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• W3036581723 abstract "Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis. TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis. Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis. TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis. Mycobacteria are endowed with an unusually thick lipid-rich cell envelope. This structure contains several families of trehalose-containing glycolipids that interact with mycolic acids attached to the arabinogalactan to form an atypical outer membrane called the mycomembrane (1Daffe M. Crick D.C. Jackson M. Genetics of capsular polysaccharides and cell envelope (glyco)lipids.Microbiol. Spectr. 2014; 2 (MGM2-0021-2013) (26104202)10.1128/microbiolspec.mgm2-0021-2013Crossref Scopus (60) Google Scholar). Among these, the ubiquitous trehalose monomycolates and trehalose dimycolates are essential for viability (2Daffe M. Marrakchi H. Unraveling the structure of the mycobacterial envelope.Microbiol. Spectr. 2019; 7 (GPP3-0027-2018) (31267927)10.1128/microbiolspec.gpp3-0027-2018Crossref PubMed Scopus (46) Google Scholar). This group also includes species-specific glycolipids, such as di- and polyacyltrehaloses (DAT and PAT) and sulfolipids (SL), which are restricted to the human pathogen Mycobacterium tuberculosis, or lipooligosacharides that have been isolated from diverse fast- and slow-growing mycobacteria (1Daffe M. Crick D.C. Jackson M. Genetics of capsular polysaccharides and cell envelope (glyco)lipids.Microbiol. Spectr. 2014; 2 (MGM2-0021-2013) (26104202)10.1128/microbiolspec.mgm2-0021-2013Crossref Scopus (60) Google Scholar). Recently, we reported that trehalose polyphleates (TPP), a family of surface-exposed glycolipids originally described in Mycobacterium phlei, are widely distributed across mycobacterial species, including Mycobacterium smegmatis and the opportunistic pathogens Mycobacterium abscessus and Mycobacterium avium (3Asselineau C. Montrozier H. Promé J.C. [Presence of polyunsaturated acids in bacteria: isolation of hexatriacontapentaene-4,8,12,16,20-oic acid and acid analogs from Mycobacterium phlei lipids].Eur. J. Biochem. 1969; 10 (4310547): 580-58410.1111/j.1432-1033.1969.tb00728.xCrossref PubMed Scopus (20) Google Scholar, 4Asselineau C.P. Montrozier H.L. Promé J.C. Savagnac A.M. Welby M. [Polyunsaturated glycolipids synthesized by Mycobacterium phlei].Eur. J. Biochem. 1972; 28 (5050253): 102-10910.1111/j.1432-1033.1972.tb01889.xCrossref PubMed Scopus (27) Google Scholar, 5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). TPP consist of octoacylated trehalose bearing seven C36:5 and C40:6 polyunsaturated fatty acids called phleic acids and a C14–C19 fatty acid residue. They share a common basic structure with SL and PAT from M. tuberculosis (6Seeliger J. Moody D.B. Monstrous mycobacterial lipids.Cell Chem. Biol. 2016; 23 (26971870): 207-20910.1016/j.chembiol.2016.02.004Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). The biological function of TPP in mycobacteria remains enigmatic. A recent study showed that TPP production in M. abscessus correlates with clump and cord formation, suggesting a potential role for these molecules in the virulence of this opportunistic human pathogen (7Llorens-Fons M. Perez-Trujillo M. Julian E. Brambilla C. Alcaide F. Byrd T.F. Luquin M. Trehalose polyphleates, external cell wall lipids in Mycobacterium abscessus, are associated with the formation of clumps with cording morphology which have been associated with virulence.Front. Microbiol. 2017; 8 (28790995): 140210.3389/fmicb.2017.01402Crossref PubMed Scopus (20) Google Scholar). Formation and export of TPP involve at least four biosynthetic enzymes and an MmpL transporter, namely MmpL10, that are encoded by genes clustered at the TPP locus (5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These proteins display sequence similarities with enzymes required for SL and PAT production, indicating that the TPP pathway can serve as an informative model to describe glycolipid production in M. tuberculosis. Biosynthesis of TPP begins in the cytoplasm with the formation of a 2,3-diacyltrehalose intermediate bearing a C14–C19 fatty acyl group and a phleic acyl substituent. The transacylase enzyme PE encoded by the MSMEG_0412 gene (previously referred to as MSMEI_0402), subsequently catalyzes transesterification of phleic acids between diacyltrehalose precursors to generate TPP. Finally, MmpL10 is involved in the translocation of TPP or/and of TPP precursors across the plasma membrane (5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 8Chalut C. MmpL transporter-mediated export of cell-wall associated lipids and siderophores in mycobacteria.Tuberculosis. 