RESUMO
LppX is an important virulence factor essential for surface localization of phthiocerol dimycocerosates (DIM) in Mycobacterium tuberculosis. Based on Concanavalin A recognition, M. tuberculosis LppX (LppX-tb) was initially proposed to be glycosylated in M. tuberculosis and more recently this glycosylation was characterized by mass spectrometry analysis on LppX-tb expressed and purified from Corynebacterium glutamicum. Here, using this model organism and Mycobacterium smegmatis, we show that S16 and T18 residues of LppX-tb are indeed glycosylated with several hexoses units. Interestingly this glycosylation is strictly dependent on the mannosyl transferase PMT which, in M. tuberculosis, has been reported to be crucial for virulence. Using a site directed mutagenesis approach, we were able to show that the absence of S16 and T18 glycosylation does not alter phthiocerol dimycocerosates (DIM) localization in M. tuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Lipídeos/análise , Lipoproteínas/metabolismo , Mycobacterium tuberculosis/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Glicosilação , Metabolismo dos Lipídeos , Lipoproteínas/química , Lipoproteínas/genética , Manosiltransferases/genética , Manosiltransferases/metabolismo , Mutagênese Sítio-Dirigida , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Virulência , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.
Assuntos
Corynebacterium glutamicum/enzimologia , Lipoproteínas/metabolismo , Transferases/metabolismo , Acilação , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/metabolismo , Teste de Complementação Genética , Maltose/metabolismo , Mutação , Mycobacterium tuberculosis/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Transferases/genéticaRESUMO
BACKGROUND: Due 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. RESULTS: In 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. CONCLUSION: Together, 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.