Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled iNKT cell gene expression during ontogeny and in peripheral subsets as part of the Immunological Genome Project (ImmGen). High-resolution comparative transcriptional analyses defined developmental and subset-specific iNKT cell gene expression programs. In addition, iNKT cells were found to share an extensive transcriptional program with natural killer (NK) cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Strikingly, the NK- iNKT program also operated constitutively in γδT cells and in adaptive T cells following activation. Together, our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.
Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.
CD1b; lipoarabinomannan; lipoglycans; Mycobacterium tuberculosis; T cells
The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of Mycobacterium leprae-specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae-specific proteins called “hypothetical unknowns.” Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. In this study, we performed cDNA-based quantitative real-time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty-six of the M. leprae-specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049), which is an important T cell antigen of low abundance in M. leprae. Fifteen of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune responses. These nine proteins may be good diagnostic reagents to improve both the sensitivity and specificity of detection of individuals with asymptomatic leprosy.
Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.
Invariant natural killer T cells (iNKT cells) play a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations where no foreign lipid antigens are present, suggesting a role for lipid self-antigen. We now demonstrate that an abundant endogenous lipid, β-D-glucopyranosylceramide (β-GlcCer), is a potent iNKT cell self-antigen in mouse and human, and that its activity depends on N-acyl chain composition. Furthermore, β-GlcCer accumulates during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of β-GlcCer by the invariant TCR translates innate danger signals into iNKT cell activation.
Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.
The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.
UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short “dead-end” intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
Hospital-based comparative effectiveness (CE) centers provide a model that clinical leaders can use to improve evidence-based practice locally. The model is used by integrated health systems outside the US, but is less recognized in the US. Such centers can identify and adapt national evidence-based policies for the local setting, create local evidence-based policies in the absence of national policies, and implement evidence into practice through health information technology (HIT) and quality initiatives. Given the increasing availability of CE evidence and incentives to meaningfully use HIT, the relevance of this model to US practitioners is increasing. This is especially true in the context of healthcare reform, which will likely reduce reimbursements for care deemed unnecessary by published evidence or guidelines. There are challenges to operating hospital-based CE centers, but many of these challenges can be overcome using solutions developed by those currently leading such centers. In conclusion, these centers have the potential to improve the quality, safety and value of care locally, ultimately translating into higher quality and more cost-effective care nationally. To better understand this potential, the current activity and impact of hospital-based CE centers in the US should be rigorously examined.
comparative effectiveness; evidence-based medicine; health technology assessment; quality of health care; cost-effectiveness; health information technology; organizational decision making
In order to generate substantial amounts of neoglycoconjugate needed for commercialization of diagnostic kits and high-throughput detection of leprosy, we developed a facile and high-yield synthesis of the corresponding disaccharide. Herein, the non-reducing disaccharide segment of phenolic glycolipid I from Mycobacterium leprae, O-(3,6-di-O-methyl-β-D-glucopyranosyl)-(1→4)-O-2,3-di-O-methyl-α-L-rhamnopyranose was synthesized by an improved procedure. The disaccharide was efficiently conjugated to bovine/human serum albumin, via acyl-azide intermediate, to form natural disaccharide-BSA/HSA neoglycoproteins that showed a high activity in serodiagnosis of leprosy. The disaccharide incorporated into the proteins was accurately measured by MALDI-ToF mass spectrometry. The serological activities of the neoglycoproteins against pooled human lepromatous leprosy were measured by ELISA and they were detectable at picogram amount.
The reemergence of tuberculosis in its present-day manifectations – single, multiple and extensive drug resistant forms and as HIV-TB coinfections – has resulted in renewed research on fundamental questions such as the nature of the organism itself, Mycobacterium tuberculosis, the molecular basis of its pathogenesis, definition of the immunological response in animal models and humans, and development of new intervention strategies such as vaccines and drugs. Foremost among these developments has been the precise chemical definition of the complex and distinctive cell wall of M. tuberculosis, elucidation of the relevant pathways and underlying genetics responsible for the synthesis of the hallmark moities of the tubercle bacillus such as the mycolic acid-arabinogalactan-peptidoglycan complex, the phthiocerol- and trehalose-containing effector lipids, the phosphatidylinositol-containing mannosides, lipomannosides and lipoarabinomannosides, major immunomodulators, and others. In this review, the laboratory personnel that have been the focal point of some to these developments review recent progress towards a comprehensive understanding of the basic physiology and functions of the cell wall of M. tuberculosis.
Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Since there is no ortholog of IspF in human cells IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.
Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of α-1,5-linked d-Araf residues with some α-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with α-1,3-branching activity on linear α-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.
arabinogalactan; arabinosyltransferase; lipoarabinomannan; Mycobacterium; tuberculosis
We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phospho-mannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.
To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.
