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.