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.
Highlights
Upon infection, bacteria must evade the arsenal of host immune defenses, including cationic antimicrobial peptides (CAMPs), designed to eliminate invading pathogens
To verify that the observed in vitro CAMP sensing by Salmonella was dependent on PhoP-mediated gene activation, ALKALINE PHOSPHATASE (AP) assays were performed with a pagJ::TnphoA PhoP− reporter fusion strain (Bader et al, 2003), where AP activity in the presence of peptides was greatly reduced in comparison to the wild type (WT) pagJ reporter strain (Figure 1)
The results confirm that sublethal concentrations of CAMPs can activate Salmonella two-component regulatory systems (TCRSs)-mediated gene expression, as reported by Bader et al (2003, 2005)
Summary
Bacteria must evade the arsenal of host immune defenses, including cationic antimicrobial peptides (CAMPs), designed to eliminate invading pathogens. The antimicrobial activity of CAMPs comes from the ability of these molecules to insert into the microbial membrane, resulting in membrane destabilization and microbial lysis (Matsuzaki et al, 1997; Radek and Gallo, 2007; Bucki et al, 2010). These peptides are rapidly produced by macrophages and epithelial cells in response to infection or injury and can mediate inflammation and stimulate the immune system upon detection of pathogens (Menendez and Brett Finlay, 2007). Typhimurium PhoPQ and PmrAB and their regulons are activated by unknown environmental signals in macrophages, in other host cells and in the intestinal lumen (Foster and Hall, 1990; Alpuche Aranda et al, 1992; Garcia Vescovi et al, 1996; Bearson et al, 1998; Wosten et al, 2000; Merighi et al, 2005)
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