Abstract
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.
Highlights
Phenolic glycolipids (PGL)[2] are produced by certain mycobacterial species, most of which are pathogenic for humans (1)
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-Dmannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/reductase, required for the formation of activated L-fucose
We investigated the structural diversity of PGL among strains of the M. tuberculosis complex and its genetic bases
Summary
The 2-kb fragment was purified using the QIAquick purification kit (Qiagen, Courtaboeuf, France) It was digested with NdeI and SpeI and inserted between the NdeI and SpeI sites of pMIP12d vector to give pWM85. PCR Amplification of the Rv1511/Rv1512 and Rv2958c Regions—Mycobacterial genomic DNA was extracted from each strain of the M. tuberculosis complex as described previously (17). We have previously shown that M. bovis BCG does not synthesize PGL-tb because mycoside B of several genetic defects. This observation sugtotal of 2500 shots were accumulated in positive ion mode, and gested that the RD4 might be a second genetic defect premass spectrometry data were acquired using the instrument venting the formation of PGL-tb in some M. bovis-related default calibration. The PGL proby using chloroform signal as an internal reference (7.23 ppm). duced by the various recombinant BCG strains were ana-
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