The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis

Abstract

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.

Keywords: Streptococcus mutans; lipidome; membrane; proteome; vesicles.

FIGURE 1

Evaluation of size and particle yield of EMVs. (A) Nanoparticle tracking analysis demonstrates particle yields of 1.15 × 10¹¹ ± 1.09 × 10¹⁰, 1.46 × 10¹¹ ± 1.10 × 10⁸, and 5.94 × 10¹⁰ ± 5.98 × 10⁹ particles/mL with mean EMV diameters of 131.8 ± 5.8, 174.5 ± 3.85, 131.45 ± 6.3 nm for WT, ΔsrtA, and Δsfp strains, respectively. (B) Dynamic light scatter (DLS) analysis demonstrates a bimodal distribution of EMVs for all three strains. DLS signal intensity is reflective of particle size, not particle number.

FIGURE 2

ζ-Potential analysis of S. mutans EMVs. (A) EMVs demonstrate a slight negative charge approaching electroneutrality. (B) Protoplasts from all three strains demonstrate a negative electrical charge of approximately –30 to –35 mV.

FIGURE 3

Comparison of protein content of EMVs with corresponding cytoplasmic membranes. (A) Venn diagram comparing proteins detected in cytoplasmic membranes (CMs) and corresponding EMVs, and in all three strains’ EMVs and CMs. (B) Volcano plots of p-values vs. fold-change to compare protein profiles of CMs (left of Y-axis) with corresponding EMVs (right of Y-axis) from each strain.

FIGURE 4

Evaluation of S. mutans membrane vesicle by SDS-PAGE and Western blot. (A) SDS-PAGE of S. mutans bacterial cell pellet (CP), crude vesicle prep (CV) and purified EMVs (PV) of WT, ΔsrtA and Δsfp mutant strains by SDS-PAGE. (B) Corresponding Western blots of the samples shown in (A). Blots were reacted with polyclonal rabbit antibodies raised against the indicated proteins. Due to limited antibody availability, only crude vesicles and Optiprep^TM gradient fractions of the WT EMVs were evaluated for GbpB.

FIGURE 5

Functional annotation analysis of proteins detected in S. mutans EMVs compared to cytoplasmic membranes using Database for Annotation, Visualization, and Integrated Discovery (DAVID). Percentages of total S. mutans WT, ΔsrtA, and Δsfp EMV and total cytoplasmic membrane (CM) proteins in the top 35 functional categories are illustrated. (1) Metabolic pathways, (2) ATP binding, (3) transferase, (4) biosynthesis of secondary metabolites, (5) hydrolase, (6) metal-binding, (7) biosynthesis of amino acids, (8) RNA-binding, (9) ligase, (10) signal, (11) oxidoreductase, (12) kinase, (13) translation, (14) purine metabolism, (15) structural constituent of ribosome, (16) lyase, (17) pyrimidine metabolism, (18) isomerase, (19) glycolysis/gluconeogenesis, (20) protease, (21) GTP binding, (22) cell cycle/cell wall, (23) starch and sucrose metabolism, (24) NAD, (25) NADP, (26) flavoprotein, (27) helicase, (28) chaperone, (29) DNA replication, (30) glycerolipid metabolism, (31) stress response, (32) dental caries, (33) fatty acid metabolism, (34) lipid metabolism, and (35) pyruvate metabolism.

FIGURE 6

Comparison of EMV lipid content with corresponding cytoplasmic membranes from S. mutans wild-type and the mutant strains. (A) Venn diagram comparing lipids detected in cytoplasmic membranes (CM) and corresponding EMVs, and in all three strains’ EMVs and CMs (right). (B) Volcano plots of p-values vs. fold-change to compare lipid profiles of CMs (left of Y-axis) with corresponding EMVs (right of Y-axis) from each strain.

FIGURE 7

Distribution of lipids detected in cytoplasmic membranes as compared to EMVs of S. mutans wild-type and the mutant strains. FA, fatty acyls; GL, glyceroplipids; GP, glycerophospholipids; PK, polyketides; PR, prenol lipids; SL, saccharolipids; SP, sphingolipids; ST, sterol lipids. Percentages of lipids in each category are indicated in the pie charts.

FIGURE 8

Categories and main classes of lipids detected in cytoplasmic membranes (CMs) compared to EMVs of S. mutans wild-type, ΔsrtA, and Δsfp strains. (1) FA, fatty acids and conjugates; (2) FA, eicosanoids; (3) FA, fatty esters; (4) FA, fatty amides; (5) GL, DAG diacylglycerol; (6) GL, MAG monoacylglyceride; (7) GL, glycosyldiradylglycerols; (8)GL, TAG triacylglycerol; (9) GP, cardiolipin; (10) GP; PI phosphatidylinositol; (11) GP, PS phosphatidylserine; (12) GP, PG glycerophosphoglycerols; (13) GP, glycerophosphoinositolglycans; (14) GP, glycosylglycerophospholipids; (15) GP, PA phosphatidic acid; (16) GP, PC phosphatidylcholine; (17) GP, PE phosphatidylethanolamine; (18) PK, flavonoids; (19) PK, non-ribosomal peptide/polyketide hybrids; (20) PR, isoprenoids; (21) PR, hopanoids; (22) PR, polyprenols; (23) PR, quinones and hydroquinones; (24) SL, acyltrehaloses; (25) SP, ceramides; (26) SP, neutral glycosphingolipids; (27) SP, phosphosphingolipids; (28) SP, sphingoid bases; (29) ST, sterols; (30) ST; bile acids and derivatives; (31) ST, secosteroids. FA, fatty acyls; GL, glyceroplipids; GP, glycerophospholipids; PK, polyketides; PR, prenol lipids; SL, saccharolipids; SP, sphingolipids; ST, sterol lipids.