Table_1_The Impacts of Sortase A and the 4′-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis.xlsx

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4′-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

细胞外膜泡（extracellular membrane vesicles，EMVs）可由多种革兰氏阳性菌产生，但目前关于其泡囊发生机制的相关信息仍不完善。本研究通过单基因缺失突变技术，评估了分选酶A（Sortase A，SrtA）与Sfp蛋白对变形链球菌（Streptococcus mutans）EMV生物发生的影响：其中SrtA可影响变形链球菌EMV的组成，而Sfp作为一种4'-磷酸泛酰巯基乙胺转移酶（4′-phosphopantetheinyl transferase），可影响枯草芽孢杆菌（Bacillus subtilis）EMV的稳定性。与Δsfp突变株和野生型（wild-type，WT）EMV相比，ΔsrtA突变株的EMV体积显著更大。从三株菌株中鉴定得到的EMV蛋白，均已知参与细胞壁生物发生与细胞结构构建、细菌黏附、生物膜细胞密度及基质形成，以及微生物竞争等生理过程。值得注意的是，ΔsrtA突变株中，AtlA自溶素（AtlA autolysin）无法被加工为成熟的活性形式。对三株菌株的蛋白质组学与脂质组学分析显示，EMV与其对应的细胞质膜（cytoplasmic membranes，CMs）之间存在多处显著差异。EMV蛋白中，被预测为通用分泌途径（general secretion pathway，GSP）底物的比例更高；相应地，GSP组分SecA蛋白被鉴定为丰度较高的EMV相关蛋白。与之相反，细胞质膜相较于EMV，含有更多共翻译转运机器作用的多次跨膜（transmembrane，TM）蛋白底物。与细胞质膜相比，野生型菌株（而非突变株）的EMV显著富集心磷脂；且所有EMV均富集聚酮类黄酮类化合物。EMV与细胞质膜均富含长链饱和、单不饱和及多不饱和脂肪酸，但Δsfp突变株的EMV仅含有多不饱和脂肪酸。相较于各自对应的细胞质膜，三株菌株的EMV中脂蛋白的占比均更低。本研究揭示了变形链球菌EMV的生物物理特征，并阐明了野生型、ΔsrtA及Δsfp菌株的EMV与对应细胞质膜之间蛋白质和脂质组分的差异化分配模式。