Cationic antimicrobial peptides serve as activation signals for the Salmonella Typhimurium PhoPQ and PmrAB regulons in vitro and in vivo - PubMed (original) (raw)

Cationic antimicrobial peptides serve as activation signals for the Salmonella Typhimurium PhoPQ and PmrAB regulons in vitro and in vivo

Susan M Richards et al. Front Cell Infect Microbiol. 2012.

Abstract

Salmonella enterica serovar Typhimurium uses two-component regulatory systems (TCRSs) to respond to environmental stimuli. Upon infection, the TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals detected in the lumen of the intestine and within host cells. TCRS-mediated gene expression leads to upregulation of genes involved in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide (CAMP) resistance. This research expands on previous studies which have shown that CAMPs can activate Salmonella TCRSs in vitro. The focus of this work was to determine if CAMPs can act as environmental signals for PhoPQ- and PmrAB-mediated gene expression in vitro, during infection of macrophages and in a mouse model of infection. Monitoring of PhoPQ and PmrAB activation using recombinase-based in vivo expression technology (RIVET), alkaline phosphtase and β-galactosidase reporter fusion constructs demonstrated that S. Typhimurium PhoQ can sense CAMPs in vitro. In mouse macrophages, the cathelecidin CRAMP does not activate the PhoPQ regulon. Acidification of the Salmonella-containing vacuole activates PhoP- and PmrA-regulated loci but blocking acidification still does not reveal a role for CRAMP in TCRS activation in mouse macrophages. However, assays performed in susceptible wild type (WT), CRAMP knockout (KO), and matrilysin (a metalloproteinase necessary for activating murine α-defensins) KO mice suggest CRAMP, but not α-defensins, serve as a putative direct TCRS activation signal in the mouse intestine. These studies provide a better understanding of the in vivo environments that result in activation of these virulence-associated TCRSs.

Keywords: CAMPs; PhoPQ; PmrAB; Salmonella Typhimurium; lipopolysaccharide modification.

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Figures

Figure 1

PhoPQ-mediated activation of S. Typhimurium phoN and pagJ in response to CAMPs. AP assays demonstrate that different CAMPs induce varying levels of PhoP-regulated gene promoter expression. CAMP sensing by PhoQ and subsequent PhoP-mediated AP reporter gene activation is abolished in the absence of PhoP. Values shown represent the mean ± standard deviation from a representative experiment performed at least twice in triplicate. Statistical significance was measured against the samples to which no peptide was added. *Indicates p < 0.05, **Indicates p < 0.005.

Figure 2

TCRS-mediated activation of S. Typhimurium pmrI and pmrC in response to CAMPs. β-galactosidase assays demonstrate that different CAMPs induce varying levels of PmrA-regulated gene promoter expression and CAMP sensing by PhoQ. Subsequent PmrA-mediated β-galactosidase reporter gene activation is abolished in the absence of PhoP. Values shown represent the mean ± standard deviation from a representative experiment performed at least twice in triplicate. Statistical significance was measured against the samples to which no peptide was added. *Indicates p < 0.05; **indicates p < 0.005.

Figure 3

Induction of pagP and pmrH in response to S. Typhimurium TCRS-mediated sensing of LL-37 and magnesium. RIVET strains containing the promoter regions of a PhoP-mediated gene, pagP, or a PmrA-regulated gene, pmrH, were incubated in vitro with (A) LL-37, (B) CRAMP or (C) Polymyxin B and high (10 mM) or low (10 μM) MgCl2 (Mg2+) for 4 h to induce resolvase production. Bacteria were diluted and plated on LB O/N at 37°C and patched on LB and LB + Tet for detection of Tet-sensitive colonies. The percentage of recovered Tet-sensitive colonies from each sample was averaged for analysis. Graphed values represent the mean ± standard deviation of one representative experiment performed at least twice in triplicate. *Indicates p < 0.05; **indicates p < 0.005.

