Identification of genetic loci that contribute to Campylobacter resistance to fowlicidin-1, a chicken host defense peptide.
Antimicrobial peptides (AMPs) are critical components of host defense limiting bacterial infections at the gastrointestinal mucosal surface. Bacterial pathogens have co-evolved with host innate immunity and developed means to counteract the effect of endogenous AMPs. However, molecular mechanisms of AMP resistance in Campylobacter, an important human food-borne pathogen with poultry as a major reservoir, are still largely unknown. In this study, random transposon mutagenesis and targeted site-directed mutagenesis approaches were used to identify genetic loci contributing Campylobacter resistance to fowlicidin-1, a chicken AMP belonging to cathelicidin family. An efficient transposon mutagenesis approach (EZ::TN™ <KAN-2> Transposome) in conjunction with a microtiter plate screening identified three mutants whose susceptibilities to fowlicidin-1 were significantly increased. Backcrossing of the transposon mutations into parent strain confirmed that the AMP-sensitive phenotype in each mutant was linked to the specific transposon insertion. Direct sequencing showed that these mutants have transposon inserted in the genes encoding two-component regulator CbrR, transporter CjaB, and putative trigger factor Tig. Genomic analysis also revealed an operon (Cj1580c-1584c) that is homologous to sapABCDF, an operon conferring resistance to AMP in other pathogens. Insertional inactivation of Cj1583c (sapB) significantly increased susceptibility of Campylobacter to fowlicidin-1. The sapB as well as tig and cjaB mutants were significantly impaired in their ability to compete with their wild-type strain 81-176 to colonize the chicken cecum. Together, this study identified four genetic loci in Campylobacter that will be useful for characterizing molecular basis of Campylobacter resistance to AMPs, a significant knowledge gap in Campylobacter pathogenesis.
Department of Animal Science, The University of Tennessee, Knoxville TN, USA.
Ky Van
KV
Hoang
Ying
Y
Wang
Jun
J
Lin
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D002167:Campylobacter / Q000187:drug effects* / Q000235:genetics*; D002645:Chickens / Q000276:immunology; D003433:Crosses, Genetic; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D056426:Genetic Loci; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA
D023181:Antimicrobial Cationic Peptides; D004251:DNA Transposable Elements; D004269:DNA, Bacterial