 or pACYC184 as described elsewhere (27).…”

“…Host cells were infected with Salmonella cultures at multiplicity of infection (MOI) of ϳ1:50 for epithelial cells and ϳ1:10 for macrophages. Infection experiments were carried out using the gentamicin protection assay as previously described (27). Salmonella invasion was determined by the number of intracellular Salmonella cells at 2 h p.i.…”

“…Purified RNA was secondarily treated with an RNase-free DNase I followed by ethanol precipitation, and 200 ng of DNase I-treated RNA was subjected to firststrand cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad Laboratories). Real-time PCRs and data analysis were performed as recently described (27).…”

Differences in Host Cell Invasion and Salmonella Pathogenicity Island 1 Expression between Salmonella enterica Serovar Paratyphi A and Nontyphoidal S . Typhimurium

“…Host cells were infected with Salmonella cultures at a multiplicity of infection (MOI) of ϳ1:50. Infection experiments were carried out using the gentamicin protection assay as previously described (15). Salmonella invasion was determined by the number of intracellular Salmonella cells at 2 h postinfection (p.i.)…”

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE

“…S10E) in the HeLa infection model. Because the invasion assay was performed after infection synchronization, the observed phenotype may be attributed partly to the down-regulation of flagellin (39,40) but also could reflect a flagellin-independent reduction of host cell adhesion in the ΔcspCE strain (SI Appendix, Fig. S10 D and E).…”