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Abstract
BackgroundDue to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), cleaved off their signal peptides by lipoprotein signal peptidase (Lsp) and, in Gram-negative bacteria, further triacylated by lipoprotein N-acyl transferase (Lnt). The existence of an active apolipoprotein N-acyltransferase (Ms-Ppm2) involved in the N-acylation of LppX was recently reported in M. smegmatis. Ms-Ppm2 is part of the ppm operon in which Ppm1, a polyprenol-monophosphomannose synthase, has been shown to be essential in lipoglycans synthesis but whose function in lipoprotein biosynthesis is completely unknown.ResultsIn order to clarify the role of the ppm operon in lipoprotein biosynthesis, we investigated the post-translational modifications of two model lipoproteins (AmyE and LppX) in C. glutamicum Δppm1 and Δppm2 mutants. Our results show that both proteins are anchored into the membrane and that their N-termini are N-acylated by Cg-Ppm2. The acylated N-terminal peptide of LppX was also found to be modified by hexose moieties. This O-glycosylation is localized in the N-terminal peptide of LppX and disappeared in the Δppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX O-glycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed.ConclusionTogether, these results show for the first time that Cg-Ppm1 (Ppm synthase) and Cg-Ppm2 (Lnt) operate in a common biosynthetic pathway in which lipoprotein N-acylation and glycosylation are tightly coupled.
Highlights
Bacterial lipoproteins are a subgroup of exported proteins anchored into the membrane surface by two or three acyl chains covalently linked to their conserved N-terminal cysteine
affinity chromatography either from the membrane fraction (AmyE) is a Bona Fide Lipoprotein of C. glutamicum According to predictions based on bioinformatics tools, the genome of C. glutamicum strain ATCC 13032 encodes 84 putative lipoproteins, representing about 2.8% of its total proteome (DOLOP [32])
We chose AmyE as a model to study the biogenesis of lipoproteins in C. glutamicum
Summary
AmyE is a Bona Fide Lipoprotein of C. glutamicum According to predictions based on bioinformatics tools, the genome of C. glutamicum strain ATCC 13032 encodes 84 putative lipoproteins, representing about 2.8% of its total proteome (DOLOP [32]). The mass difference between the diacylglyceryl and the triacyl Nterminal peptide is 238 Da, which corresponds to a palmitoyl moiety This result suggests that AmyE is diacylated in Dppm but contains an additional modification with a palmitic acid in the wild-type and complemented strains. The m/z 3540.89 peak (diacylated peptide) is observed neither in the wild type nor in the ppm background suggesting that, in these conditions, LppX is fully triacylated exactly as AmyE This peak corresponds to the non-glycosylated diacylated LppX1–29 Nterminal peptide. Only the non-glycosylated form of the LppX6–29 peptide was detected (data not shown), further confirming that LppX is not glycosylated when expressed in the Dppm mutant This could be an indication that protein glycosylation occurs downstream of protein acylation or that the absence of Cg-Ppm affects the stability and/or the activity of enzymes that directly contribute to lipoprotein glycosylation. This result confirms that glycosylation does not require Nacylation and suggestsa functional interplay between protein Nacylation and glycosylation in C. glutamicum
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