Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium

The two-component regulatory system PmrA-PmrB confers resistance of Salmone lla spp. to cationic antimicrobial peptides (AP) such as polymyxin (PM), ba ctericidal/permeability-increasing protein, and azurocidin. This resistance occurs by transcriptional activation of two loci termed pmrE and pmrNFIJKL M, Both pmrE and pmrHFIJKLM produce products required for the biosynthesis of lipid A with 4-aminoarabinose (Ara4N). Ara4N addition creates a more pos itively charged lipopolysaccharide (LPS) and thus reduces cationic AP bindi ng. Experiments were conducted to further analyze the regulation of the pmr HFIJKLM operon and the role of this operon and the surrounding genomic regi on in LPS modification and antimicrobial peptide resistance. The pmrHFIJKLM genes are cotranscribed and over 3,000-fold regulated by PmrA-PmrB. The pm rHFIJKLM promoter bound PmrA, as determined by gel shift analysis, as did a 40-bp region of the PmrA-PmrB-regulated pmrCAB promoter, Construction of n onpolar mutations in the pmrHFIJKLM genes showed that all except pmrM were necessary for the Ara4N addition to lipid A and PM resistance. The flanking genes of the operon (pmrG and pmrD) were not necessary for PM resistance, but pmrD was shown to be regulated by the PhoP-PhoQ regulatory system. BALB /c mice inoculated with pmrA and pmrHFIJKLM mutant strains demonstrated vir ulence attenuation when the strains were administered orally but not when t hey were administered intraperitoneally, indicating that Ara4N addition may be important for resistance to host innate defenses within intestinal tiss ues.