Systematic Identification of Genetic Loci Required for Polymyxin Resistance in Campylobacter jejuni Using an Efficient In Vivo Transposon Mutagenesis System
Systematic Identification of Genetic Loci Required for Polymyxin Resistance in Campylobacter jejuni Using an Efficient In Vivo Transposon Mutagenesis System
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DOI：
10.1089/fpd.2008.0177
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发表时间：
2009-03-01
影响因子：
2.8
通讯作者：
Van Hoang, Ky
中科院分区：
文献类型：
--
作者：
Lin, Jun;Wang, Ying;Van Hoang, Ky
本研究的目的是确定所需的遗传位点的空肠弯曲菌多粘菌素（PM）的耐药，使用一个有效的体内随机诱变系统。抗菌肽是宿主先天免疫的主要效应物，也是新一代抗生素的候选者，已被广泛用作研究细菌对抗菌肽耐药性机制的模型肽。在这项研究中，商业上可获得的转座子诱变方法（EZ-Tn 5（TM）< KAN-2 >Transposome（TM）; Epicentre，麦迪逊，WI）进行评价，并用于系统地鉴定对PM具有增加的易感性的弯曲杆菌突变体。这种简单而有效的转座子诱变方法鉴定了12个突变体，代表了C.空肠81-176参与获得性PM抗性。将转座子突变回交到亲本菌株中证实了每个突变体中的PM敏感表型与具有特定转座子插入的基因相关联。这些基因被鉴定为参与细胞表面碳水化合物的合成、细胞内靶标的修饰、信号转导和跨膜电位的调节。对PM具有最高易感性的突变体在推定的galU基因中包含转座子插入，该基因对于产生尿苷二磷酸葡萄糖（UDP）-葡萄糖（脂寡糖（LOS）合成所需的前体）是必需的。通过tricine SDS-PAGE的LOS分析显示在galU突变体中LOS核心结构的显著截短。药敏试验也表明GalU对C.空肠对一些天然AMP的抗性。互补的galU突变体的反式完全恢复LOS合成和抗性的亲本菌株的水平。这些结果共同定义了7个C。空肠的遗传位点，这将是有用的表征弯曲杆菌耐PM和天然AMP的分子基础，也突出了有用的体内诱变方法的功能重要的弯曲杆菌基因的系统表征。
The aim of this study was to identify genetic loci required for polymyxin (PM) resistance in Campylobacter jejuni using an efficient in vivo random mutagenesis system. PM has been widely used as a model peptide to examine mechanisms of bacteria] resistance to antimicrobial peptides (AMPs), the major effectors of host innate immunity and also candidates for a new generation of antibiotics. In this study, a commercially available transposon mutagenesis approach (EZ-Tn5 (TM) < KAN-2 > Transposome (TM); Epicentre, Madison, WI) was evaluated and used to systematically identify Campylobacter mutants with increased susceptibility to PM. This simple, yet efficient, transposon mutagenesis approach identified 12 mutants representing seven different genes of C. jejuni 81-176 involved in acquired PM resistance. Backcrossing of the transposon mutations into the parent strain confirmed that the PM-sensitive phenotype in each mutant was linked to the gene with a specific transposon insertion. The genes are identified as being involved in the synthesis of cell-surface carbohydrates, modification of intracellular targets, signal transduction, and modulation of transmembrane potential. The mutant with the highest susceptibility to PM contains a transposon insertion in a putative galU gene that is essential for production of uridine diphosphate glucose (UDP)-glucose, a precursor required for lipooligosaccharide (LOS) synthesis. LOS analysis by tricine SDS-PAGE showed significant truncation of the LOS core structure in the galU mutant. Susceptibility assays also indicated that GalU contributed C. jejuni resistance to some natural AMPs. Complementation of the galU mutant in trans fully restored LOS synthesis and resistance to the levels of the parent strain. Together, these results define seven C. jejuni genetic loci that will be useful for characterizing the molecular basis of Campylobacter resistance to PM and natural AMPs, and also highlight the usefulness of the in vivo mutagenesis approach for systematic characterization of functionally important Campylobacter genes.