Glycopeptidolipids Research Articles

Inactivation of MSMEG_0412 gene drastically affects surface related properties of Mycobacterium smegmatis.

BackgroundThe outermost layer of mycobacterial cell wall is rich in lipids and glycolipids, surface molecules which differ among species. Mycobacterium smegmatis, an attractive model for the study of both pathogenic and non-pathogenic mycobacteria, presents glycopeptidolipids (GPLs). All the genes necessary for the biosynthesis of such molecules are clustered in a single region of 65 kb and among them, the msmeg_0412 gene has not been characterized yet. Here we report the isolation and subsequent analysis of a MSMEG_0412 null mutant strain.ResultsThe inactivation of the msmeg_0412 gene had a drastic impact on bacterial surface properties which resulted in the lack of sliding motility, altered biofilm formation and enhanced drug susceptibility. The GPLs analysis showed that the observed mutant phenotype was due to GPLs deficiencies on the mycobacterial cell wall. In addition, we report that the expression of the gene is enhanced in the presence of lipidic substrates and that the encoded protein has a membrane localization.Conclusion msmeg_0412 plays a crucial role for GPLs production and translocation on M. smegmatis surface. Its deletion alters the surface properties and the antibiotic permeability of the mycobacterial cell barrier.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0888-z) contains supplementary material, which is available to authorized users.

The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

Structural insights into the regulation of NADPH binding to reductase domains of nonribosomal peptide synthetases: A concerted loop movement model.

The termination module of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) offloads the final product as an acid (occasionally also accompanied by cyclization) upon hydrolysis by employing thioesterase domains (TE-domains). Reductase domains (R-domains) of short-chain dehydrogenase/reductase (SDR) family offer an alternative offloading mechanism by reducing 4'-phosphopantetheine (4'-PPant) arm-tethered peptidyl chain, a thioester, to an aldehyde or an alcohol. Recent studies have highlighted their functional importance, for instance in the glycopeptidolipid (GPL) biosynthesis of Mycobacterium smegmatis, where the resulting alcoholic group is the site for subsequent modifications such as glycosylations. The mechanistic understanding of how these R-domains function in the context of multi-modular NRPS and PKS is poorly understood. In this study, conformational differences in functionally important loops, not reported previously, were identified in a new crystal form of R-domain which may be relevant to functioning in the context of assembly-line NRPS and PKS enzymology. Here, we propose a concerted loop movement model that allows gating of cofactor binding to these enzymes, enabling the release of the final product only after the substrate has reached the active site during biosynthesis, and therefore distinct from a canonical single domain SDR family of enzymes.

Mycobacterial glycopeptidolipids (GPL) are transported by the MmpL4a membrane protein. Genetic studies have identified critical residues within the transmembrane segments of MmpL proteins postulated to be couple the proton motive force (PMF) to MmpL transport activity. For details, see the article by Bernut et al. on pp. 866–883 of this issue

Mycobacterial glycopeptidolipids (GPL) are transported by the MmpL4a membrane protein. Genetic studies have identified critical residues within the transmembrane segments of MmpL proteins postulated to be couple the proton motive force (PMF) to MmpL transport activity. For details, see the article by Bernut <i>et al</i>. on pp. 866–883 of this issue

Mechanistic insight into mycobacterial MmpL protein function.

Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. () present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance-nodulation-cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth-to-rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters.

Diagnostic performance of measuring antibodies to the glycopeptidolipid core antigen specific to Mycobacterium avium complex in patients with rheumatoid arthritis: results from a cross-sectional observational study.

IntroductionThe aim of this study was to investigate the diagnostic performance of measuring antibodies to the glycopeptidolipid (GPL) core antigen specific to Mycobacterium avium complex (MAC) in patients with rheumatoid arthritis (RA).MethodsWe cross-sectionally investigated anti-GPL antibodies and radiographs of 396 patients with RA. A diagnosis of MAC pulmonary disease (MAC-PD) was made according to the criteria by the American Thoracic Society and the Infectious Diseases Society of America. Serum immunoglobulin A antibodies to MAC-specific GPL core antigen were measured by an enzyme immunoassay. All patients with RA with abnormal shadows on chest x-rays underwent chest computed tomography (CT). Bronchoscopy was performed on patients with negative cultures for MAC by expectorated sputum and positive CT findings compatible with MAC-PD.ResultsTen patients were newly diagnosed with MAC-PD. Eight individuals who already had diagnoses of MAC-PD at the time of enrollment and nineteen who had negative expectorated sputum cultures for MAC and positive CT images compatible with MAC-PD and who refused bronchoscopy were excluded from the following analysis. Anti-GPL antibodies were detected in 12 of 369 patients. Eight of the ten patients with MAC-PD and 4 of 359 patients without MAC-PD tested positive for the anti-GPL antibodies. The specificity and sensitivity were 99 % and 80 %, respectively. Positive and negative predictive values were 67 %, and 97 %, respectively. When we analyzed diagnostic performance of the antibodies in 57 patients with RA who had abnormal shadows on chest x-rays, the positive and negative predictive values were 100 %, and 96 %, respectively. Twelve patients underwent bronchoscopy. Bronchoalveolar lavage fluid (BALF) samples from six patients were positive for MAC, and BALF samples from the remainder were negative. Anti-GPL antibodies were detected in the sera of all six patients with positive results for MAC by BALF sampling, whereas the antibodies were not detected in the sera from the remainder with negative results for MAC by BALF sampling.ConclusionsThe measurement of anti-GPL antibodies is useful as a supplementary diagnostic tool for MAC-PD in patients with RA and may provide a new strategy, in combination with chest x-ray and CT, for differentiating MAC-PD from other pulmonary comorbidities in patients with RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0787-y) contains supplementary material, which is available to authorized users.

