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Resolvase-in vivo expression technology analysis of the Salmonella enterica serovar Typhimurium PhoP and PmrA regulons in BALB/c mice.

ABSTRACT: Salmonella enterica modulates resistance to antimicrobial peptides in part via covalent modifications of the lipopolysaccharide (LPS). The two-component systems PhoP/PhoQ and PmrA/PmrB are activated during infection and regulate several genes involved in LPS modifications by responding to signals such as pH, iron, magnesium, and antimicrobial peptides. A recombination-based in vivo expression technology approach was adopted to analyze the spatial-temporal patterns of in vivo expression of genes of the PhoP and PmrA regulons and to identify the in vivo signals modulating their transcription. In vitro, we showed PhoP- and/or PmrA-dependent induction of pmrH (LPS aminoarabinose modification operon) by acidic pH, low levels of magnesium, or high levels of Fe(III). Upregulation in cultured J774A.1 macrophages was shown for pmrH, pagP (LPS palmitate addition), and ssaB (pathogenicity island II secretion) but not for prgH (pathogenicity island I secretion). Increased levels of pmrH, phoP, and prgH transcription but not ssaB were observed in bacteria isolated from the lumen of the distal ileum. Bacteria isolated from spleens of orally inoculated mice showed no further induction of prgH but had the highest expression of pmrH, pagP, and ssaB. In vivo induction of pmrH was fully dependent on pmrA and phoP, and buffering stomach acidity, iron chelation, or low-iron diets did not affect the expression of pmrH in the intestinal lumen. The observation of pmrH and pagP expression in the intestine refutes the paradigm of PhoP/PhoQ and PmrA/PmrB in vivo expression as solely intracellularly induced and supports previous data demonstrating peroral virulence attenuation of pmrH mutants.

SUBMITTER: Merighi M

PROVIDER: S-EPMC1272988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Salmonella must rapidly adapt to various niches in the host during infection. Relevant virulence factors must be appropriately induced, and systems that are detrimental in a particular environment must be turned off. Salmonella infects intestinal epithelial cells using a type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The system is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which form a complex feed-forward loop to activate expression of hilA, encoding the main transcriptional regulator of T3SS structural genes. This system is tightly regulated, with many of the activating signals acting at the level of hilD translation or HilD protein activity. Once inside the phagosomes of epithelial cells, or in macrophages during systemic stages of disease, the SPI1 T3SS is no longer required or expressed. Here, we show that the PhoPQ two-component system, critical for intracellular survival, appears to be the primary mechanism by which Salmonella shuts down the SPI1 T3SS. PhoP negatively regulates hilA through multiple distinct mechanisms: direct transcriptional repression of the hilA promoter, indirect transcriptional repression of both the hilD and rtsA promoters, and activation of the small RNA (sRNA) PinT. Genetic analyses and electrophoretic mobility shift assays suggest that PhoP specifically binds the hilA promoter to block binding of activators HilD, HilC, and RtsA as a mechanism of repression.IMPORTANCESalmonella is one of the most common foodborne pathogens, causing an estimated 1.2 million illnesses per year in the United States. A key step in infection is the activation of the bacterial invasion machinery, which induces uptake of the bacterium into epithelial cells and leads to induction of inflammatory diarrhea. Upon entering the vacuolar compartments of host cells, Salmonella senses an environmental transition and represses the invasion machinery with a two-component system relevant for survival within the vacuole. This adaptation to specific host niches is an important example of how signals are integrated for survival of the pathogen.

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Resolvase-in vivo expression technology analysis of the Salmonella enterica serovar Typhimurium PhoP and PmrA regulons in BALB/c mice.

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