前噬菌体ɸSa3XN通过Hlb调控金黄色葡萄球菌毒力的作用研究

Prophages that widely distributed in bacterial genomes have interactions with host bacteria through lysogenic conversion or active lysogeny, and play an important role in the regulation of bacterial virulence. However, the mechanisms of most prophages regulating bacterial virulence are poorly understood. Recently, a prophage named ɸSa3XN was identified in the genome of S. aureus isolate XN108, resulting in the truncation of the β-hemolysin encoding gene hlb through integration and virulence attenuation. ɸSa3XN could excise from XN108 genome and restore the Hlb expression of XN108, indicating the possibility that ɸSa3XN could regulate the virulence of S. aureus via active lysogeny. This project intends to further characterize prophage ɸSa3XN; determine its excision frequency in vivo and horizontal transfer ability between strains; express and purify recombinant Hlb protein in vitro, and construct knockout and complementary mutant of integrase encoding gene int; detect the effect of ɸSa3XN excision on the virulence of S. aureus using a murine lung infection model. Comparative genomic analysis revealed that 90% of S. aureus strains carry ɸSa3XN prophage, this study is expected to reveal a new virulence regulation mechanism that is universal in S. aureus, and may provide a new target and idea for the control of S. aureus infections.

前噬菌体在细菌基因组中广泛存在，可通过溶原性转换或活性溶原与宿主菌产生相互作用，在细菌毒力调控中扮演重要角色。然而，目前对多数前噬菌体调控细菌毒力的机制不清。课题组前期在金黄色葡萄球菌分离株XN108中鉴定出一前噬菌体ɸSa3XN，其整合能截断β-溶血素编码基因hlb，降低菌株毒力。实验证实ɸSa3XN可从XN108基因组中切离并恢复XN108的Hlb表达，提示ɸSa3XN可能通过活性溶原机制调控金黄色葡萄球菌的毒力。本项目拟进一步鉴定ɸSa3XN，测定其体内切离频率和菌株间水平转移能力；体外重组表达和纯化Hlb蛋白，构建整合酶基因int敲除株及其回补株，以小鼠肺部感染为模型，检测ɸSa3XN切离对XN108毒力的影响。比较基因组学发现90%的金黄色葡萄球菌携带有ɸSa3XN前噬菌体，本研究结果将揭示一种普遍存在的金葡菌毒力调控新机制，并为控制该菌感染提供新的靶点和思路。

前噬菌体在细菌基因组中广泛存在，可通过溶原性转换或活性溶原与宿主菌相互作用，在细菌毒力调控中发挥重要作用。金黄色葡萄球菌中溶原性转换已报道较多，而活性溶原研究匮乏。本项目在金葡菌临床分离株XN108中鉴定了一属于Sa3int家族的前噬菌体ΦSa3XN，其位点特异性整合导致β-溶血素编码基因hlb因截断而失活。ΦSa3XN能从XN108基因组中自发和诱导切离，但不产生子代噬菌体，表明其具有活性溶原特性。ΦSa3XN切离后菌株XN108恢复Hlb表达，溶血活性增强。葡萄糖可诱导ΦSa3XN切离，且高表达Hlb显著促进高血糖小鼠皮下脓肿形成。进一步研究发现，在葡萄糖或丝裂霉素C诱导条件下，ΦSa3XN编码的基因SAXN108_2102上调表达，且过表达SAXN108_2102显著增加ΦSa3XN的切离频率。分析金葡菌临床分离株，证实在糖尿病足感染条件下Sa3int噬菌体丢失增加。本研究表明ΦSa3XN通过活性溶原调控Hlb表达进而促进金葡菌糖尿病足感染，揭示了金葡菌毒力调控新机制。另，在项目资助下，我们还阐明了全局调节子CcpA通过负调控Sak表达进而控制金葡菌生物被膜形成的新通路，以及筛选并鉴定了金葡菌单感染和与鲍曼不动杆菌共感染条件下的必需基因，为控制金葡菌感染提供了新的认识和潜在靶点。