Mycobacterium massiliense Type II genotype leads to higher level of colony forming units and TNF-α secretion from human monocytes than Type I genotype.

Recently, we introduced a novel Mycobacterium massiliense Type II genotype from Korean patients, in which all isolates showed only a rough (R) colony morphotype. In this study, we sought to compare clinical factors and virulence potentials of two genotypes of M.massiliense, Type I and Type II. Patients infected with Type II tend to be younger at infection than those infected with Type I (56.7 vs 62.3, p=0.051). Type II was more significantly related to R colony type than Type I (34.1% vs 94.1%, p<0.001). The Type II strain showed significantly more colony forming units (CFUs) and higher levels of TNF-α secretion in infection of human monocytes than the Type I strain. The challenge of extracted glycopeptidolipid (GPL) into human monocytes indicated that the loss of GPL from the cell wall of the Type II genotype led to a higher level of TNF-α secretion in a toll-like receptor 2(TLR2)-dependent manner. Taken together, our data suggest that the M.massiliense Type II genotype shows higher virulence than Type I, which may be due to the induction of TNF-α via the loss of GPL from the Type II cell wall.

Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates.

The smooth-to-rough colony morphology shift in Mycobacterium abscessus has been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistent M. abscessus infection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level of fadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed as M. abscessus subspecies abscessus and were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations in mps1 and mps2, 13% displayed previously unreported mutations in mmpL4a and mmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression of fadD23, sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection with M. abscessus. GPL loss alone may not account for all traits associated with rough morphology.

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells.

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc2155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc2155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc2155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

Novel functions of (p)ppGpp and Cyclic di-GMP in mycobacterial physiology revealed by phenotype microarray analysis of wild-type and isogenic strains of Mycobacterium smegmatis.

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (ΔrelMsm) or c-di-GMP synthesis (ΔdcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the ΔrelMsm and ΔdcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Sliding Motility, Biofilm Formation, and Glycopeptidolipid Production in Mycobacterium colombiense Strains.

Mycobacterium colombiense is a novel member of the Mycobacterium avium complex, which produces respiratory and disseminated infections in immunosuppressed patients. Currently, the morphological and genetic bases underlying the phenotypic features of M. colombiense strains remain unknown. In the present study, we demonstrated that M. colombiense strains displaying smooth morphology show increased biofilm formation on hydrophobic surfaces and sliding on motility plates. Thin-layer chromatography experiments showed that M. colombiense strains displaying smooth colonies produce large amounts of glycolipids with a chromatographic behaviour similar to that of the glycopeptidolipids (GPLs) of M. avium. Conversely, we observed a natural rough variant of M. colombiense (57B strain) lacking pigmentation and exhibiting impaired sliding, biofilm formation, and GPL production. Bioinformatics analyses revealed a gene cluster that is likely involved in GPL biosynthesis in M. colombiense CECT 3035. RT-qPCR experiments showed that motile culture conditions activate the transcription of genes possibly involved in key enzymatic activities of GPL biosynthesis.

Serodiagnosis of Mycobacterium avium complex disease in humans: translational research from basic mycobacteriology to clinical medicine.