Figure 4

TCRS-mediated activation of pagP and pmrH in WT and CRAMP KO macrophages. BALB/c and CRAMP-deficient mouse bone marrow-derived macrophages were infected with the S. Typhimurium (A) pagP or (B) pmrH RIVET strain. Gentamycin-treated macrophages were lysed at 2.5, 6, 12 and 24 h p.i., Lysates containing intracellular bacteria were plated on LB and incubated O/N at 37°C. Resulting colonies were patched onto LB and LB Tet to detect loss of Tet-resistance due to promoter activation. Graphed values represent the mean ± standard deviation of one representative experiment performed at least twice in triplicate. *Indicates p < 0.05.

Figure 5

TCRS-mediated pagP and pmrH activation in bafilomycin-treated WT and CRAMP KO macrophages. BALB/c and CRAMP-deficient mouse bone marrow-derived macrophages were pre-treated with bafilomycin in DMSO and infected with the S. Typhimurium (A) pagP or (B) pmrH RIVET strain. Gentamicin-treated macrophages were lysed at 2.5, 6, 12 and 24 h p.i., Lysates containing intracellular bacteria were plated on LB. Resulting colonies were patched onto LB and LB Tet to detect loss of Tet-resistance due to promoter activation. Graphed values represent the mean ± standard deviation of one representative experiment performed at least twice in triplicate.

Figure 6

TCRS-mediated pagP and pmrH activation in NH4Cl-treated WT and CRAMP KO macrophages. BALB/c and CRAMP-deficient mouse bone marrow-derived macrophages were pre-treated with NH4Cl in IMDM and infected with the S. Typhimurium (A) pagP or (B) pmrH RIVET strain. Gentamicin-treated macrophages were lysed at 2.5, 6, 10 and 24 h p.i. Lysates containing intracellular bacteria were plated on LB. Resulting colonies were patched onto LB and LB Tet to detect loss of Tet-resistance due to promoter activation. Graphed values represent the mean ± standard deviation of one representative experiment performed three times in triplicate.

Figure 7

TCRS-mediated activation of S. Typhimurium pagP and pmrH in the WT and CRAMP KO murine intestinal lumen and Peyer's patches. BALB/c and CRAMP KO mice were infected orally with 108 CFU of the S. Typhimurium pagP or pmrH RIVET strains. Mice were sacrificed at 4, 12, 24 and 48 h p.i. for removal of infected organs. Tissue samples were homogenized, diluted, plated on LB and grown overnight at 37°C to recover intracellular bacteria. One hundred colonies recovered from each sample were patched on LB and LB Tet to to detect loss of Tet-resistance due to promoter activation in vivo. Graphed values represent the mean percentage ± standard deviation of Tet-sensitive colonies recovered from the (A) and (C) intestinal lumen and (B) and (D) Peyer's patches of three BALB/c and three CRAMP KO mice infected with the pagP or pmrH RIVET strain for each time point in one representative experiment performed at least twice in triplicate. One asterisk (*) indicates p < 0.005 and two asterisks (**) indicate p < 0.001.

Figure 8

TCRS-mediated activation of S. Typhimurium pagP and pmrH in the WT and Matrilysin-deficient murine intestine. C57BL/6 and MMP7 KO mice were infected orally with 108 CFU of the S. Typhimurium pagP and pmrH RIVET strains. Mice were sacrificed at 6, 24 and 48 h p.i., for removal of two-three inches of the small intestine (measured from the distal ileum). Tissue samples were homogenized, diluted, plated on LB and grown overnight at 37°C to recover intracellular bacteria. One hundred colonies recovered from each sample were patched on LB and LB Tet to detect loss of Tet-resistance due to promoter activation in vivo. Graphed values represent the mean percentage ± standard deviation of Tet-sensitive colonies recovered from the intestine of three BALB/c and three CRAMP KO mice infected with the pagP or pmrH RIVET strain for each time point in one representative experiment.

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Cationic antimicrobial peptides serve as activation signals for the Salmonella Typhimurium PhoPQ and PmrAB regulons in vitro and in vivo - PubMed (original) (raw)