Microarray Analysis of Acid-Stress Induced Changes in EHEC O157:H7

While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections. Bacteria were grown in LB broth overnight, then subcultured into DMEM and grown at 37C, 5%Co2. Bacteria were then subjected to one of three acid stress protocols: 1) UA30: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 30 minutes; 2) AA30: growth in DMEM pH 5.0 (adaptation) followed by growth in DMEM pH 3.0; 3) UA15: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 15 minutes. DMEM was supplemented with 25 mM MES (pH 5.0) and in the case of the control (unadapted, unshocked) 25 mM MOPS (pH 7.4) and the adaptation step was again carried out at 37C and 5% CO2. Acid shocking was done at pH 3.0 (unbuffered) at room temperature for all treatments

尽管肠出血性大肠杆菌（Enterohemorrhagic Escherichia coli, EHEC）的致病机制研究已取得显著进展，但目前仍未完全明确环境应激对其毒力的影响。在感染进程中，EHEC必须规避或克服多重生物屏障，首当其冲的便是经胃时遭遇的胃酸环境。EHEC耐受胃酸的能力尤为突出，大肠杆菌O157:H7共拥有四种不同的酸抗性系统，可帮助其抵御pH 2~2.5的低酸环境。

有趣的是，EHEC在食品与牛肠道内的生存会差异化地使用这些酸抗性系统。据推测，谷氨酸依赖型酸抗性系统在pH低于3时可提供最佳保护。由于EHEC的感染剂量极低（仅50~100个菌体），酸抗性遂成为一项重要的毒力性状。现有针对EHEC酸应激响应的研究主要聚焦于酸耐受水平及其分子基础，而酸应激对EHEC毒力的影响则尚未得到充分阐释。

在相关病原体肠致病性大肠杆菌（Enteropathogenic E. coli, EPEC）中，质粒编码的调节因子Per可调控多种EPEC毒力因子的表达，其在pH 5.5时被负向调控，在pH 8.0时被正向调控，这表明毒力基因的表达在轻度酸应激下会被抑制，而在小肠典型的碱性pH环境中则会增强。已有研究显示，参与黏附消除（A/E）损伤形成的EPEC III型分泌因子的表达，会受培养基成分、铁与钙水平等因素影响，且在pH 6和pH 8环境下蛋白分泌会受到抑制。另有研究发现，gadE（编码酸抗性调节因子）的突变会增强大肠杆菌O157:H7对结肠上皮细胞的黏附能力，这提示gadE对一种或多种黏附素存在负调控作用。

其他研究亦表明，志贺毒素的产生会受包括pH在内的培养条件影响。然而，目前尚无针对EHEC在重度酸应激后毒力变化的研究，也未有将应激后EHEC的毒力表型与转录变化相关联的报道。本研究旨在明确酸应激如何影响EHEC的毒力特性，并通过基因芯片（microarray）分析阐明这些变化的遗传基础。解析酸应激如何调控该病原体的毒力潜力，对于阐明EHEC感染所致疾病的致病机制至关重要，同时也可为EHEC感染的防治提供全新思路。

实验中，细菌先于LB肉汤中过夜培养，随后转接至达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）中，于37℃、5%CO₂条件下培养。随后将细菌分为三组，分别施以三种酸应激处理方案：1）UA30：先在pH 7.4的DMEM中培养，再转接至pH 3.0的DMEM中处理30分钟；2）AA30：先在pH 5.0的DMEM中培养以适应环境，再转接至pH 3.0的DMEM中处理；3）UA15：先在pH 7.4的DMEM中培养，再转接至pH 3.0的DMEM中处理15分钟。

DMEM培养基中添加了25 mM吗啉乙磺酸（MES，用于pH 5.0体系），对照组（未适应、未酸击）则添加25 mM吗啉丙磺酸（MOPS，用于pH 7.4体系），适应步骤同样在37℃、5%CO₂条件下进行。所有处理组的酸击实验均在未缓冲的pH 3.0环境、室温条件下完成。