2016; 100 (27553408): 32-4510.1016/j.tube.2016.06.004Crossref PubMed Scopus (41) Google Scholar). Despite this knowledge, the late stages of the TPP biosynthetic pathway and the relationship between biosynthesis and transport of these compounds to the cell envelope remain elusive. The PE protein is predicted to be a membrane-anchored protein harboring a putative N-terminal signal peptide and a C-terminal α/β-serine hydrolase domain, consistent with its proposed role as an acyltransferase in TPP assembly (Fig. 1) (9Adindla S. Guruprasad L. Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes.J. Biosci. 2003; 28 (12711809): 169-17910.1007/BF02706216Crossref PubMed Scopus (41) Google Scholar, 10Sultana R. Tanneeru K. Guruprasad L. The PE-PPE domain in mycobacterium reveals a serine α/β hydrolase fold and function: an in-silico analysis.PLoS ONE. 2011; 6 (21347309): e1674510.1371/journal.pone.0016745Crossref PubMed Scopus (37) Google Scholar). Interestingly, Chp1 and Chp2, two membrane-bound acyltransferases, respectively involved in SL and PAT production in M. tuberculosis, share overall domain organization with PE (Fig. 1). It has been proposed that these enzymes catalyze the last step of SL and PAT assembly by a mechanism that is tightly coupled to lipid transport across the plasma membrane, but the molecular details of this process are not fully understood, partly because the in vivo subcellular localization of their catalytic domains remains controversial. Seeliger et al. (11Seeliger J.C. Holsclaw C.M. Schelle M.W. Botyanszki Z. Gilmore S.A. Tully S.E. Niederweis M. Cravatt B.F. Leary J.A. Bertozzi C.R. Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis.J. Biol. Chem. 2012; 287 (22194604): 7990-800010.1074/jbc.M111.315473Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) indeed demonstrated that the catalytic domain of Chp1 is located in the cytoplasm, but two studies reported contradictory results on the orientation of Chp2, the catalytic domain being either cytoplasmic or periplasmic (12Belardinelli J.M. Larrouy-Maumus G. Jones V. Sorio de Carvalho L.P. McNeil M.R. Jackson M. Biosynthesis and translocation of unsulfated acyltrehaloses in Mycobacterium tuberculosis.J. Biol. Chem. 2014; 289 (25124040): 27952-2796510.1074/jbc.M114.581199Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Touchette M.H. Holsclaw C.M. Previti M.L. Solomon V.C. Leary J.A. Bertozzi C.R. Seeliger J.C. The rv1184c locus encodes Chp2, an acyltransferase in Mycobacterium tuberculosis polyacyltrehalose lipid biosynthesis.J. Bacteriol. 2015; 197 (25331437): 201-21010.1128/JB.02015-14Crossref PubMed Scopus (15) Google Scholar). Likewise, the orientation of PE in the plasma membrane and thereby the subcellular location of its catalytic domain, is unknown, raising questions on the cell compartment in which the transacylation step takes place and on the identity of the compounds that are transported by MmpL10. The goal of this study was to shed light on the last step of TPP biosynthesis by investigating the topological organization and subcellular location of the PE protein. Using a combination of genetic, biochemical, and proteomic approaches, we established that PE has a C-terminal periplasmic domain and found that proteolytic cleavage of PE is required for efficient formation of TPP. Based on these data, we present here a refined model of the TPP biosynthesis pathway. This work provides clues for a better understanding of the molecular mechanisms involved in DAT/PAT production in M. tuberculosis. In silico analysis using the