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

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). Upregullation 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.