During the last decade, the combination of rapid whole genome sequencing capabilities, application of genetic and computational tools, and establishment of model systems for the study of a range of species for a spectrum of biological questions has enhanced our cumulative knowledge of mycobacteria in terms of their growth properties and requirements. The adaption of the corynebacterial surrogate system has simplified the study of cell wall biosynthetic machinery common to actinobacteria. Comparative genomics supported by experimentation reveals that superimposed on a common core of ‘mycobacterial’ gene set, pathogenic mycobacteria are endowed with multiple copies of several protein families that encode novel secretion and transport systems such as mce and esx; immunomodulators named PE/PPE proteins, and polyketide synthases for synthesis of complex lipids. The precise timing of expression, engagement and interactions involving one or more of these redundant proteins in their host environments likely play a role in the definition and differentiation of species and their disease phenotypes. Besides these, only a few species specific ‘virulence’ factors i.e., macromolecules have been discovered. Other subtleties may also arise from modifications of shared macromolecules. In contrast, to cope with the broad and changing growth conditions, their saprophytic relatives have larger genomes, in which the excess coding capacity is dedicated to transcriptional regulators, transporters for nutrients and toxic metabolites, biosynthesis of secondary metabolites and catabolic pathways. In this review, we present a sampling of the tools and techniques that are being implemented to tease apart aspects of physiology, phylogeny, ecology and pathology and illustrate the dominant genomic characteristics of representative species. The investigation of clinical isolates, natural disease states and discovery of new diagnostics, vaccines and drugs for existing and emerging mycobacterial diseases, particularly for multidrug resistant strains are the challenges in the coming decades.
Genomics; Evolution; Mycobacteria; Virulence; COGs
Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensive drug resistant (XDR)-TB. Novel anti-TB drugs are urgently required. In this context, the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of Mycobacterium tuberculosis has drawn attention; it is one of several pathways vital for M. tuberculosis viability and the human host lacks homologous enzymes. Thus, the MEP pathway promises bacterium-specific drug targets and the potential for identification of lead compounds unencumbered by target-based toxicity. Indeed, fosmidomycin is now known to inhibit the second step in the MEP pathway. This review describes the cardinal features of the main enzymes of the MEP pathway in M. tuberculosis and how these can be manipulated in high throughput screening campaigns in the search for new anti-infectives against TB.
Tuberculosis; 2C-methyl-D-erythritol 4-phosphate pathway; high throughput screening campaigns; anti-infectives
Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.
Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in Mycobacterium spp. Here, the crystallization and preliminary crystallographic analysis of GpgS from M. tuberculosis and of its complex with UDP are reported.
Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty-acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active Enzymes Database; see http://www.cazy.org). Here, the purification, crystallization and preliminary crystallographic analysis are reported of GpgS from Mycobacterium tuberculosis and of its complex with UDP. GpgS crystals belonged to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c = 127.64 Å, and diffracted to 2.6 Å resolution. GpgS–UDP complex crystals belonged to space group I4, with unit-cell parameters a = 98.32, b = 98.32, c = 127.96 Å, and diffracted to 3.0 Å resolution.
glycosyltransferases; methylglucose lipopolysaccharides; Mycobacterium tuberculosis
Synopsis Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in Mycobacterium spp. Here we report the crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP at 2.6 Å and 3.0 Å resolution, respectively.
Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active enZymes data base; see www.cazy.org). Here we report the purification, crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP. GpgS crystals belong to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c= 127.64 Å, and diffract to 2.6 Å resolution. GpgS-UDP complex crystals belong to space group I4 with unit-cell parameters a= 98.32, b= 98.32, c= 127.96 Å, and diffract to 3.0 Å resolution.
glycosyltransferase; methylglucose lipopolysaccharides; Mycobacterium; X ray structure
Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides - the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) - the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.
We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.
This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.
Comparative genomics analysis of the Tamil Nadu strain of Mycobacterium leprae has uncovered several polymorphic sites with potential as epidemiological tools. In this study we compared the stability of two different markers of genomic biodiversity of M. leprae in several biopsy samples isolated from the same leprosy patient. The first type comprises five different variable-number tandem repeats (VNTR), while the second is composed of three single nucleotide polymorphisms (SNP). Contrasting results were obtained, since no variation was seen in the SNP profiles of M. leprae from 42 patients from 7 different locations in Mali whereas the VNTR profiles varied considerably. Furthermore, since variation in the VNTR pattern was seen not only between different isolates of M. leprae but also between biopsy samples from the same patient, these VNTR may be too dynamic for use as epidemiological markers for leprosy.
Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2′-methyl-3′-hydroxy-4′-methoxy-pentanoyl-amido-3,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→2)-6-deoxy-α-l-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.
Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual β-(1→4) and β-(1→5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.
The peptidoglycan structure of Mycobacterium spp. has been investigated primarily with the readily cultivable Mycobacterium smegmatis and Mycobacterium tuberculosis and has been shown to contain unusual features, including the occurrence of N-glycolylated, in addition to N-acetylated, muramic acid residues and direct cross-linkage between meso-diaminopimelic acid residues. Based on results from earlier studies, peptidoglycan from in vivo-derived noncultivable Mycobacterium leprae was assumed to possess the basic structural features of peptidoglycans from other mycobacteria, other than the reported replacement of l-alanine by glycine in the peptide side chains. In the present study, we have analyzed the structure of M. leprae peptidoglycan in detail by combined liquid chromatography and mass spectrometry. In contrast to earlier reports, and to the peptidoglycans in M. tuberculosis and M. smegmatis, the muramic acid residues of M. leprae peptidoglycan are exclusively N acetylated. The un-cross-linked peptide side chains of M. leprae consist of tetra- and tripeptides, some of which contain additional glycine residues. Based on these findings and genome comparisons, it can be concluded that the massive genome decay in M. leprae does not markedly affect the peptidoglycan biosynthesis pathway, with the exception of the nonfunctional namH gene responsible for N-glycolylmuramic acid biosynthesis.