Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in; Corynebacterium glutamicum;

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

Triage and biogenesis of the lipoproteins in Corynebacterium glutalicum

En raison de leur contribution à la virulence bactérienne, les lipoprotéines et les membres de la voie de biogenèse des lipoprotéines représentent des cibles prometteuses pour la recherche de nouveaux antibiotiques. À la suite de translocation à travers la membrane interne la future lipoprotéine ancrée dans la membrane par l’intermédiaire de son peptide signal, va subir en premier lieu l’addition de sn-1,2-diacylglyceryle sur la fonction sulfhydryle de la future cystéine N-terminale de la lipoprotéine mature. Cette modification est catalysée par Lgt (prolipoprotéin diacylglycérol transférase) avant même que le peptide signal de la lipoprotéine ne soit clivé par Lsp (lipoprotéine signal peptidase). L’action de la peptidase permet de libérer l’amine terminale de la cystéine qui pourra alors, chez les bactéries à Gram-négatif, être acylée par Lnt (lipoprotéin aminoacyl transférase). La présence d’un apolipoprotéine N-acyltransférase (Ppm2-Ms) impliquées dans la N-acylation de LppX a récemment été montrée chez M. smegmatis. Ppm2-Ms fait partie de l'opéron ppm dans laquelle ppm1, une synthase polyprénol-monophosphomannose, a été révélée essentielle dans la synthèse lipoglycans mais dont la fonction dans la biosynthèse des lipoprotéines est totalement inconnue. Afin de clarifier le rôle de l'opéron ppm dans la biosynthèse des lipoprotéines, nous avons étudié les modifications post-traductionnelles de deux modèles (lipoprotéines AmyE et LppX) dans les mutants Δppm1 et Δppm2 chez C. glutamicum.Nos résultats montrent que les deux lipoprotéines modèles sont ancrées dans la membrane et que leurs extrémités N-terminales sont N-acylés par Ppm2-Cg. Le peptide N-teminal acylé de LppX a été également modifié par des groupements d'hexose. Cette O-glycosylation est localisée dans le peptide N-terminal de LppX mais absente dans le mutant Δppm1. Tandis compromise en l'absence de Cg-PPM2, O-glycosylation LppX pourrait être rétabli lorsque Cg-PPM1, Cg-PPM2 ou l'homologue Mt-ppm1 de M. tuberculosis a été surexprimée. Ensemble, ces résultats montrent pour la première fois que Ppm1-Cg (Ppm synthase) et Ppm2-Cg (Lnt) fonctionnent dans une voie de biosynthèse commune dans laquelle la glycosylation et la N-acylation des lipoprotéines sont étroitement couplésDue 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. 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 ∆ppm1 and ∆ppm2 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 Ntermina 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 ∆ppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX Oglycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed. 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

Étude de la biogénèse des lipoprotéines chez Corynebacterium glutamicum

En raison de leur contribution à la virulence bactérienne, les lipoprotéines et les membres de la voie de biogenèse des lipoprotéines représentent des cibles prometteuses pour la recherche de nouveaux antibiotiques. À la suite de translocation à travers la membrane interne la future lipoprotéine ancrée dans la membrane par l intermédiaire de son peptide signal, va subir en premier lieu l addition de sn-1,2-diacylglyceryle sur la fonction sulfhydryle de la future cystéine N-terminale de la lipoprotéine mature. Cette modification est catalysée par Lgt (prolipoprotéin diacylglycérol transférase) avant même que le peptide signal de la lipoprotéine ne soit clivé par Lsp (lipoprotéine signal peptidase). L action de la peptidase permet de libérer l amine terminale de la cystéine qui pourra alors, chez les bactéries à Gram-négatif, être acylée par Lnt (lipoprotéin aminoacyl transférase). La présence d un apolipoprotéine N-acyltransférase (Ppm2-Ms) impliquées dans la N-acylation de LppX a récemment été montrée chez M. smegmatis. Ppm2-Ms fait partie de l'opéron ppm dans laquelle ppm1, une synthase polyprénol-monophosphomannose, a été révélée essentielle dans la synthèse lipoglycans mais dont la fonction dans la biosynthèse des lipoprotéines est totalement inconnue. Afin de clarifier le rôle de l'opéron ppm dans la biosynthèse des lipoprotéines, nous avons étudié les modifications post-traductionnelles de deux modèles (lipoprotéines AmyE et LppX) dans les mutants ppm1 et ppm2 chez C. glutamicum.Nos résultats montrent que les deux lipoprotéines modèles sont ancrées dans la membrane et que leurs extrémités N-terminales sont N-acylés par Ppm2-Cg. Le peptide N-teminal acylé de LppX a été également modifié par des groupements d'hexose. Cette O-glycosylation est localisée dans le peptide N-terminal de LppX mais absente dans le mutant ppm1. Tandis compromise en l'absence de Cg-PPM2, O-glycosylation LppX pourrait être rétabli lorsque Cg-PPM1, Cg-PPM2 ou l'homologue Mt-ppm1 de M. tuberculosis a été surexprimée. Ensemble, ces résultats montrent pour la première fois que Ppm1-Cg (Ppm synthase) et Ppm2-Cg (Lnt) fonctionnent dans une voie de biosynthèse commune dans laquelle la glycosylation et la N-acylation des lipoprotéines sont étroitement couplésDue 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. 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 ppm1 and ppm2 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 Ntermina 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 ppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX Oglycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed. 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.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