SignalP software predicts that PE possesses a putative signal sequence with a AXA signal peptidase I cleavage motif (PE1-28, cleavage site between positions 28 and 29: AAA-DD, Fig. 1) (14Auclair S.M. Bhanu M.K. Kendall D.A. Signal peptidase I: cleaving the way to mature proteins.Protein Sci. 2012; 21 (22031009): 13-2510.1002/pro.757Crossref PubMed Scopus (112) Google Scholar); it is thus possible that this protein is associated with the plasma membrane, facing either the periplasm or the cytoplasm, or exported to the bacterial cell wall after cleavage of the signal sequence. To discriminate between these possibilities, we first investigated the topological organization of PE in M. smegmatis. Plasmids expressing PE fused at the C terminus with either the GFP or the alkaline phosphatase (PhoA) (Table S1) were introduced in the WT strain of M. smegmatis, and the GFP and PhoA activities were monitored to determine the subcellular localization of the reporter proteins. GFP exhibits fluorescence only when localized to the cytosolic compartment, whereas PhoA is only active inside the periplasmic space (15Wiker H.G. Wilson M.A. Schoolnik G.K. Extracytoplasmic proteins of Mycobacterium tuberculosis—mature secreted proteins often start with aspartic acid and proline.Microbiology. 2000; 146 (10878117): 1525-153310.1099/00221287-146-7-1525Crossref PubMed Scopus (39) Google Scholar, 16Belardinelli J.M. Jackson M. Green fluorescent protein as a protein localization and topological reporter in mycobacteria.Tuberculosis. 2017; 105 (28610783): 13-1710.1016/j.tube.2017.04.001Crossref PubMed Scopus (10) Google Scholar). Expression of PE-GFP or PE-PhoA in an M. smegmatis pE knockout mutant (PMM229) restored TPP production, indicating that the fusion proteins are correctly folded and display proper subcellular localization in bacteria (Fig. S1). Additional M. smegmatis strains producing KatG1, a cytosolic protein, MmpL10, a transmembrane protein with a cytoplasmic C-terminal end, or MmpS4, a transmembrane protein with the C‐terminal end facing the periplasm, fused to the same topology reporter proteins were generated to serve as controls in the experiments (16Belardinelli J.M. Jackson M. Green fluorescent protein as a protein localization and topological reporter in mycobacteria.Tuberculosis. 2017; 105 (28610783): 13-1710.1016/j.tube.2017.04.001Crossref PubMed Scopus (10) Google Scholar, 17Deshayes C. Bach H. Euphrasie D. Attarian R. Coureuil M. Sougakoff W. Laval F. Av-Gay Y. Daffé M. Etienne G. Reyrat J.M. MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis.Mol. Microbiol. 2010; 78 (21062372): 989-100310.1111/j.1365-2958.2010.07385.xCrossref PubMed Scopus (59) Google Scholar). Bacteria producing PE-GFP displayed no increase in average fluorescence when compared with WT or control bacteria expressing MmpS4-GFP (Fig. 2A). Conversely, as expected, high fluorescence signals were detected for strains expressing KatG1-GFP or MmpL10-GFP. M. smegmatis strains producing the PhoA fusions were first assayed for alkaline phosphatase activity on LB agar plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Production of PE-PhoA resulted in blue colonies after incubation at 37 °C, indicative of PhoA activity (Fig. 2B). A similar pattern was observed for the control strain expressing MmpS4-PhoA, whereas no color change was observed for strains expressing either KatG1-PhoA or MmpL10-PhoA. To confirm these data, the PhoA activity of fusion proteins was assessed on cells grown in LB using the chromogenic substrate p-nitrophenylphosphate (pNPP). Results obtained were consistent with those obtained on BCIP agar plates: bacteria that express PE-PhoA, as well as those producing MmpS4-PhoA, exhibited high levels of PhoA activity compared with the WT strain and control strains expressing KatG1-PhoA or MmpL10-PhoA (Fig. 2B). Collectively, these data indicated that the C-terminal domain of PE is exposed on the outside of the plasma membrane. In these studies, we also sought to confirm the topology of Chp1 and to resolve the contradictory research findings regarding the topological organization of Chp2, using M. smegmatis