The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles

Abstract

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.

Fig. 1.

Lpps are released into the growth medium as high molecular weight structures. S. pyogenes strain M1–3348 was grown until midexponential phase in THB medium. Growth medium (S1) was high-speed centrifuged producing supernatant (S2) and pellet (P). Aliquots from S1, S2, and P were normalized to the same volume and separated by SDS-PAGE. Proteins were then transferred onto a nitrocellulose membrane and analyzed by Western blot using mouse polyclonal antibodies antiSPy1882, SPy2000, and SPy1390.

Fig. 2.

Lpps are released as LMVs. A, S. pyogenes strain M1–3348 was grown until midexponential phase in THB medium. The culture volume was twofold diluted with prewarmed THB medium without or with penicillin at 0.7 μg/ml final concentration. After 80 min, culture medium was high-speed centrifuged, and pellets was solubilized in PBS and analyzed by SDS-PAGE. Proteins from the major Blue Coomassie stained bands were identified by Peptide Mass Fingerprint. Lpps are reported in bold. B, Pellet derived from penicillin treatment (LMVs) was also prepared for negative staining and viewed by electron microscopy. C, LMVs were stained with the 10-n-nonyl-acridine orange to evidence anionic lipids (central panel) and with antiLpp peptidylprolyl isomerase (SPy1390) polyclonal antibody (left panel); the merge of the two staining is reported in the right panel, corroborating the association between Lpps and LMVs. D, Lpp SPy1390 accessibility on the LMV surface was confirmed by FACS analysis. * Protein identified from the MGAS5005 genome.

Fig. 3.

S. pyogenes membrane and vesicle lipid composition is different. Lipids were extracted by chloroform/methanol from total extract of bacteria grown in presence (lane 2) or in absence (lane 3) of penicillin or from the purified vesicles (lane 4) and separated by thin layer chromatography. Lipids were stained with primulin and visualized by UV lamp. Circles highlight difference in lipid proportion. Lipid standard mixture (5 μg each) was loaded in lane 1: l-α-phosphatidyl-dl-glycerol (PG); 3-sn-phosphatidylethanolamine (PE); l-α-phosphatidic acid (PA); l-α-phosphatidylcholine (PC); 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS); sphingomyelin (S); l-α-lysophosphatidylcholine (LC).

Fig. 4.

The Δlgt mutant strain releases vesicles in the growth medium. A, Vesicles purified from the same culture volume of M1–3348 wild type and Δlgt mutant strains grown in presence of penicillin were analyzed by SDS-PAGE. B, The same volumes of vesicles were analyzed by FACS. C, Δlgt mutant strain was grown in presence of penicillin and culture medium (S1) was then high speed-centrifuged producing supernatant (S2) and vesicle containing pellet (P). Aliquots from S1, S2, and P were normalized to the same volume and separated by SDS-PAGE. Proteins were then transferred onto a nitrocellulose membrane and analyzed by Western blot using mouse polyclonal antibodies antiSPy1892, SPy2000, and SPy1390.

Fig. 5.

S. pyogenes LMVs do not activate TLR2. HEK293-cells stable transfected with FLAG-human TLR2 were stimulated with different dilutions of cell-free culture medium and LMVs purified from M1–3348 wild type and Δlgt mutant strains grown in presence or absence of penicillin. Luciferase expression was measured after 6 h of stimulation. All samples were normalized to the equivalent volume of starting material. TLR2 activation is expressed as percentage of the luciferase expression measured for the LMVs purified from M1–3348 wild type grown in presence of penicillin. S1, S2, and P refer to the starting material and ultracentrifuge fractions as reported in Figs. 1 and 4.