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. 2005 Nov;187(21):7407-16.
doi: 10.1128/JB.187.21.7407-7416.2005.

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

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

Massimo Merighi et al. J Bacteriol. 2005 Nov.

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.

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Figures

FIG. 1.
FIG. 1.
Expression of pmrH in vitro. S. enterica serovar Typhimurium JSG2428 carrying a cointegrated pmrH-tnpRmut135 fusion was grown in LB (pH 7.7) in the presence of Ap and Tc and then was diluted 1:100 in NMM (pH 7.7) with 10 mM MgCl2, NMM (pH 5.6)- 10 μM MgCl2, or NMM (pH 5.6)-10 μM MgCl2 with 100 μM FeSO4. At various time points, aliquots were diluted and plated on LB-Ap, and 100 colonies were patched to LB-Tc from each sample. The proportion of Tets colonies was calculated, and the background resolution in the original inoculum was subtracted.
FIG. 2.
FIG. 2.
Expression of pmrH during infection of BALB/c mice. S. enterica serovar Typhimurium JSG2428 carrying a cointegrated pmrH-tnpRmut135 fusion was grown in LB (pH 7.7) in the presence of Ap and Tc. Female BALB/c mice were infected with 108 CFU each by oral gavage. At various time points, groups of two mice were sacrificed, and selected organs were dissected and homogenized. The proportion of Tets colonies was determined as described in Materials and Methods.
FIG. 3.
FIG. 3.
Expression of tnpR fusions within murine macrophage cell lines. Salmonella strains carrying a cointegrated tnpRmut135 fusion to various genes (pmrH, pagP, prgH, and ssaB) were grown in LB (pH 7.7) in the presence of Ap and Tc to prepare the inoculum for cell culture infections. The mouse macrophage cell line J774A.1 was infected at an multiplicity of infection of 10 and, following the invasion period, was exposed to gentamicin. After 16 h, macrophage cells were lysed with 1% Triton X-100 and aliquots were diluted and plated on LB-Ap. Approximately 100 colonies were patched to LB-Tc from each sample. As a medium control, bacteria were grown in DMEM for 16 h. The proportion of Tets colonies was calculated, and the background resolution in the original inoculum was subtracted.
FIG. 4.
FIG. 4.
In vivo expression of pmrH is fully dependent on both PhoP and PmrA. The phoP::Tn10d-Cm or pmrA::kan allele was transduced into S. enterica serovar Typhimurium JSG2428 (pmrH-tnpRmut135) to generate JSG2437 and JSG2436, respectively. The strains were grown in LB (pH 7.7) in the presence of Ap and Tc to prepare the inoculum. Two female BALB/c mice were infected with 108 CFU each by oral gavage. After 48 h, the mice were sacrificed and selected organs were dissected and homogenized. Aliquots were diluted and plated on LB-Ap, and up to 100 colonies were patched to LB-Tc from each sample. The proportion of Tets colonies was calculated, and the background resolution in the original inoculum was subtracted. Averages with the corresponding standard deviations are shown. WT, wild type.
FIG. 5.
FIG. 5.
Orally fed sodium bicarbonate does not affect the induction of pmrH in the gastrointestinal lumen. S. enterica serovar Typhimurium JSG2428 carrying a cointegrated pmrH-tnpRmut135 fusion was grown in LB (pH 7.7) in the presence of Ap and Tc to prepare the inoculum. Two female BALB/c mice were infected with 108 CFU each by oral gavage. Three oral doses of 100 μl 10% NaHCO3 were administered at −60, 0, and +30 min from the inoculation time. After 5, 24, and 72 h, the mice were sacrificed and selected organs were dissected and homogenized. Aliquots were diluted and plated on LB-Ap, and up to 100 colonies were patched to LB-Tc from each sample. The proportion of Tets colonies was calculated, and the background resolution in the original inoculum was subtracted. Averages with the corresponding standard deviations are shown.
FIG. 6.
FIG. 6.
Expression of pmrH in the gastrointestinal lumen is not dependent on high concentrations of bioavailable iron. Female BALB/c mice were fed iron-deficient or normal diets for 3 weeks. S. enterica serovar Typhimurium strain JSG2428 (pmrH-tnpRmut135) was grown in LB (pH 7.7) in the presence of Ap and Tc. Mice were infected with 108 CFU each by oral gavage. The iron-deficient group was also administered water containing the iron(III) chelator desferrioxamine (DFO) 24 h before inoculation until the end of the experiment. After 48 h, the mice were sacrificed and selected organs were dissected and homogenized. Aliquots were diluted and plated on LB-Ap, and up to 100 colonies were patched to LB-Tc from each sample. The proportion of Tets colonies was calculated, and the background resolution in the original inoculum was subtracted. Bioavailable iron was measured using a Ferrozine protocol, and the concentrations ranged from 7.5 to 40 μmol/kg lumen content in the normal diet treatment to <0 to 1.4 μmol/kg lumen content in the low-iron diet treatment.
FIG. 7.
FIG. 7.
In vivo transcription of the PhoP-activated gene pagP, the PhoP-repressed gene prgH, and the SPI-2 gene ssaB. S. enterica serovar Typhimurium strains carrying cointegrated fusions to tnpRmut135 were grown in LB (pH 7.7) in the presence of Ap and Tc. Female BALB/c mice were infected with 108 CFU each by oral gavage. At 48 h postinoculation, two mice per treatment were sacrificed, and the luminal contents, Peyer's patches, and spleens were homogenized. Aliquots were diluted and plated on LB-Ap, and up to 100 colonies were patched to LB-Tc from each sample. The proportion of Tets colonies was calculated, and the background resolution in the original inoculum was subtracted. Averages with the corresponding standard deviations are shown. JSG2502, pagP-tnpRmut135; JSG2504, prgH-tnpRmut135; JSG2524, ssaB-tnpRmut135. ND, not determined.

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