PhoU同源物调控金黄色葡萄球菌代谢通路与持留菌形成机制的研究

Antibiotics resistant of Staphylococcus aureus is not only related to expression of drug resistant genes, but also related to persister formation, invading and surviving in the host cells. Persister formation is one of factors resulting in the failure of treatment of infections. Multiple factors involve in persister formation, and PhoU homologue is an important regulator. The bacteria shared distinct mechanisms of PhoU, but its function is currently unknown. With bioinformatics analysis, we have identified two phoU homologues（phoU-1 and phoU-2）in the genomes of S. aureus and S. epidermidis, which is different from that of E. coli (only one phoU exits). In our preliminary study, phoU1 knockout reduced persister formation of S. aureus, but no effect on the Staphylococcus epidermidis. For investigating S. aureus’s phoU homologues functions and mechanisms in the persister formation, virulence, invasiveness and intracellular survival, we shall use a S. aureus virulent strain (USA500) to construct phoU homologous genes knock-out strains, as ∆phoU1, ∆phoU2 or ∆phoU1-phoU2. By comparison the transcriptomes of the gene knock-out strains with that of the parent strains, we shall identified the metabolism pathways which may regulated by PhoU homologues, and further validate by detection of metabolic products or subtracts in the pathway. Then the interaction of PhoU homologue with two component system, PhoRP /or YycG will be investigated. This study shall elucidate mechanism of PhoU homologue in regulation of S. aureus metabolism pathways involving persister formation and bacterial virulence.

金黄色葡萄球菌的耐药性不仅与耐药基因表达有关，还与持留菌形成、入侵及细胞内存活有关，是治疗失败的重要因素之一。多因素参与细菌持留菌的形成，其中PhoU是重要的调控因子。经生物信息学分析，我们发现金葡菌和表葡菌基因组中存在2个phoU同源基因（phoU1和phoU2），而大肠埃希菌含1个phoU。初步研究显示phoU1敲除可减少金葡菌持留菌的形成，而对表葡菌无影响，提示PhoU同源物在不同细菌中作用机制不同。因此，本课题将构建金葡菌USA500 phoU1或phoU2单基因敲除株和phoU1-phoU2双基因敲株，研究其对金葡菌持留菌形成、毒力、侵袭力及胞内存活的影响；比较表达谱，分析金葡菌PhoU同源物可能调控的代谢通路，并通过其代谢产物或底物进行验证；研究PhoRP /YycGF对PhoU同源物表达的调控，阐明PhoU同源物调控金黄色葡萄球菌相关代谢途径与持留菌形成及细菌毒力的机制。

课题前期研究发现表皮葡萄球菌基因组含2个phoU（phoU-1 和 phoU-2），其中phoU-2参与持留菌的形成，但金黄色葡萄球菌PhoU同源物的调控功能尚不清楚。本研究分别构建了金黄色葡萄球菌USA500 2395单基因敲除株（ΔphoU1和ΔphoU2）及双基因敲除株（ΔphoU1ΔphoU2），检测了其生长能力、抗生素敏感性、持留菌形成能力及环境压力的耐受力、细胞入侵和胞内存活能力。通过构建USA300 FPR3757菌株（生物膜阴性）和SA113菌株（生物膜阳性）phoU1或phoU2沉默株进行验证。phoU1和phoU2敲除对USA500 2395的生长无明显影响，但均可导致持留菌形成能力下降，互补株的持留菌数量恢复。沉默phoU2可减少细菌生物膜的形成。突变株在7 mM H2O2压力下持留菌形成能力下降，而在7 mM H2O2下细菌生长无差异。提示金黄色葡萄球菌PhoU同源物调控的生物学功能与表皮葡萄球菌有所不同。.进而，基于phoU1和phoU2敲除株与野生株的转录谱和差异表达基因分析，对PhoU同源基因突变株（ΔphoU1、ΔphoU2和ΔphoU1ΔphoU2）/野生株的磷酸盐代谢（polyP、Pi含量、ATP合成）以及葡萄糖代谢、乳酸代谢、磷酸戊糖等通路进行生物学验证。敲除株胞内Pi含量相似，phoU2敲除可增强胞内PolyP水平。phoU突变株胞外葡萄糖含量降低；胞内丙酮酸和ATP含量升高。phoU1或phoU2敲除可消耗更多葡萄糖并生成ATP，与持留菌形成有关。然而，突变株的乳酸代谢以及磷酸戊糖通路与野生株无明显差异。提示金黄色葡萄球菌phoU1和/或phoU2产物调控的代谢通路与表皮葡萄球菌有所不同。.基于突变株的毒力因子表达改变，我们研究了phoU敲除株（ΔphoU1、ΔphoU2和ΔphoU1ΔphoU2）的毒力。突变株在细胞中存活能力下降，但入侵A549细胞的菌量无差异。ΔphoU2（94.03%）和ΔphoU1ΔphoU2(84.62%)的α-溶血能力与比USA5002395（70.96%）增强且α-溶血素蛋白表达量升高，回复突变可使溶血表型恢复，证明phoU2产物可调控金黄色葡萄球菌毒力。EMAS显示PhoP/PhoR可结合与到phoU1、phoU2及phoP/phoR的启动子。