SppI Forms a Membrane Protein Complex with SppA and Inhibits Its Protease Activity in Bacillus subtilis.

The membrane protease SppA of Bacillus subtilis was first described as a signal peptide peptidase and later shown to confer resistance to lantibiotics. Here, we report that SppA forms octameric complexes with YteJ, a membrane protein of thus-far-unknown function. Interestingly, sppA and yteJ deletion mutants exhibited no protein secretion defects. However, these mutant strains differed significantly in their resistance to antimicrobial peptides. In particular, sppA mutant cells displayed increased sensitivity to the lantibiotics nisin and subtilin and the human lysozyme-derived cationic antimicrobial peptide LP9. Importantly, YteJ was shown to antagonize SppA activity both in vivo and in vitro, and this SppA-inhibitory activity involved the C-terminal domain of YteJ, which was therefore renamed SppI. Most likely, SppI-mediated control is needed to protect B. subtilis against the potentially detrimental protease activity of SppA since a mutant overexpressing sppA by itself displayed defects in cell division. Altogether, we conclude that the SppA-SppI complex of B. subtilis has a major role in protection against antimicrobial peptides.IMPORTANCE Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and SppI form a complex in the Gram-positive model bacterium B. subtilis and that SppI inhibits SppA protease activity in vitro and in vivo Furthermore, we demonstrate that the C-terminal domain of SppI is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-SppI complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses.

Analysis of Cell Wall Teichoic Acids in Staphylococcus aureus.

Most bacterial cells are surrounded by a surface composed mainly of peptidoglycan (PGN), a glycopolymer responsible for ensuring the bacterial shape and a telltale molecule that betrays the presence of bacteria to the host immune system. In Staphylococcus aureus, as in most gram-positive bacteria, peptidoglycan is concealed by covalently linked molecules of wall teichoic acids (WTA)-phosphate rich molecules made of glycerol and ribitol phosphates which may be tailored by different amino acids and sugars.In order to analyze and compare the composition of WTA produced by different S. aureus strains, we describe methods to: (1) quantify the total amount of WTA present at the bacterial cell surface, through the determination of the inorganic phosphate present in phosphodiester linkages of WTA; (2) identify which sugar constituents are present in the assembled WTA molecules, by detecting the monosaccharides, released by acid hydrolysis, through an high-performance anion exchange chromatography analysis coupled with pulsed amperometric detection (HPAEC-PAD) and (3) compare the polymerization degree of WTA found at the cell surface of different S. aureus strains, through their different migration in a polyacrylamide gel electrophoresis (PAGE).