strains expressing each of these proteins fused to either the GFP or PhoA. Attempts to detect GFP fluorescence or PhoA activity in bacteria producing the Chp1 fusion proteins failed (data not shown). For Chp2, we observed a significant PhoA activity in cells expressing Chp2-PhoA on both solid and liquid media, but no increase in fluorescence in bacteria producing Chp2-GFP, compared with the WT or the negative control MmpS4-GFP (Fig. 2, A and B), thus establishing that like PE, the Chp2 protein has its C-terminal domain exposed to the periplasmic space. Our topology studies revealed that the C-terminal domain of PE is located outside of the cytoplasm. To examine whether PE remains attached to the plasma membrane or is released into the bacterial cell wall, we performed a comprehensive proteomic analysis of the subcellular fractions of M. smegmatis. The proteomes of four independent cultures of M. smegmatis were split in four subcellular fractions: plasma membrane, culture filtrate (secretome), mycomembrane-containing cell-wall (MMCW), and soluble proteins from cytosol and periplasm. Plasma membrane and MMCW were isolated from the cell lysates by differential ultracentrifugation on a sucrose gradient, as described (18Chiaradia L. Lefebvre C. Parra J. Marcoux J. Burlet-Schiltz O. Etienne G. Tropis M. Daffé M. Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane.Sci. Rep. 2017; 7 (28993692): 1280710.1038/s41598-017-12718-4Crossref PubMed Scopus (115) Google Scholar). The homogeneity of both membrane fractions has been demonstrated previously, using biochemical markers: arabinose and galactose (arabinogalactan), glucosamine, muramic acid, and diaminopimelic acid (peptidoglycan) as markers of MMCW and NADH oxidase and ATP synthase as markers of the plasma membrane. Only traces of MMCW markers were detected in the plasma membrane fraction, and conversely, very little NADH oxidase activity (3.6% of that measured in the plasma membrane) and no ATP synthase was detected in the MMCW fraction (18Chiaradia L. Lefebvre C. Parra J. Marcoux J. Burlet-Schiltz O. Etienne G. Tropis M. Daffé M. Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane.Sci. Rep. 2017; 7 (28993692): 1280710.1038/s41598-017-12718-4Crossref PubMed Scopus (115) Google Scholar). After concentration on SDS-PAGE, proteins from each fraction were digested in gel using trypsin. Peptides were then extracted from the gel and analyzed in quadruplicate injections by nano-LC–MS/MS using an Orbitrap Fusion™ Tribrid™ mass spectrometer. MS data were searched against the Uniprot M. smegmatis mc2155 database for protein identification, and the quantitative comparison of relative protein abundances was performed using MS1-based label-free quantification. This data set contains 3,510 proteins and their relative quantities in the different fractions. We first confirmed the presence of proteins of known subcellular localization in the different fractions and their clustering based on MS signal (see Fig. S2 and Table S2 for the list of marker proteins). The PE protein was not detected in the cytosol or in the plasma membrane (Fig. 3A). It was detected with a very good sequence coverage in the MMCW (Fig. 3B). Although its signal was less intense, two specific peptides of PE were identified in the culture filtrate (see Fig. S3 for annotated spectra). Of note, the quantitative data relative to the subcellular specific proteomes generated in this study are available in Table S3. These data constitute an informative resource on the subcellular distribution of M. smegmatis proteins. As mentioned, PE possesses a putative signal sequence with a cleavage site. Therefore, we next examined whether the signal sequence is cleaved and whether the mature protein is exported to the MMCW using biochemical approaches. An HA-tagged PE protein (PE-HA) was expressed in the PMM229 mutant (ΔpE) of M. smegmatis for estimating its relative molecular weight in cells, based on SDS-PAGE migration and Western blotting analysis. Control strains producing either a mutated form of PE (PEΔCS-HA), in which a stretch of amino acids (Ala26–Asp29) surrounding the putative cleavage site