The ppm Operon Is Essential for Acylation and Glycosylation of Lipoproteins in Corynebacterium glutamicum

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

S16 and T18 mannosylation sites of LppX are not essential for its activity in phthiocerol dimycocerosates localization at the surface of Mycobacterium tuberculosis

International audienceLppX 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

Lipoprotein AmyE is triacylated.

<p><b>A.</b> AmyE localization in <i>C. glutamicum</i> wild-type cells expressing AmyE (pAmyE) or a variant of AmyE with a point mutation substituting the cysteine +1 by a leucine (pAmyE^C1L). An empty vector (pCGL482) was used as a control. Membrane and secreted proteins were analyzed by SDS-PAGE followed by immunoblotting using monoclonal anti-his antibodies. The band labeled with an asterisk corresponds to a shorter form of AmyE. <b>B.</b> AmyE (left panel) and AmyE^C1L (right panel) were purified and analyzed by SDS PAGE before (lane 1) or after Triton X114 extraction. Proteins from both aqueous (lane 2) and detergent (lane 3) phases were precipitated and loaded on the gel. <b>C.</b> MALDI mass measurements of intact purified AmyE and AmyE^C1L proteins. Estimated mass accuracy is 150 Da. <b>D.</b> MALDI PMFs of AmyE and AmyE^C1L proteins purified from <i>C. glutamicum</i> wild-type strain. The <i>m/z</i> 3200–4800 region of the mass spectra of AmyE and AmyE^C1L tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are indicated. Upper panel: <i>m/z</i> 4097.10 (bold) corresponds to the triacylated AmyE_1–29 peptide while <i>m/z</i> 3451.65, 3821.53 and 4045.88 match to internal tryptic peptides, AmyE_334–365, AmyE_366–397 and AmyE_55–91 respectively. <i>m/z</i> 4061.74 and 4077.54 peaks could correspond to mono- and di- oxidized AmyE_55–91 peptides. Bottom panel: <i>m/z</i> 3292.60 (bold) corresponds to the C1L-mutated AmyE_1–29 peptide while <i>m/z</i> 3451.65 and 3821.72 match to AmyE_334–365 and AmyE_366–397 peptides. The <i>m/z</i> 4061.79, 4077.80, 4093.86 and 4109.83 peaks match to mono-, di-, tri- and tetra-oxidized AmyE_55–91 peptides. The <i>m/z</i> 4342.89 peak corresponds to the di-oxidized AmyE_277–315 peptide. It’s worth noting that AmyE^C1L peptides were more often detected in the oxidized state than AmyE peptides. Insets aim at emphasizing the specificity of the <i>m/z</i> 4097.10 signal detected only in the AmyE spectrum. Asterisks indicate that <i>m/z</i> assignments are not accurate because of low mass resolution and weak signal/noise ratio. <b>E.</b> Sequence of the recombinant purified wild-type AmyE protein. Identified unmodified peptides after trypsin digestion and DDM/CHCl₃-CH₃OH treatment are shown in boldface type on the amino acid sequence of recombinant AmyE.</p

Cg-Ppm1 is required for LppX glycosylation.

<p>Comparison of MALDI PMF profiles of LppX protein purified from <i>C. glutamicum</i> wild-type and Δ<i>ppm1</i> strains. The <i>m/z</i> 3500–4550 region of the mass spectra of LppX tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are annotated. Upper panel: <i>m/z</i> peaks correspond to glycosylated and triacylated LppX_1–29 peptides are indicated in bold (<i>m/z</i> 4103.09, 4203.03, 4427.11 and 4526.95). Three peaks (<i>m/z</i> 3624.65, 3794.74 and 3808.88) were detected but not identified. Bottom panel: the <i>m/z</i> peak corresponding to the non-glycosylated triacylated LppX_1–29 peptide was specifically observed in the Δ<i>ppm1</i>mutant (<i>m/z</i> 3779.28, in bold). The <i>m/z</i> 3795.28 peak was not identified. • = 1 hexose (Δm = 162 Da). • = unknown modification (Δm = 262 Da). “ni” means not identified and asterisks indicate that <i>m/z</i> assignments are not very accurate. Inset: SDS PAGE of the purified LppX proteins from the wild type and the Δ<i>ppm1</i> strains.</p

