Myrbraten, I. S. | Search bioRxiv

bioRxiv2023

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus

To maintain cell integrity and facilitate cell division in Staphylococcus aureus, a well-coordinated interplay between membrane biogenesis, peptidoglycan formation, and teichoic acid synthesis is crucial. However, the molecular mechanisms and regulatory pathways that underpin their coordination are still poorly understood. CozE constitute a conserved family of membrane proteins implicated in cell division via regulation of penicillin binding proteins. It has been shown that the two staphylococcal cozE genes (cozEa and cozEb) constitute a synthetic lethal gene pair. Depletion of CozEa and CozEb simultaneously in S. aureus resulted in severely defective cell division phenotypes, reminiscent of cell lacking lipoteichoic acid (LTA). Indeed, we demonstrate that there is an intricate interplay between CozE, biosynthesis of LTA, and membrane homeostasis in S. aureus. By screening for potential genetic links, we establish that there is synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. On the contrary, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozEa and CozEb was alleviated. Furthermore, by immunoblotting, we found that CozEb plays a unique role in controlling LTA polymer length and stability. Using reconstituted proteoliposomes, we also demonstrated that CozE proteins modulate the glycolipid flipping activity of LtaA in vitro. Together, the results demonstrate a new function of CozE proteins, facilitating proper membrane homeostasis and LTA biosynthesis in S. aureus.

microbiology↗

bioRxiv2021

SmdA is a novel cell morphology determinant in Staphylococcus aureus

Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining their nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively, however, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. We here identified and investigated a protein with major influence on cell morphology in Staphylococcus aureus. The protein, named SmdA (for staphylococcal morphology determinant A), is a membrane-protein with septum-enriched localization. By CRISPRi knockdown and overexpression combined with different microscopy techniques, we demonstrate that proper levels of SmdA is necessary for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that are critical for its functionality. Pulldown- and bacterial two-hybrid interaction experiments showed that SmdA interacts with several known cell division- and cell wall synthesis proteins, including penicillin binding proteins (PBPs) and EzrA. Notably, SmdA also affects susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results show that S. aureus is dependent on balanced amounts of membrane-attached SmdA in order to carry out proper cell division. ImportanceStaphylococcus aureus is an important human and animal pathogen. Antibiotic resistance is a major problem in treatment of staphylococcal infections, and cell division and cell wall synthesis factors have previously been shown to modulate susceptibility to antibiotics in this species. In the current work we investigated the function of an essential protein named SmdA, which was identified based on its septal localization and knockdown phenotype resulting in defective cellular morphologies. We demonstrate that this protein is critical for normal cell division in S. aureus. Depletion of SmdA sensitize resistant staphylococci to {beta}-lactam antibiotics. This work thus reveals a new staphylococcal cell division factor and a potential future target for narrow spectrum antimicrobials or compounds to resensitize antibiotic resistant staphylococcal strains.

microbiology↗