Rapid and accurate diagnosis of infectious diseases, including mycobacterial disease such as tuberculosis (TB) and diseases due to nontuberculous mycobacteria (NTM), is a very important element of global health. The gold standard in diagnosis of mycobacterial diseases remains clinical examination, combined with direct microscopic examination of sputum and culture of bacteria. Culture of slowly growing mycobacteria, including Mycobacterium tuberculosis and NTM (such as M. avium complex: MAC), can take up to 4 to 6 weeks, and in 10-20% of cases the bacillus is not successfully cultivated. Diagnosis of MAC pulmonary disease (MAC-PD) is complicated and time-consuming (usually at least 1 month). I have characterized the nature of MAC antigens and immune responses from the aspect of basic mycobacteriology, and then translated to clinical science. My multicenter study in Japan has demonstrated the usefulness of a serodiagnostic test to determine serum IgA antibodies against mycobacterial glycopeptidolipid (GPL) core antigen for diagnosing MAC-PD within a few hours. To validate in a larger number of patients, at diverse geographic locations, and among other races, the test was also assessed the usefulness internationally in the United States and Taiwan. In this review, I discuss development of serodiagnosis of MAC-PD by translational research and international collaboration study.

Sero-Diagnosis of Mycobacterium avium Complex Lung Disease Using Serum Immunoglobulin A Antibody against Glycopeptidolipid Antigen in Taiwan

BackgroundLung disease (LD) due to non-tuberculous mycobacteria is an important clinical concern. Mycobacterium avium complex (MAC) is one of the most common causative agents but the diagnosis of MAC-LD remains challenging. Detection of serum IgA antibody against MAC glycopeptidolipid (GPL) has recently been shown to improve the diagnosis of MAC-LD, but has yet to be validated worldwide.MethodsThis prospective study was conducted in a tertiary referral center in northern Taiwan and enrolled patients with MAC-LD, MAC contamination, other lung diseases, and control subjects. Serum immunoglobulin A (IgA) antibody against MAC-GPL was detected in the participants and its specificity and sensitivity was assessed.ResultsThere were 56 patients with MAC-LD, 11 with MAC contamination, 13 M. kansasii-LD, 26 LD due to rapidly-growing mycobacteria (RGM), 48 pulmonary tuberculosis, and 42 household contacts of patients with TB. Patients with MAC-LD were older and 32% of them had an underlying co-morbidity. By logistic regression, serum MAC-GPL IgA level was an independent predictor of MAC-LD among the study subjects and those with culture-positive specimens for MAC. By the receiver operating characteristic curve, serum MAC-GPL IgA had a good power to discriminate MAC-LD from MAC contamination. Under the optimal cut-off value of 0.73 U/mL, its sensitivity and specificity were 60% and 91%, respectively. Among MAC-LD patients, presence of co-morbidity was associated with MAC-GPL <0.73 U/ml in logistic regression analysis.ConclusionsMeasurement of serum anti-MAC-GPL IgA level is useful for the diagnosis of MAC-LD. However, its implement in clinical practice for immuno-compromised hosts needs careful consideration.

Identification and characterization of the genetic changes responsible for the characteristic smooth‐to‐rough morphotype alterations of clinically persistent Mycobacterium abscessus

Mycobacterium abscessus is an emerging pathogen that is increasingly recognized as a relevant cause of human lung infection in cystic fibrosis patients. This highly antibiotic-resistant mycobacterium is an exception within the rapidly growing mycobacteria, which are mainly saprophytic and non-pathogenic organisms. M. abscessus manifests as either a smooth (S) or a rough (R) colony morphotype, which is of clinical importance as R morphotypes are associated with more severe and persistent infections. To better understand the molecular mechanisms behind the S/R alterations, we analysed S and R variants of three isogenic M. abscessus S/R pairs using an unbiased approach involving genome and transcriptome analyses, transcriptional fusions and integrating constructs. This revealed different small insertions, deletions (indels) or single nucleotide polymorphisms within the non-ribosomal peptide synthase gene cluster mps1-mps2-gap or mmpl4b in the three R variants, consistent with the transcriptional differences identified within this genomic locus that is implicated in the synthesis and transport of Glyco-Peptido-Lipids (GPL). In contrast to previous reports, the identification of clearly defined genetic lesions responsible for the loss of GPL-production or transport makes a frequent switching back-and-forth between smooth and rough morphologies in M. abscessus highly unlikely, which is important for our understanding of persistent M. abscessus infections.

Structure and function of mycobacterium glycopeptidolipids from comparative genomics perspective

Glycopeptidolipids (GPLs) attached to the outer surface of the greasy cell envelope, are a class of important glycolipids synthesized by several non-tuberculosis mycobacteria. The deletion or structure change of GPLs confers several phenotypical changes including colony morphology, hydrophobicity, aggregation, sliding motility, and biofilm formation. In addition, GPLs, particular serovar specific GPLs, are important immunomodulators. This review aims to summarize the advance on the structure, function and biosynthesis of mycobacterium GPLs.

