Deciphering the Genetic Bases of the Structural Diversity of Phenolic Glycolipids in Strains of the Mycobacterium tuberculosis Complex

Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnosyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-d-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/reductase, required for the formation of activated l-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex. Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnosyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-d-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/reductase, required for the formation of activated l-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex. Phenolic glycolipids (PGL) 2The abbreviations used are: PGL, phenolglycolipid; DIM, phthiocerol dimycocerosates; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; LOS, lipooligosaccharides. are produced by certain mycobacterial species, most of which are pathogenic for humans (1Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar). These substances are located in the outermost layers of the mycobacterial envelope where they play a key role in the pathogenicity of mycobacteria. For instance, PGL-1, the major PGL synthesized by Mycobacterium leprae, is important for the unique tropism of this bacterium to peripheral nerves through binding to laminin 2 (2Ng V. Zanazzi G. Timpl R. Talts J.F. Salzer J.L. Brennan P.J. Rambukkana A. Cell. 2000; 103: 511-524Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Additionally, PGL-1 synthesis in the leprosy bacillus affects the resistance to intracellular killing by macrophages (3Neill M.A. Klebanoff S.J. J. Exp. Med. 1988; 167: 30-42Crossref PubMed Scopus (67) Google Scholar) and modifies binding to complement receptors (4Schlesinger L.S. Horwitz M.A. J. Exp. Med. 1991; 174: 1031-1038Crossref PubMed Scopus (69) Google Scholar), which may be important for the pathogenesis of leprosy. PGL-1 can also modulate the immune response (5Charlab R. Sarno E.N. Chatterjee D. Pessolani M.C.V. Lepr. Rev. 2001; 72: 63-69PubMed Google Scholar, 6Mehra V. Brennan P.J. Rada E. Convit J. Bloom B.R. Nature. 1984; 308: 194-196Crossref PubMed Scopus (139) Google Scholar). In Mycobacterium tuberculosis, recent findings reveal that the production of PGL is associated with hypervirulence in mice (7Reed M.B. Domenech P. Manca C. Su H. Barczak A.K. Kreiswirth B.N. Kaplan G. Barry III, C.E. Nature. 2004; 431: 84-87Crossref PubMed Scopus (618) Google Scholar) and in a rabbit model of meningitis (8Tsenova L. Ellison E. Harbacheuski R. Moreira A.L. Kurepina N. Reed M.B. Mathema B. Barry III, C.E. Kaplan G. J. Infect. Dis. 2005; 192: 98-106Crossref PubMed Scopus (201) Google Scholar). This glycolipid seems to inhibit the release of pro-inflammatory mediators (7Reed M.B. Domenech P. Manca C. Su H. Barczak A.K. Kreiswirth B.N. Kaplan G. Barry III, C.E. Nature. 2004; 431: 84-87Crossref PubMed Scopus (618) Google Scholar). PGL consist of a lipid core formed by a long-chain β-diol, occurring naturally as diester of polymethyl-branched fatty acids (1Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar) (Figs. 1 and 3). The lipid core is ω-terminated by an aromatic nucleus that is glycosylated. The sugar moiety of PGL consists of one to four sugar residues, depending on the species, and most are O-methylated deoxysugars (9Daffé M. Lemassu A. Doyle R.J. Glycomicrobiology. Plenum Publishing Corp, New York2000: 225-273Google Scholar, 10Brennan P.J. Ratledge C. Wilkinson S.G. Microbial Lipids. Academic Press, London1988: 203-298Google Scholar). Only a few M. tuberculosis strains synthesize PGL-tb (11Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). In such strains, the carbohydrate domain of the major form of PGL-tb is 2,3,4-tri-O-methyl-l-fucopyranosyl-(α1->3)-l-rhamnopyranosyl-(α1->3)-2-O-methyl-l-rhamnopyranosyl-(α1->) (12Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar) (Fig. 1). This structure seems specific to the M. tuberculosis and Mycobacterium canettii lineages; other members of the M. tuberculosis complex, which gathers mycobacterial strains very closely related to M. tuberculosis, do not produce the same saccharidic domain (1Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar). For instance, M. bovis and Mycobacterium microti produce a truncated form of PGL-tb, called mycoside B, in which the carbohydrate domain is restricted to 2-O-methylrhamnose (1Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar, 13Thurman P.F. Chai W. Rosankiewicz J.R. Rogers H.J. Lawson A.M. Draper P. Eur. J. Biochem. 1993; 212: 705-711Crossref PubMed Scopus (7) Google Scholar). In other members of M. tuberculosis complex, such as Mycobacterium africanum and Mycobacterium pinnipedii, production of PGL has not been reported to date.FIGURE 1Identification of the genes required for the production of PGL-tb in M. bovis BCG.A, TLC analysis of lipid extracts from the bacterial cell of recombinant M. bovis BCG. Crude lipid extract was from M. bovis BCG (lane 1), M. bovis BCG::pPET52 (Rv2958c) (lane 2), M. bovis BCG::pPET52:pWM85 (Rv1511, Rv1512, and Rv2958c) (lane 5), M. tuberculosis H37Rv:pPET1 (pks15/1) (lane 6), M. canettii, a natural mycobacterial producer of PGL-tb (lane 7), mycoside B purified from M. bovis BCG (lane 3), PGL-tb purified from M. bovis BCG::pPET52:pWM85 (Rv1511, Rv1512, and Rv2958c) (lane 4). Lipid extracts were dissolved in CHCl3 and run in CHCl3/CH3OH (95:5, v/v). Glycolipids were visualized by spraying the plates with 0.2% anthrone (w/v) in concentrated H2SO4 followed by heating. PGL-2S corresponds to mono-O-methyl-diglycosyl-phenolphthiocerol dimycocerosate as described previously (15Perez E. Constant P. Lemassu A. Laval F. Daffe M. Guilhot C. J. Biol. Chem. 2004; 279: 42574-42583Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The arrowheads indicate the positions of the purified PGLs. B, structure of the putative PGLs produced by the recombinant M. bovis BCG strains. R1 corresponds to the phthiocerol dimycocerosate core of PGL. Genes transferred in M. bovis BCG are indicated beside the arrows. Other genes such as Rv2957 (encoding the third glycosyltransferase) and those encoding the methyltransferases are required for the synthesis of the complete saccharide moiety of PGL-tb, but our findings demonstrated that they are all already functional in M. bovis BCG.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The biosynthesis of PGL-tb involves more than 25 enzymatic steps. Genes required for PGL biosynthesis and translocation to the mycobacterial cell surface are clustered on a 73-kb fragment of the M. tuberculosis chromosome (14Guilhot C. Chalut C. Daffé M. Daffé M. Reyrat J.M. The Mycobacterial Cell Envelope. American Society for Microbiology, Washington, DC2008: 273-285Google Scholar). The genetic basis for the inability of most M. tuberculosis isolates to synthesize PGL-tb was found to be a mutation within the pks15/1 gene that encodes an enzyme involved in the formation of a precursor of PGL-tb (11Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Howe

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