| Abstract: | Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.The genus Mycobacterium has a unique feature in the cell envelope that contains a multilayered structure consisting of peptidoglycan, mycolyl-arabinogalactan complex, and surface glycolipids (8, 12). It is known that these components play a role in protection from environmental stresses, such as antimicrobial agents and host immune responses (8, 12). Some of them are recognized as pathogenic factors related to mycobacterial diseases, such as tuberculosis and leprosy (8, 12). In case of nontuberculous mycobacteria that are widely distributed in the natural environment as opportunistic pathogens, glycopeptidolipids (GPLs) are abundantly present on the cell envelope as surface glycolipids (34). GPLs have a core structure in which a fatty acyl-tetrapeptide is glycosylated with 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha) (2, 5, 13). This structure is common to all types of GPLs, and GPLs with this structure that have not undergone further glycosylation are termed non-serovar-specific GPLs (nsGPLs) (2, 5, 13). Structural diversity generated by further glycosylations, such as rhamnosylation, fucosylation, and glucosylation, is observed for the oligosaccharide portion linked to the 6-d-Tal residue of nsGPLs from Mycobacterium avium complex (MAC), a member of the nontuberculous mycobacteria consisting of two species, M. avium and M. intracellulare (2, 5, 34). Consequently, these nsGPLs with varied oligosaccharides lead to the formation of the serovar-specific GPLs (ssGPLs) that define around 30 types of MAC serovars (10).The properties of MAC serovars are known to be notably different from each other and also to be closely associated with the pathogenicity of MAC (3, 6, 18, 30, 31, 32). Various epidemiological studies indicate that serovar 4 is the most prevalent type and is also one of the serovars frequently isolated from AIDS patients (1, 20, 33, 36). Additionally, pulmonary MAC disease caused by serovar 4 is shown to exhibit a poorer prognosis than that caused by other serovars (23). With respect to host immune responses to MAC infection, serovar 4-specific GPL is reported to have characteristic features that are in contrast to those of other ssGPLs (21, 30). Structurally, serovar 4-specific GPL contains a unique oligosaccharide in which the oligosaccharide of serovar 2-specific GPL is further glycosylated with 4-O-methyl-rhamnose (4-O-Me-Rha) residue through a 1→4 linkage (Table ) (24). Therefore, it is thought that the presence of 4-O-Me-Rha and its linkage position are important in exhibiting the specificity of biological activities. The biosynthesis of the oligosaccharide portion in several ssGPLs is currently being clarified (15, 16, 17, 25, 26), while that of serovar 4-specific GPL is still unresolved. In this study, we have focused on the genomic region predicted to be associated with GPL biosynthesis in the serovar 4 strain and explored the key genes responsible for the formation of 4-O-Me-Rha that might determine the specific properties of MAC serovar 4.TABLE 1.Oligosaccharide structures of serovar 2- and 4-specific GPLs| Serovar | Oligosaccharide | Reference |
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| 2 | 2,3-di-O-Me-α-l-Fuc-(1→3)-α-l-Rha-(1→2)-l-6-d-Tal | 9 | | 4 | 4-O-Me-α-l-Rha-(1→4)-2-O-Me-α-l-Fuc-(1→3)-α-l-Rha-(1→2)-l-6-d-Tal | 24 |
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