Identification of genetic loci that contribute to Campylobacter resistance to fowlicidin-1, a chicken host defense peptide.
Identification of genetic loci that contribute to Campylobacter resistance to fowlicidin-1, a chicken host defense peptide.
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DOI：
10.3389/fcimb.2012.00032
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发表时间：
2012
影响因子：
5.7
通讯作者：
Lin J
中科院分区：
文献类型：
--
作者：
Hoang KV;Wang Y;Lin J
抗菌肽（AMPs）是限制胃肠道粘膜表面细菌感染的宿主防御的关键成分。细菌病原体与宿主先天免疫共同进化，并发展出对抗内源性amp影响的手段。然而，弯曲杆菌（Campylobacter）耐AMP的分子机制仍然很大程度上未知。弯曲杆菌是一种重要的人类食源性病原体，以家禽为主要宿主。本研究采用随机转座子诱变和靶向定点诱变两种方法，鉴定了弯曲杆菌对家禽licidin-1（鸡抗菌肽）耐药的基因位点。一种高效的转座子诱变方法（EZ::TN™<KAN-2>转座子）与微滴板筛选相结合，鉴定出三种对fowlicidin-1易感性显著增加的突变体。转座子突变回交到亲本菌株中证实，每个突变体的amp敏感表型与特定的转座子插入有关。直接测序显示，这些突变体在编码双组分调节因子CbrR、转运蛋白CjaB和推定的触发因子Tig的基因中插入转座子。基因组分析还发现了一个与sapABCDF同源的操纵子（Cj1580c-1584c）， sapABCDF在其他病原体中具有AMP抗性。Cj1583c （sapB）的插入失活显著增加弯曲杆菌对fowlicidin-1的敏感性。sapB以及tig和cjaB突变体与野生型菌株81-176竞争鸡盲肠定植的能力明显受损。总之，本研究确定了弯曲杆菌的四个遗传位点，这将有助于表征弯曲杆菌对AMPs耐药的分子基础，这是弯曲杆菌发病机制的重要知识空白。
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.
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