表皮葡萄球菌生物膜中Persister形成分子机制及调控

The persisters with phenotype of anti-microbicidal antibiotics exist in bacterial populations. Persister cell formation and presence are associated with tolerant antibiotic treatment of bacterial biofilm-associated diseases as well as chronic and recurrent infections. S. epidermidis, an opportunistic pathogen, has become one of the most prevalent causes of nosocomial infections, especially in patients with prosthetic medical devices. Pathogenesis of S. epidermidis is closely associated with its ability of biofilm formation. Bacterial cells in biofilms are resistant to multiple antibiotics and host defenses. So far there is no cure antibiotics for bacterial biofilm associated diseases. It has been found that in E.coli, multiple genes are related to persister cell formation, while phoU plays an important role which is also found in M. tuberculosis (PhoU homologue as PhoY2). However there is no much information about the mechanisms and regulations of persister cell formation in Staphylococcus, though persister was first found in Staphylococcus and presents in biofilms. Our group has investigation of roles of genes or two component signal transduction in S. epidermidis biofilms formation. In the study of role of VraSR in S. epidermidis, preliminary results showed that VraSR knock out affects S. epidermidis biofilm formation and cell survival in the biofilms (not published data). Therefore we intend to investigate roles of PhoU and VraSR played in regulation of persister cell formation and survival in the biofilm of S. epidermidis, with phoU knock out and VraSR knock out through homologous recombination, and confirm the phenomena with related complementary strains and overexpressed strains. With custom-made S. epidermidis GeneChips, the genes regulated by PhoU would be analyzed through comparing the transcriptional profile of knock out strain with wild type strain. We will futher investigate the regulation factors including for PhoU, and downstream genes regulated by VraSR, which could be related with persister cell formation or survive. The study will elucidate molecular mechanism and regulation of phoU and VraSR in the persister formation and suvival in the S. epidermidis biofilm, for understanding the intrinsic resistant antibiotics of bacterial biofilms and discovering potential anti-persister or anti-biofilm targets and contribute to the discovery of new antibiotics against Staphylococcal biofilm infections.

Persister是细菌生长过程种形成的具有耐药表型的细胞，其形成和存在与细菌生物膜及细菌慢性复发性感染的耐药性密切相关。目前医院感染日趋严重，表皮葡萄球菌感染位于前列，其致病性与生物膜形成能力密切相关，目前尚无有效控制细菌生物膜疾病药物。在E.coli中发现多基因参与Persister的形成，其中phoU发挥重要作用。葡萄球菌的Persister形成分子机制和调控机制尚不清楚。本课题拟在前期研究的基础上，以表皮葡萄球菌生物膜中Persister形成为切入点，利用同源重组方法构建phoU及VraSR敲除株、回复突变株、过量表达株，研究PhoU和VraSR对生物膜中Persister形成的影响；研究调控PhoU表达的相关因素以及VraSR调控的下游基因，从而阐明表皮葡萄球菌生物膜Persiser形成的分子机制及相关调控机制，为探索生物膜内细菌的耐药机制及发现新型抗菌/抗生物膜药靶奠定基础。

phoU是负调节蛋白，调节参与磷酸盐代谢基因的表达。在革兰阴性埃希大肠杆菌中发现PhoU位于与磷酸盐代谢相关的操纵子中，称为pst-phoU操纵子。2007年Yin Zhang等发现phoU参与药物耐受菌persister（原称为持留菌）的形成，但phoU在细菌生物膜形成的作用尚不清楚，虽然对铜绿假单胞菌研究结果提示phoU对生物膜的形成无调控作用。我们在表皮葡萄球菌ATCC12228和ATCC35984菌株基因组中发现pst-phoU操纵子中含有phoU同源基因，进行phoU的功能区同源性分析，结果显示相似2个与E.coli Phou同源的蛋白。因此，进而对表皮葡萄球菌基因组中的phoU同源物调控药物耐受菌persister和细菌生物膜形成机制进行了研究。通过基因敲除技术构建了∆phoU1和∆phoU2 以及phoU1和phoU2回复互补突变株，生物学试验结果显示：与野生株相比∆phoU2株生长减缓、生物膜形成能力下降、氧化应激能力下降、药物耐受性等，而回复互补突变株的表型则与野生株相似。进而分析和比较了∆phoU2株和野生株的转录谱，并进一步通过生物学验证结果提示：在表皮葡萄球菌中phoU蛋白调控多个代谢通路，对药物耐受性(persister)、细菌生长、生物膜形成、呈现正向调控，对磷酸盐代谢为负调控，如phoU2敲除则导致相应的表型改变。除了已有报道的磷酸盐代谢通路外，本研究主要发现了磷酸戊糖代谢通路也受PhoU的调控，进而影响药物耐受性(persister)、细菌生长和生物膜形成，以及细菌对应激应答等。此外，还发现了PhoU与表皮葡萄球菌双组分信号系统中生长和生物膜形成相关的YycFG呈互相调控。然而，PhoU的调控机制及其功能还有待深入研究。