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Identification and characterization of two novel methyltransferase genes that determine the serotype 12-specific structure of glycopeptidolipids of Mycobacterium intracellulare.

ABSTRACT: The Mycobacterium avium complex is distributed ubiquitously in the environment. It is an important cause of pulmonary and extrapulmonary diseases in humans and animals. The species in this complex produce polar glycopeptidolipids (GPLs); of particular interest is their serotype-specific antigenicity. Several reports have described that GPL structure may play an important role in bacterial physiology and pathogenesis and in the host immune response. Recently, we determined the complete structure of the GPL derived from Mycobacterium intracellulare serotype 7 and characterized the serotype 7 GPL-specific gene cluster. The structure of serotype 7 GPL closely resembles that of serotype 12 GPL, except for O methylation. In the present study, we isolated and characterized the serotype 12-specific gene cluster involved in glycosylation of the GPL. Ten open reading frames (ORFs) and one pseudogene were observed in the cluster. The genetic organization of the serotype 12-specific gene cluster resembles that of the serotype 7-specific gene cluster, but two novel ORFs (orfA and orfB) encoding putative methyltransferases are present in the cluster. Functional analyses revealed that orfA and orfB encode methyltransferases that synthesize O-methyl groups at the C-4 position in the rhamnose residue next to the terminal hexose and at the C-3 position in the terminal hexose, respectively. Our results show that these two methyltransferase genes determine the structural difference of serotype 12-specific GPL from serotype 7-specific GPL.

SUBMITTER: Nakata N

PROVIDER: S-EPMC2223587 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Identification and characterization of two novel methyltransferase genes that determine the serotype 12-specific structure of glycopeptidolipids of Mycobacterium intracellulare.

Nakata Noboru N Fujiwara Nagatoshi N Naka Takashi T Yano Ikuya I Kobayashi Kazuo K Maeda Shinji S

Journal of bacteriology 20071116 3

The Mycobacterium avium complex is distributed ubiquitously in the environment. It is an important cause of pulmonary and extrapulmonary diseases in humans and animals. The species in this complex produce polar glycopeptidolipids (GPLs); of particular interest is their serotype-specific antigenicity. Several reports have described that GPL structure may play an important role in bacterial physiology and pathogenesis and in the host immune response. Recently, we determined the complete structure ...[more]

PMID: 18024513

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Project description:BackgroundMycobacterium intracellulare is a representative etiological agent of emerging pulmonary M. avium-intracellulare complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary M. avium-intracellulare complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for M. avium; however, the genomic characteristics of M. intracellulare remain to be elucidated.ResultsIn this study, we performed comparative genomic analysis of 55 M. intracellulare and related strains such as M. paraintracellulare (MP), M. indicus pranii (MIP) and M. yonogonense. Based on the average nucleotide identity, the clinical M. intracellulare strains were phylogenetically grouped in two clusters: (1) the typical M. intracellulare (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., fadE3, fadE33), transporters (e.g., mce3), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. M. intracellulare was shown to be pan-genomic at the species and subspecies levels. The mce genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies.ConclusionsOur data suggest that genomic diversity among M. intracellulare, M. paraintracellulare, M. indicus pranii and M. yonogonense remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as mce genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for M. intracellulare and related strains.

Project description:During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (between d-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The dd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used by ld-transpeptidases as substrates to form 3-3 cross-links. Therefore, dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of dd-CPases in mycobacteria is relatively unexplored. In this study, we deleted two dd-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single dd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz, a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the dd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCE The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including Mycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.

Dataset Information

Identification and characterization of two novel methyltransferase genes that determine the serotype 12-specific structure of glycopeptidolipids of Mycobacterium intracellulare.

Publications

Identification and characterization of two novel methyltransferase genes that determine the serotype 12-specific structure of glycopeptidolipids of Mycobacterium intracellulare.

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