High-throughput transposon sequencing highlights cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors

Staphylococcus aureus is an opportunistic pathogen that causes a variety of diseases. It presents a problem in hospitals as well as communities partly due to the acquisition of multiple antibiotic resistances such as methicillin and oxacillin (MRSA strains), which makes infections difficult to treat. S. aureus is also a frequent cause of food borne infections and one contributing factor is its high salt tolerance, which allows this organism to survive commonly used methods of food preservation. How this resistance is mediated is poorly understood. In this study, we used TN-seq-based high throughput screens to find genes that are involved in the salt tolerance of S. aureus and identified the previously uncharacterized DUF2538 domain containing gene SAUSA300_0957 (gene 957) as highly essential under salt stress. Further experiments revealed that a 957 mutant strain is less susceptible to oxacillin and shows increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by point mutations in the transglycosylase domain of the penicillin binding protein gene pbp2, and these mutations also restored the peptidoglycan crosslinking to WT levels. These results indicate that too tight crosslinking of the peptidoglycan can be detrimental and highlights the role of the bacterial cell wall for osmotic stress resistance. To gain more information on the general osmotic stress response of S. aureus and how responses differ between different osmotic stressors, additional TN-seq studies were performed with KCl and sucrose. Although it is generally assumed that an initial generic osmotic stress response exists, our results revealed that the long-term response to NaCl, KCl and sucrose stress is very different and distinct from each other. Using a global and genome-wide TN-seq approach, we were able to link numerous previously unknown factors to the osmotic stress response in S. aureus. This study will also serve as a starting point for future research in osmotic stress and might help us develop strategies to tackle food borne staphylococcal infections.

金黄色葡萄球菌（Staphylococcus aureus）是一种可引发多种疾病的机会致病菌。该菌在医院与社区环境中均构成公共卫生难题，部分原因在于其获得了针对甲氧西林（methicillin）、苯唑西林（oxacillin）等多种抗生素的耐药性，即耐甲氧西林金黄色葡萄球菌（MRSA）菌株，这使得相关感染的治疗难度大幅提升。金黄色葡萄球菌亦是食源性感染的常见致病菌，其高盐耐受性是重要致病诱因之一——该特性可使该菌在食品常用的盐渍保存方式下存活。目前学界对该耐药性的介导机制尚不清楚。本研究采用基于转座子测序（Transposon sequencing, TN-seq）的高通量筛选技术，挖掘与金黄色葡萄球菌耐盐性相关的基因，并鉴定出此前未被表征的、含DUF2538结构域（DUF2538 domain）的基因SAUSA300_0957（简称基因957），该基因在盐胁迫条件下的生存必需性极高。进一步实验显示，该基因的缺失突变株对苯唑西林的敏感性降低，且肽聚糖交联程度升高。青霉素结合蛋白（penicillin binding protein, PBP）基因pbp2的转糖基酶结构域发生点突变可逆转该盐敏感表型，同时此类突变还可将肽聚糖交联水平恢复至野生型标准。上述结果表明，肽聚糖交联过强反而会对细菌产生不利影响，同时凸显了细菌细胞壁在渗透胁迫抗性中的作用。为进一步解析金黄色葡萄球菌的通用渗透胁迫应答机制，以及不同渗透胁迫源诱导的应答差异，本研究分别以氯化钾（KCl）和蔗糖作为胁迫源开展了额外的转座子测序研究。尽管学界普遍认为存在初始的通用渗透胁迫应答通路，但本研究结果显示，金黄色葡萄球菌对氯化钠（NaCl）、氯化钾（KCl）与蔗糖三种胁迫源的长期应答模式彼此差异显著、各具特征。本研究通过全基因组范围的转座子测序分析，将众多此前未被关联的因子与金黄色葡萄球菌的渗透胁迫应答联系起来。本研究可为后续渗透胁迫相关研究提供起点，亦有望为开发防控食源性葡萄球菌感染的策略提供参考。