宿主免疫抑制状态对MRSA毒力因子表达的影响及其调控机制研究

Staphylococcus aureus is one of the key reasons leading to hospital acquired infection and higher mortality of inpatients.The emergence of heterogeneous vancomycin intermediate staphylococcus aureus(hVISA) and vancomycin resistant staphylococcus aureus(VRSA) brought a greater obstacle for the prevention,controlling and treatment of staphylococcus aureus.Besides the targeted antibiotics,any other methods or agents which can decrease or weaken the staphylococcus aureus virulence may be of great interests to clinicians and patients..The virulence of staphylococcus aureus was significantly influenced by the iron content in the host environment,whose influential effects was achieved through ferric uptake regulator(fur),which can induce the changing signals of iron content in host environment and initiate modulation mechanism immediately.It was revealed that the immunosuppressive state was closely correlated with the evolution of MRSA into hVISA,and our previous studies revealed that the iron content in peripheral blood of the patients with malignant tumors decreased significantly after administration of chemotherapy drugs,and MRSA colonization rate of these patients was up to 7.55%.However,whether the staphylococcus aureus virulence can be influenced by the immunosuppressive state or not is still uncertain..In this project,we aim to construct rat immunosuppression model and MRSA colonization model using the nosocomial prevalent strain ST-239-III type of MRSA isolated in the region of Inner Mongolia as the objective MRSA strain,and further to analyze the influential effects of immunosuppressive state on the expression of virulence factors and fur regulator in extracellular and intracellular MRSA using multiplex fluorescent quantitative PCR method and western blotting technique ,respectively.Furthermore,gene homologous recombination technique will be used to knock-out MRSA fur encoding gene to further explore the role of fur in the process of immunosuppressive state modulating virulence of MRSA. .The point of innovation in this project lies in combing host and pathogen together to explore the effects of environmental changes of host on MRSA virulence and their interaction mechanism,and to further provide new ideas and experimental data to elucidate the mechanism of variation of staphylococcus aureus virulence and develop of new types of anti-MRSA drugs.

金黄色葡萄球菌是造成住院病人医院感染及高病死率的主要病原之一，其毒力因子表达受宿主环境铁含量变化的显著影响，影响机制主要由铁摄取调节蛋白fur感应铁含量的变化信号而完成。前期研究发现，免疫抑制病人外周血结合铁含量显著降低，且该病人群体耐甲氧西林金黄色葡萄球菌（MRSA）定植率高达7.55%。fur调控子是否可感应其宿主环境铁含量变化来影响MRSA毒力因子表达尚不明确。本课题拟以我国医院内流行株ST-239-III型MRSA构建免疫抑制大鼠MRSA定植模型，分析免疫抑制状态对宿主细胞内、外MRSA毒力因子和fur调控子表达的影响；进一步采用基因敲除技术研究fur调控子在此调控过程中的作用。本课题创新之处在于结合宿主和病原两个方面研究宿主环境变化对MRSA毒力的影响及其机制，为进一步阐明金黄色葡萄球菌毒力变异机制及研发新型抗MRSA感染药物提供新的思路和实验数据。

本项目通过PFGE及MLST分子分型方法筛选内蒙古地区ST239型MRSA临床分离株，在基因水平和蛋白水平对该型菌株多种毒力因子表达情况进行了研究，发现该型菌株高表达sea等毒力基因并表现出较高的体外侵袭能力。我们进一步对1株ST239型MRSA临床分离株进行了全基因组测序，并对其毒力和耐药基因分布特征进行了比对和分析，为进一步探究内蒙古地区ST239型MRSA流行及致病机制提供了基础。我们进一步构建了金黄色葡萄球菌fur基因缺失株及免疫抑制动物模型,并就宿主不同免疫状态对金黄色葡萄球菌毒力基因表达水平的影响进行了研究。结果发现金黄色葡萄球菌Newman株在免疫抑制血清中基因表达密度最高。我们对表达水平存在显著差异基因进行检索和分析后发现，表达显著下调的包括多种外毒素编码基因，但与细菌增殖、代谢和免疫保护相关基因的表达显著增强，而fur缺失株在正常血清及免疫抑制血清中外毒素基因mRNA表达水平无明显差异，提示fur可能是免疫抑制状态调控金黄色葡萄球菌毒力过程的一个关键因素，其具体调控机制和信号通路有待进一步探究。