Serological assay by use of glycopeptidolipid core antigen for Mycobacterium avium complex

We evaluated the clinical usefulness of glycopeptidolipid (GPL) core antigen for diagnosing Mycobacterium avium complex (MAC) pulmonary disease (MAC-PD), including patients with clinically suspected MAC-PD. GPL core antibody levels were measured in 57 patients with MAC-PD satisfying the American Thoracic Society (ATS) criteria for pulmonary disease due to non-tuberculous mycobacteria (NTM), and in 18 patients with clinically suspected MAC-PD, 10 with MAC contamination, 18 with pulmonary tuberculosis (TB), 9 with other NTM disease, 18 with other lung diseases, and 20 healthy subjects. The positive response rate was 77% for MAC-PD, 39% for suspected MAC-PD, 10% for MAC contamination, and 0% for pulmonary TB, other NTM diseases, other lung diseases, and healthy subjects. GPL core antibody levels were significantly higher in patients with MAC-PD than in those of the other groups (p < 0.01). The sensitivity and specificity of the GPL core antibody assay for MAC were 77% and 99%, respectively. Among 13 patients with MAC-PD who showed false-negative results, 5 had immunosuppressive underlying diseases. No significant correlations between the antibody level and species of MAC, clinical disease types, and extent of the disease on chest computed tomography were found in patients with MAC-PD. This enzyme immunoassay kit is a useful supportive method for the rapid and convenient diagnosis of MAC-PD using a small dose of serum, and for the differentiation of MAC-PD from other lung diseases. However, underlying diseases need to be considered in the interpretation of negative results.

Rough colony morphology of Mycobacterium massiliense Type II genotype is due to the deletion of glycopeptidolipid locus within its genome

BackgroundRecently, we introduced the complete genome sequence of Mycobacterium massiliense clinical isolates, Asan 50594 belonging to Type II genotype with rough colony morphology. Here, to address the issue of whether the rough colony morphotype of M. massiliense Type II genotype is genetically determined or not, we compared polymorphisms of the glycopeptidolipid (GPL) gene locus between M. massiliense Type II Asan 50594 and other rapidly growing mycobacteria (RGM) strains via analysis of genome databases.ResultsWe found deletions of 10 genes (24.8 kb), in the GPL biosynthesis related gene cluster of Asan 50594 genome, but no deletions in those of other smooth RGMs. To check the presence of deletions of GPL biosynthesis related genes in Mycobacterium abscessus − complex strains, PCRs targeting 12 different GPL genes (10 genes deleted in Asan 50594 genome as well as 2 conserved genes) were applied into 76 clinical strains of the M. abscessus complex strains [54 strains (Type I: 33, and Type II: 21) of M. massiliense and 22 strains (rough morphoype: 11 and smooth morphotype: 11) of M. abscessus]. No strains of the Type II genotype produced PCR amplicons in a total of 10 deleted GPL genes, suggesting loss of GPL biosynthesis genes in the genome of M. massiliense type II genotype strains.ConclusionsOur data suggested that the rough colony morphotype of the M. massiliense Type II genotype may be acquired via deletion events at the GPL gene locus for evolutionary adaptation between the host and pathogen.

Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high‐resolution multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

Glycopeptidolipids (GPLs) are abundant in the cell walls of different species of mycobacteria and consist of tripeptide-amino-alcohol core of D-Phe-D-allo-Thr-D-Ala-L-alaninol linked to 3-hydroxy or 3-methoxy C(26-34) fatty acyl chain at the N-terminal of D-Phe via amide linkage, and a 6-deoxytalose (6-dTal) and an O-methyl rhamnose residues, respectively, attach to D-allo-Thr and the terminal L-alaninol. They are important cell-surface antigens that are implicated in the pathogenesis of opportunistic mycobacteria belonging to the Mycobacterium avium complex. In this contribution, we described multiple-stage linear ion trap in conjunction with high-resolution mass spectrometry towards structural characterization of complex GPLs as [M + Na](+) ions isolated from Mycobacterium smegmatis, a fast-growing and non-pathogenic mycobacterial species. Following resonance excitation in an ion trap, MS(n) spectra of the [M + Na](+) ions of GPLs contained mainly b and y series ions that readily determine the peptide sequence. Fragment ions from MS(n) also afford locating the 6-dTal and O-methyl rhamnose residues linked to the D-allo-Thr and terminal L-alaninol of the peptide core, respectively, as well as recognizing the modifications of the glycosides, including their acetylation and methylation states and the presence of succinyl group. The GPL families consisting of 3-hydroxy fatty acyl and of 3-methoxy fatty acyl substituents are readily distinguishable. The MS profiles of the GPLs from cells are dependant on the conditions they were grown, and several isobaric isomers were identified for many of the molecular species. These multiple-stage mass spectrometric approaches give detailed structures of GPL in complex mixtures of which the isomeric structures are difficult to define using other analytical methods.

Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages.

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.

Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C(26)-C(34) fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs.