Identification of Salmonella enterica serovar typhimurium genes important for survival in the swine gastric environment
Identification of Salmonella enterica serovar typhimurium genes important for survival in the swine gastric environment
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DOI：
10.1128/aem.72.4.2829-2836.2006
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发表时间：
2006-04-01
影响因子：
4.4
通讯作者：
Rasmussen, MA
中科院分区：
文献类型：
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作者：
Bearson, SMD;Bearson, BL;Rasmussen, MA
由于胃是宿主抵御摄入微生物的第一道防线，因此建立了一种离体猪胃内容物（SSC）测定方法，以寻找对肠道沙门氏菌血清型鼠伤寒沙门氏菌在恶劣的胃环境中存活重要的基因。使用先前鉴定的酸敏感型鼠伤寒菌突变体进行SSC试验（pH 3.87）的初步表征显示，phoP突变体在攻击20分钟后存活率降低了10倍，rpoS或fur突变体没有存活。为了鉴定其他基因，构建了一个标记诱变库，并在SSC试验中进行了筛选。鉴定了19个突变体，并分别在SSC和耐酸反应试验中进行了分析；与野生型菌株相比，13个突变体在SSC检测中表现出10倍或更高的敏感性，但只有3个突变体在pH 3.0的酸性刺激下表现出10倍或更高的存活率下降。进一步的研究确定，SSC的致死作用与pH值有关，但低pH值并不是唯一的致死机制。SSC的气相色谱分析显示乳酸水平为126 mM。在研究乳酸对合成胃液中血清型鼠伤寒菌存活的影响时，不仅观察到浓度和时间依赖性的致死效应，而且还发现phoP、rpoS、fur和pnp基因参与了对乳酸暴露的保护。这些研究表明了几种血清型鼠伤寒杆菌基因在胃存活中的作用，并暗示胃环境不仅仅是由低pH值定义的。
Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.