Abstract
BackgroundThe cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria.MethodsIn this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen Klebsiella pneumoniae were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of pmr genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and in vitro phosphor-transfer assay.ResultsDeletion of the rcsB, which encoded an activator for the production of capsular polysaccharide, had a minor effect on K. pneumoniae resistance to polymyxin B. On the other hand, deletion of ugd or pmrF gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in Salmonella, expression of pmrD in K. pneumoniae was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of pmr for the resistance to polymyxin B.ConclusionsThe study reports a role of the capsular polysaccharide level and the pmr genes for K. pneumoniae resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of pmr expression was demonstrated. In comparison to the pmr regulation in Salmonella, PhoP in K. pneumoniae plays a major regulatory role in polymyxin B resistance.
Highlights
The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria
In order to verify the role of capsular polysaccharide (CPS) in polymyxin B resistance, the Δugd and Δwza mutants were generated by allelic exchange strategy, and their phenotype as well as the amount of CPS produced were compared with the parental strain CG43S3 and ΔrcsB mutant [14]
The colony morphology of the ΔrcsB mutant was indistinguishable from CG43S3, the CPS-deficient phenotype was evident as assessed using sedimentation assay and the amount of K2 CPS produced (Figure 1B)
Summary
The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria. Polymyxins, originally isolated from Bacillus polymyxa, have emerged as promising candidates for the treatment of infections [5]. The assembly and transport of Klebsiella CPS followed the E. coli Wzydependent pathway [11], in which mutations at wza encoding the translocon protein forming the complex responsible for CPS polymer translocation and export resulted in an inability to assemble a capsular layer on the cell surface [12]. The CPS biosynthesis in K. pneumoniae was transcriptionally regulated by the two-component system (2CS) RcsBCD [13] where the deletion of the response regulator encoding gene rcsB in K. pneumoniae caused a loss of mucoid phenotype and reduction in CPS production [14]
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