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Many cell wall anchored surface proteins of Gram-positive bacteria harbor a highly conserved YSIRK/G-S signal peptide (SP YSIRK+ ), which deposits surface protein precursors at the cell division septum where they are subsequently anchored to septal peptidoglycan. Previously we identified that LtaS-mediated lipoteichoic acid (LTA) synthesis regulates septal trafficking of YSIRK+ proteins in S. aureus . Interestingly, both LtaS and SP YSIRK+ are cleaved by the signal peptidase SpsB, but the biological implications remain unclear. Here we show that SpsB is required for cleaving SP SpA(YSIRK+) of staphylococcal surface protein A (SpA). Depletion of spsB not only diminished SP SpA processing but also abolished SpA septal localization. The mis-localization is attributed to the cleavage activity of SpsB, as an A37P mutation of SP SpA that disrupted SpsB cleavage also abrogated SpA septal localization. Strikingly, depletion of spsB led to aberrant cell morphology, cell cycle arrest and daughter cell separation defects. Localization studies showed that SpsB predominantly localized at the septum of dividing staphylococcal cells. Finally, we show that SpsB spatially regulates LtaS as spsB depletion enriched LtaS at the septum. Collectively, the data suggest a new dual-mechanism model mediated by SpsB: the abundant YSIRK+ proteins are efficiently processed by septal localized SpsB; SpsB cleaves LtaS at the septum, which spatially regulates LtaS activity contributing to YSIRK+ proteins septal trafficking. The study identifies SpsB as a novel and key regulator orchestrating protein secretion, cell cycle and cell envelope biogenesis.

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle., (© 2021 John Wiley & Sons Ltd.)

Surface proteins of Staphylococcus aureus and other Gram-positive bacteria play essential roles in bacterial colonization and host-microbe interactions. Surface protein precursors containing a YSIRK/GXXS signal peptide are translocated across the septal membrane at mid-cell, anchored to the cell wall peptidoglycan at the cross-wall compartment, and presented on the new hemispheres of the daughter cells following cell division. After several generations of cell division, these surface proteins will eventually cover the entire cell surface. To understand how these proteins travel from the bacterial cytoplasm to the cell surface, we describe a series of immunofluorescence microscopy protocols designed to detect the stepwise subcellular localization of the surface protein precursors: surface display (protocol A), cross-wall localization (protocol B), and cytoplasmic/septal membrane localization (protocol C). Staphylococcal protein A (SpA) is the model protein used in this work. The protocols described here are readily adapted to study the localization of other surface proteins as well as other cytoplasmic or membrane proteins in S. aureus in general. Furthermore, the protocols can be modified and adapted for use in other Gram-positive bacteria. Graphic abstract: Tracking the subcellular localization of surface proteins in S. aureus ., Competing Interests: Competing interestsThe authors declare that there are no conflicts of interest or competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)