<i>M. tuberculosis</i> LppX is O-glycosylated in <i>C. glutamicum.</i>

<p><b>A.</b> Localization of LppX in <i>C. glutamicum</i> wild-type strain: Membrane (M) and secreted (S) proteins were analyzed by SDS-PAGE, followed by immunoblotting using monoclonal anti-his antibodies (left). Purification of LppX: LppX was purified and analyzed by SDS PAGE before (lane 1) or after Triton X114 extraction. Proteins from both aqueous (lane 2) and detergent (lane 3) phases were precipitated and loaded on the gel (right). LC-ESI-MS/MS analysis of LppX peptides: Identified peptides generated by standard extraction procedures are shown in boldface type on the amino acid sequence of recombinant LppX. * indicates hydroxyl amino acid residues of the LppX_6–29 peptide. <b>B.</b> LC-MS analysis of LppX_6–29 glycopeptides. The deconvoluted LC-MS chromatogram is shown (21.5+1 ions corresponding to the unmodified peptide (<i>m/z</i> 2462.17), glycosylated forms with 1 to 4 hexose units (<i>m/z</i> 2624.21, 2786.26, 2948.30, 3110.35) and Δ262- glycosylated forms with 0 to 3 hexose units (<i>m/z</i> 2724.23, 2886.28, 3048.34, 3210.37) are shown. • = 1 hexose (Δm = 162). • = unknown modification (Δm = 262). <b>C.</b> Deconvoluted MS/MS spectra of unmodified and tetraglycosylated peptides. Fragmentation patterns of the triply charged ions corresponding to unmodified (left) and tetraglycosylated (right) LppX_6–29 peptides are shown. b8, y8 and y16 most intense fragment ions are annotated in both MS/MS spectra. Fragmentation pattern of the tetraglycosylated LppX_6–29 peptidereveals 4 neutral losses of 162 Da coming from the intact tetraglycosylated peptide (<i>m/z</i> 3110.38) and from the tetraglycosylated y16 fragment ion (<i>m/z</i> 2285.03). • = 1 hexose (Δm = 162 Da).</p

Cg-Ppm2 activity affects LppX glycosylation.

<p>Comparison of MALDI PMF profiles of LppX protein purified from ΔΔ<i>ppm2</i>, Δ<i>ppm2</i> (pCg-<i>ppm2</i>) and Δ<i>ppm2</i> (pCg-<i>ppm1</i>). The <i>m/z</i> 3500–4550 region of the mass spectra of LppX tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are annotated. Upper spectrum: in the the Δ<i>ppm2</i> strain, only the <i>m/z</i> peak corresponding to the non-glycosylated diacylated LppX_1–29 peptide is identified (<i>m/z</i> 3540.89, bold). Five peaks were assigned but not identified (<i>m/z</i> 3624.64, 3635.96, 3794.77, 3808.89 and 4518.28). These peaks do not match to internal tryptic LppX peptides. Middle spectrum: in the Δ<i>ppm2</i> (pCg-<i>ppm2</i>) strain, <i>m/z</i> peaks corresponding to different glycosylated forms of the triacylated LppX_1–29 peptide are observed (<i>m/z</i> 3941.15, 4041.13 and 4103.22, in bold) as well as the peak corresponding to the non-glycosylated triacylated LppX_1–29 peptide (<i>m/z</i> 3779.23, in bold). The <i>m/z</i> 3624.63 peak was detected but not identified. Bottom spectrum: in the Δ<i>ppm2</i> (pCg-<i>ppm1</i>) strain different glycosylated forms of the diacylated LppX_1–29 peptide are detected (<i>m/z</i> 3864.99, 3964,86, 4188.82 and 4288.85, in bold). • = 1 hexose (ΔΔm = 162 Da). • = unknown modification (Δm = 262 Da). “ni” means not identified and asterisks indicate that <i>m/z</i> assignments are not very accurate. Inset: SDS PAGE of the purified LppX proteins from the wild type, the Δ<i>ppm2</i> and the complemented strains.</p

Cg-Ppm2 exhibits apolipoprotein N-acyltransferase activity. A.

<p>Localization of AmyE in <i>C. glutamicum</i> Δ<i>ppm2</i> or Δ<i>ppm2</i> (Cg-<i>ppm2</i>) strains. Membrane (M) and secreted (S) proteins were analyzed by SDS-PAGE followed by immunoblotting using monoclonal anti-his antibodies. The band labeled with an asterisk corresponds to a shorter form of AmyE. <b>B.</b> MALDI PMFs of AmyE protein purified from ΔCg-<i>ppm2</i> and ΔCg-<i>ppm2</i> (Cg-<i>ppm2</i>) strains. The <i>m/z</i> 3200–4800 region of the mass spectra of AmyE tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are annotated. Upper panel: <i>m/z</i> 3858.98 (bold) corresponds to the diacylated AmyE_1–29 peptide while <i>m/z</i> 3451.65, 3547.59 and 4045.88 match to internal tryptic peptides, AmyE_334–365, AmyE_60–91 and AmyE_55–91, respectively. Bottom panel: <i>m/z</i> 4097.21 (bold) corresponds to the triacylated AmyE_1–29 peptide while <i>m/z</i> 3451.65, 3547.61 and 4045.88 match to AmyE_334–365, AmyE_60–91 and AmyE_55–91 peptides.</p