Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

Abstract

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors.

Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

“…The transcriptional activity of these reporters was measured in vitro in defined LPM minimal medium, which provides an environment in which SsrB and upstream regulators of virulence genes, PhoPQ and SlyA, are active (10). We assessed the transcriptional activities of our reporters in backgrounds deficient in these proteins involved in intracellular pathogenesis (56). We observed altered profiles of transcriptionally active regions upstream of sciF, sciG, and vrgS (Fig.…”

“…To determine whether the expression of the SPI-6 T6SS is repressed by the activity of regulators of intracellular virulence, we assessed the levels of chromosomal fusion protein VrgS::HA expressed under the control of its native promoter in backgrounds deficient in regulators of intracellular pathogenesis (56). These included the two-component systems PhoPQ, OmpR/EnvZ, QseBC, PmrAB, and SsrAB and the RNA binding protein Hfq, regulators important for intracellular pathogenesis (Fig.…”

“…Given our observation of increased transcriptional activity upstream of sciF and the previously reported increase in sciS transcripts in a ssrB mutant background, we asked whether the loss of these regulatory inputs has a significant effect on VrgS protein expression through regulatory deregulation. Other regulatory proteins aside from PhoP-PhoQ, SsrASsrB, and SlyA are necessary for intracellular survival during host infection (56). VrgS levels were not affected in the phoP, ssrB, qseC, ompR, or hfq mutant background, although the disruption of these proteins results in extensive changes in S. Typhimurium gene expression and leads to abrogation of pathogenesis (56).…”

Dual RNA-seq of pathogen and host

“…This factor has previously been suggested to impact Salmonella virulence by affecting both SPI-1 (Chen et al, 2010) and SPI-2 (Yoon et al, 2009), via unknown mechanisms. Whereas the presented data also cannot provide a mechanistic link between CRP expression and SPI activity, it suggests that the CRP hub in the regulatory network would provide a temporal gap between PhoP-dependent SPI-2 induction and its indirect, STnc440-mediated repression (Fig.…”

“…Since GrxA and CRP affect intracellular replication by activating Salmonella virulence genes (Bjur et al, 2006;Chen et al, 2010;Yoon et al, 2009), they are considered putative factors through which STnc440 might signal to SPI-2. Likewise, EcnB levels have recently been shown to be regulated between proliferating and resting Salmonella in vivo (Claudi et al, 2014).…”