Genome-wide screen identifies novel factors for surface protein cross-wall trafficking and cell envelope homeostasis in Staphylococcus aureus

Cell wall anchored surface proteins are integral components of the Gram-positive bacterial cell envelope and are vital for bacterial survival in different environmental niches. The trafficking of many surface proteins carrying a YSIRK/G-S signal peptide is synchronized with cell envelope biogenesis during cell division, whereby YSIRK proteins traffic to the septal membrane and anchor to septal peptidoglycan (cross-wall). Previous work demonstrated that LtaS-mediated lipoteichoic acid (LTA) synthesis restricts YSIRK proteins septal trafficking. Here we did a comprehensive immunofluorescence microscopy screen of the entire S. aureus Nebraska Transposon Mutant Library (NTML) for additional factors regulating cross-wall trafficking of staphylococcal protein A (SpA), an archetype of YSIRK proteins. We characterized the top nine major hits that drastically diminished SpA cross-wall localization, including ypfP and ltaA (LTA glycolipid anchor synthesis genes), lcpB (LytR-CpsA-Psr family protein), mprF (lysyl-phosphatidylglycerol synthase), lytH (cell wall hydrolase), scdA (nitrite reductase), yjbH (protease adaptor protein ), cbiO (cobalt transporter) and SAUSA300_2311 (LytTR regulatory system) along with {Delta}tagO (wall teichoic acid synthesis). Interestingly, unlike the ltaS mutant that delocalizes SpA at both the septal membrane and peptidoglycan (PG) layer, all the hits only delocalized SpA at the PG layer, suggesting that these mutants affect the late-stage SpA trafficking. In addition, mutants of lcpB, yjbH, cbiO and 2311 exhibit both transcriptional and spatial regulation. All the hits showed defects in cell cycle, cell morphology and spatially dysregulated PG synthesis. The shared phenotypes among the mutants suggest that impaired PG homeostasis and cell cycle defects are the mechanisms underlying dysregulated SpA localization. Overall, this work not only expands our understanding of YSIRK protein cross-wall trafficking but also identifies new leads that have a broader impact on the dynamics of cell cycle and cell envelope homeostasis. ImportanceSurface proteins of gram-positive bacteria are key virulence factors in human pathogen S. aureus. Most surface proteins carry a YSIRK/G-S type signal peptide that promotes cross-wall trafficking and attachment to septal cell wall during cell division. This study identified several new factors regulating this process through a comprehensive screen. The mutants identified here display dysregulated cell wall synthesis along with cell cycle defects. The results provide new insight into virulence factor trafficking and cell envelope homeostasis, which lays the foundation for development of new antimicrobial therapeutics.