致腹泻奇异变形杆菌毒力因子研究及应用

Proteus mirabilis has been recognized as an opportunistic pathogen resulting in urinary tract infection, but its importance as a diarrhea pathogen is unclear. Proteus mirabilis produces several well characterized virulence determinants, encoded by the urease(ureC), metalloproteinase (zapA), metabolic regulation (rsmA) and hemolysin(hpmA) genes; however the association of the virulence factor to diarrhea has not been demonstrated. We have isolated P.mirabilis as the sole microorganism from the outbreak cases of diarrhea and remaining food products; these isolates had the same pulsed-field gel electrophoresis (PFGE) pattern and could cause mice diarrhea or death which suggesting that these outbreaks were caused by P.mirabilis. Furthermore, the PFGE patterns of isolates recovered from outbreak patients differed from those recovered from the stools of healthy individuals, supporting the causality of diarrhea and indicating the potential for genome differences between strains. This has led us to hypothesize that some strains of P.mirabilis may contain virulence factors increasing their pathogenic. According to our preliminary studies, representative strains isolated from outbreak patients, healthy individual stools and the environment were selected for whole genomic sequencing to locate new virulence genes that might function with diarrhea,some new virulence genes such as virB coding operon of type IV secretion system(T4SS) was found only in diarrhea strains.We propose to discover the mechanism that P.mirabilis strains use to cause diarrhea by creating knockout mutants in this new virulence gene studying their ability to cause diarrhea in cell and animal models.Real-time PCR based on those specific genes will be developed for the rapid detection of P.mirabilis to ensure food safety and human health.

奇异变形杆菌是一种能引起泌尿系统感染的常见致病菌，但该菌已知的毒力基因与腹泻关系尚不明确，其是否为腹泻致病菌尚无定论。我们在部分食物中毒事件中，从病人腹泻标本和剩余食品中都分离到奇异变形杆菌，未分离到其他致病菌。这些菌株皆具有相同的脉冲场凝胶电泳（PFGE）型别，感染小鼠后能造成腹泻或死亡。而从健康人群与外环境分离的菌株不引起小鼠出现以上症状，其PFGE图谱间差异较大。由此推论，引起腹泻的奇异变形杆菌可能存在其它特异的毒力因子。在前期研究中，我们通过基因组测序比较食物中毒株与对照株的差异，发现致腹泻菌株中存在特有的毒力基因virB，编码IV型分泌系统，可能与腹泻密切相关，国内外未见报道。本课题拟通过构建该基因的缺失株和回补株，采用分子生物学、细胞实验和动物实验等技术，阐明新发现的毒力基因的功能。并利用其为分子诊断靶标，建立荧光PCR诊断方法，用于食物中毒快速检测，保障食品安全和人体健康。

奇异变形杆菌是一种常见条件致病菌，该菌是否引起腹泻尚无定论。我们在部分食物中毒事件中，从病人腹泻标本和剩余食品中都分离到同一型别的奇异变形杆菌。提出假设，除了已知的毒力基因外，致腹泻的奇异变形杆菌的基因组中可能还存在着其它特异的毒力因子，是造成食物中毒的主要原因。.为了明确这些问题，我们开展了相关研究并获得以下结果：.（一）奇异变形杆菌毒力因子研究.1、分析比较了不同来源的奇异变形杆菌的病原学特征和分子特征，发现分别存在着1~3个不同大小的质粒。为奇异变形杆菌耐药及致病性研究提供了理论依据。.2、成功建立了小鼠腹泻动物模型，证实了自食物中毒病人分离的奇异变形杆菌对小鼠的毒力强于健康人群及外环境分离株。.3、成功建立了合适的细胞实验模型，证实了自食物中毒病人分离的奇异变形杆菌具有更强的侵袭性和黏附性。.4、首次完成2株奇异变形杆菌的腹泻病人分离株和2株健康人群(外环境)分离株的全基因组测序。通过全基因组比较发现，致腹泻菌株存在很多特异的基因。尤其是在食物中毒分离株中发现了编码IV 型分泌系统的特异基因（virB1~virB11基因），在调控不同大小和性质底物结合蛋白的分泌过程中发挥了极其重要的作用。.5、成功构建了奇异变形杆菌virB9以及virB1-11的敲除株和回补株。.6、分析比较了野生株、敲除株和回补株的毒力，发现缺失virB9及缺失virB1-11后的菌株对细胞的黏附力和侵袭力均下降，对动物的毒性作用也减弱。说明virB1-11基因是致腹泻奇异变形杆菌的重要的毒力基因。.（二）奇异变形杆菌快速检测方法建立与应用.在新发现的特异的毒力因子基础上，筛查选取了4个在食物中毒菌株中普遍存在的毒力基因作为靶基因，建立了1管同时检测奇异变形杆菌的多重实时荧光PCR方法。可广泛用于食物中毒或食品污染物的快速检测，区分正常人群携带株（外环境污染菌株）和致腹泻菌株，简化和完善现有的国标诊断方法，节省人力物力，满足食物中毒快速准确的诊断和传染来源追踪，减少误判和漏检，保障食品安全和人体健康。