Isolation and expression of a gene cluster responsible for biosynthesis of the glycopeptidolipid antigens of Mycobacterium avium

Abstract

Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS. Serovars of the M. avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces. A genomic library of DNA from serovar 2 of the M. avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M. avium serovar 2-specific glycopeptidolipid. The responsible gene cluster was mapped to a 22-to 27-kb functional region of the M. avium genome. The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M. smegmatis, whereas the oligosaccharide segment arose from the cloned M. avium genes. This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes.

The impact of the absence of glycopeptidolipids on the ultrastructure, cell surface and cell wall properties, and phagocytosis of Mycobacterium smegmatis

“…Compared to the wild-type smooth morphotype strain, the C-type-GPL-deficient mutant strains TM99, 9-1045 and 8-245 exhibited a rough colony morphology (this work, Recht et al, 2000). This observation was in agreement with previous studies on GPL-deficient M. avium-intracellulare (Barrow & Brennan, 1982 ;Belisle et al, 1991). The absence of C-type GPLs also affects the surface properties of strain TM99.…”

“…Strain TM99 displayed a rough colony morphology, compared to the smooth aspect of the mc#155 parent strain (Fig. 2) ; these data were consistent with previous observations that showed a correlation between the absence of C-type GPL and the rough morphotype (Barrow & Brennan, 1982 ;Belisle et al, 1991 ;Recht et al, 2000).…”

Synthesis of an unusual polar glycopeptidolipid in glucose-limited culture of Mycobacterium smegmatis

“…However, M. smegmatis, a fast-growing non-pathogenic species, has been reported to produce only the apolar nsGPL, structurally similar to those produced by M. avium (Billman-Jacobe et al, 1999 ;Daffe et al, 1983 ;Patterson al., 2000). Based on the fact that M. smegmatis does not produce multiglycosylated polar GPLs, it has been genetically exploited as a surrogate host for identification of M. avium genes encoding specific glycosyltransferases, which add sugars to 6-dTal (Belisle et al, 1991(Belisle et al, , 1993Eckstein et al, 1998). Through this strategy a ser2 locus was identified in M. avium that had genes encoding rhamnosyl transferase and fucosyl transferase (Belisle et al, 1991).…”

“…Based on the fact that M. smegmatis does not produce multiglycosylated polar GPLs, it has been genetically exploited as a surrogate host for identification of M. avium genes encoding specific glycosyltransferases, which add sugars to 6-dTal (Belisle et al, 1991(Belisle et al, , 1993Eckstein et al, 1998). Through this strategy a ser2 locus was identified in M. avium that had genes encoding rhamnosyl transferase and fucosyl transferase (Belisle et al, 1991). In this study we report a conditional synthesis of a polar GPL in glucose-limited culture of M. smegmatis.…”

Modification of glycopeptidolipids by an O-methyltransferase of Mycobacterium smegmatis a aThe GenBank accession number for the sequence determined in this work is AY138899.

“…The GPLs of M. smegmatis, which are not glycosylated at 6-dTal, are non-serovar-specific GPLs and represent the biosynthetic precursor of the ssGPLs of M. avium (Eckstein et al, 1998). The M. avium genomic region encoding glycosylation of serovar-2-specific GPLs has been identified (Belisle et al, 1991(Belisle et al, , 1993Mills et al, 1994). Deletion of the ser2 gene cluster results in GPL-deficient mutants that have rough colony morphology (Eckstein et al, 2000 D. Jeevarajah and others and a potential transporter protein (TmptC) (BillmanJacobe et al, 1999 ;Patterson et al, 2000 ;Recht & Kolter, 2001 ;Recht et al, 2000).…”