has been deleted to interfere with signal peptide removal, or the predicted mature form of PE (mPE-HA, residues 29-383) were used to distinguish between cleaved and uncleaved forms of PE (Fig. 4A). Two protein bands that migrate at positions corresponding to mPE-HA and to the noncleavable PEΔCS-HA variant, respectively, were detected in the cell extract of M. smegmatis expressing PE-HA, indicating that the PE protein underwent partial cleavage in this strain (Fig. 4A). To further establish that PE contains a cleavable signal sequence, its N terminus region with or without the AAA motif and its local peptide environment was fused to the mature form of the Escherichia coli β-lactamase BlaTEM-1 (mBlaT, residues 24-286), generating fusion proteins PE1-25-mBlaT and PE1-30-mBlaT (Fig. 4B). BlaTEM-1 has been used before as a reporter for protein export with Sec and Tat substrates (19McCann J.R. McDonough J.A. Pavelka M.S. Braunstein M. β-Lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells.Microbiology. 2007; 153 (17906134): 3350-335910.1099/mic.0.2007/008516-0Crossref PubMed Scopus (23) Google Scholar). PE1-30-mBlaT and PE1-25-mBlaT were expressed in PMM299, a M. smegmatis mutant lacking the major β-lactamase BlaS (see Fig. S4), and bacteria were tested for susceptibility toward ampicillin using a disk diffusion assay and the broth dilution method for MIC determination. Production of PE1-30-mBlaT in PMM299 restored resistance to ampicillin beyond the WT level (more than 64-fold and 2-fold increases in MIC relative to the PMM299 mutant and the parent strain, respectively). In contrast, production of PE1-25-mBlaT conferred only moderate resistance to ampicillin on both solid and liquid media (2–4-fold increase in MIC compared with the PMM299 mutant) (Fig. 4B). To confirm that the difference we observed between the two proteins reflected a real difference in protein cleavage, we performed Western blotting analysis with an anti-BlaTEM-1 antibody on cell lysates prepared from PMM299 expressing either PE1-30-mBlaT or PE1-25-mBlaT. We found that the two proteins were produced in similar quantities in bacteria (Fig. 4C). In accordance with their calculated molecular weights (CMW), PE1-25-mBlaT (CMW 31.8 kDa) migrated as a single band protein with an apparent molecular mass slightly higher than that of the full-length β-lactamase BlaTEM-1 (CMW 31.6 kDa), confirming that the protein was not processed in PMM299 (Fig. 4C). By contrast, two bands of similar intensity were visible for PE1-30-mBlaT. Their migration on SDS-PAGE was consistent with the CMW of the full-length unprocessed form of the protein (32.3 kDa) and of the mature form of BlaTEM-1 with an additional DDKL amino acid sequence (DDKL-mBlaT, 29.4 kDa) (Fig. 4C). It can thus be concluded that PE1-25-mBlaT remains attached to the plasma membrane due to the lack of signal sequence cleavage, causing a moderate increase in antibiotic resistance, whereas PE1-30-mBlaT undergoes proteolytic processing, leading to mBlaT excretion in the cell envelope and high-level ampicillin resistance. Consistently, expression of BlaTEM-1 proteins with an altered cleavage signal sequence in E. coli confers intermediate-level resistance to ampicillin compared with the WT form of BlaTEM-1 (20Palzkill T. Le Q.Q. Wong A. Botstein D. Selection of functional signal peptide cleavage sites from a library of random sequences.J. Bacteriol. 1994; 176 (8300511): 563-56810.1128/jb.176.3.563-568.1994Crossref PubMed Google Scholar). Altogether, data obtained with the BlaTEM-1 reporter protein, in addition to those obtained with the HA-tagged fusion proteins, confirmed that PE is excreted in the periplasmic compartment or the MMCW of M. smegmatis following signal peptide cleavage. We finally explored whether formation of TPP in M. smegmatis depends on proteolytic cleavage of PE. Strain PMM229 (ΔpE) expressing either PE-HA or PEΔCS-HA (Fig. 4A), as well as the WT and the PMM229 mutant strains, were grown to mid-exponential phase. The cellular and surface-exposed lipid fractions were extracted from each strain and separated by TLC. As reported previously, disruption of pE impaired