ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis

Abstract

Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen’s ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella -containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3′UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program. IMPORTANCE Salmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella .

The FinO/ProQ-like protein PA2582 impacts antimicrobial resistance in Pseudomonas aeruginosa

“…However, it remains to be shown how the protein affects their transcript abundance. For some ProQ homologs, it has been suggested that the protein binds to the 3′-ends of mRNAs and stabilizes these transcripts ( Holmqvist et al, 2018 ; Bauriedl et al, 2020 ; Gulliver et al, 2022 ; Bergman et al, 2024 ). Alternatively or in addition, ProQ Pae might be involved in sRNA-mediated regulation of these genes.…”

“…In addition, the proQ mutant strain was deficient in biofilm formation ( Sheidy and Zielke, 2013 ), and showed a decrease in virulence ( Wang et al, 2023 ). In S. enterica , ProQ modulates the expression of genes involved in motility and virulence ( Westermann et al, 2019 ; Bergman et al, 2024 ), and impacts persister cell formation ( Rizvanovic et al, 2022 ). Furthermore, ProQ affects the growth rate of S. enterica in full broth and minimal media with succinate as the sole C-source under microaerobic conditions ( El Mouali et al, 2021 ), and was shown to be involved in sRNA-mediated riboregulation in this organism ( Smirnov et al, 2017 ).…”