Biology subjects

Scaffidi, S. J.

Publications and source records attributed to Scaffidi, S. J..

3 recordsLinked to original sources

bioRxiv2025

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.

microbiology↗

bioRxiv2025

Mis-localization of PBPs in Staphylococcus aureus gdpP mutant contributes to β-lactam resistance and surface protein cross-wall trafficking

Mutations in gdpP are frequently associated with {beta}-lactam resistance in Staphylococcus aureus. GdpP is a phosphodiesterase that degrades the second messenger c-di-AMP, which plays a key role in osmoregulation and antibiotic resistance in many Gram-positive bacteria. However, the mechanisms of {beta}-lactam resistance remain unknown. We show here that deletion of gdpP disrupted penicillin-binding proteins (PBPs)-mediated deposition of YSIRK+ surface protein SpA to septal peptidoglycan (cross-wall). In contrast to their septal localization in WT staphylococcal cells, all four PBPs (PBP1-4) were drastically mis-localized as distinct single foci in {Delta}gdpP. The mis-localization of PBPs is attributed to c-di-AMP accumulation as overexpression of c-di-AMP synthetase dacA phenocopied {Delta}gdpP. In addition, {Delta}gdpP exhibited severe cell cycle retardation deficient in cell division initiation. The aberrant foci formation of PBPs altered the spatial distribution of cell wall synthesis but did not affect the overall cell wall cross-linking. The foci formation of PBPs correlated with {beta}-lactam resistance: the foci gradually decreased with increased concentrations of penicillin. We concluded that the aberrant spatial distribution of PBPs and the cell division defects of {Delta}gdpP contribute to {beta}-lactam resistance and YSIRK+ protein cross-wall trafficking.

microbiology↗

bioRxiv2024

Signal peptidase SpsB coordinates staphylococcal cell cycle, surface protein septal trafficking and LTA synthesis

Many cell wall anchored surface proteins of Gram-positive bacteria harbor a highly conserved YSIRK/G-S signal peptide (SPYSIRK+), 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 SPYSIRK+ are cleaved by the signal peptidase SpsB, but the biological implications remain unclear. Here we show that SpsB is required for cleaving SPSpA(YSIRK+) of staphylococcal surface protein A (SpA). Depletion of spsB not only diminished SPSpA processing but also abolished SpA septal localization. The mis-localization is attributed to the cleavage activity of SpsB, as an A37P mutation of SPSpA 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. ImportanceSurface proteins containing a YSIRK/G-S positive signal peptide are widely distributed in Gram-positive bacteria and play essential roles in bacterial pathogenesis. They are highly expressed proteins that are enriched at the septum during cell division. The biogenesis of these proteins is coordinated with cell cycle and LTA synthesis. The current study identified the staphylococcal signal peptidase SpsB as a key determinant in regulating surface protein septal trafficking. Furthermore, this study highlights the novel functions of SpsB in coordinating LtaS-mediated LTA production and regulating staphylococcal cell cycle. As SpsB, YSIRK+ proteins and LTA synthesis are widely distributed and conserved, the mechanisms identified here may be shared across Gram-positive bacteria.

microbiology↗