TPP production and resulted in accumulation of a relatively polar compound that corresponds to the diacyl trehalose precursor of TPP, in both the cellular and surface compartments (5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) (Fig. 5A). Production of PE-HA in PMM229 restored the WT phenotype. In this complemented strain, TPP were found in the cellular and surface-exposed lipid fractions, and the intermediate product was undetectable in these lipid fractions. By contrast, PMM229 cells expressing the PEΔCS-HA protein were defective in TPP production and accumulated the precursor in the cellular and surface compartments. These data demonstrate that the PEΔCS-HA protein is unable to catalyze TPP formation, likely because the enzyme is anchored to the plasma membrane and has limited access to its substrate that is mainly located in the outer layers of the cell envelope. We cannot totally rule out that the small deletion present in PEΔCS-HA may affect protein stability. However, amino acid residues surrounding the signal peptidase cleavage motif are located away from the predicted serine hydrolase core domain of PE (residues 90–324) and are probably not required for the correct folding of this domain. Supporting this, it has been shown that the Ala30–Gly359 domain of Chp2, which starts 5 residues after the putative AXA cleavage motif (Fig. 1), displays acyltransferase activity in vitro (12Belardinelli J.M. Larrouy-Maumus G. Jones V. Sorio de Carvalho L.P. McNeil M.R. Jackson M. Biosynthesis and translocation of unsulfated acyltrehaloses in Mycobacterium tuberculosis.J. Biol. Chem. 2014; 289 (25124040): 27952-2796510.1074/jbc.M114.581199Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In a previous study, we proposed a model for the biosynthesis of TPP, but some steps of this biosynthetic pathway remain poorly characterized, including the one involving the acyltransferase PE (5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Here, we shed new light on the molecular mechanisms that underlie the latter stages of TPP production in M. smegmatis. Based on a series of genetic, biochemical, and proteomic approaches, we established that (i) the C-terminal domain of PE, and therefore its catalytic domain, resides outside the plasma membrane, (ii) PE is cleaved and excreted in the MMCW, and (iii) proteolytic processing of PE is required for efficient production of TPP in bacteria. These data, in combination with our previous findings, allow us to propose an updated version of the TPP biosynthetic pathway (Fig. 6): the 2,3-diacyltrehalose precursor is synthetized through a series of cytoplasmic steps catalyzed by enzymes encoded by the TPP locus (FadD23, Pks, and PapA3) (5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and is translocated across the plasma membrane by the MmpL10 transporter. The intermediate molecule is then exported to the cell surface, where PE transacylates phleic acids between precursors to yield TPP. Several lines of evidence support this scenario. First, a mmpL10 mutant of M. smegmatis is completely devoid of TPP, indicating that the transacylase reaction depends on the translocation process and likely occurs after this step (5Burbaud S. Laval F. Lemassu A. Daffé M. Guilhot C. Chalut C. Trehalose polyphleates are produced by a glycolipid biosynthetic pathway conserved across phylogenetically distant mycobacteria.Cell Chem. Biol. 2016; 23 (27028886): 278-28910.1016/j.chembiol.2015.11.013Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Second, accumulation of the intermediate product in the surface-exposed lipid fraction of the PMM229 (ΔpE) mutant indicates that this compound is translocated through the plasma membrane by MmpL10 and transferred to the cell surface. Third, we established that only the mature form of PE catalyzes TPP formation, supporting the notion that transacylation takes place in the cell envelope. Presumably, the full-length unpro" @default.
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• W3036581723 title "The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope" @default.
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