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Intracellular pathogens represent a challenge for therapy because the antibiotics used need to diffuse into the cytoplasm to target the pathogens. The situation is more complicated in the mycobacteria family because members of this family infect and multiply within macrophages, the cells responsible for clearing microorganisms in the body. In addition, mycobacteria members are enclosed inside pathogen-containing vesicles or phagosomes. The treatments of these pathogens are aggravated when these pathogens acquire resistance to antibiotic molecules. As a result, new antimicrobial alternatives are needed. Niosomes are vesicles composed of cholesterol and nonionic surfactants that can be used for antibiotic encapsulation and delivery. The current study developed a systematic formulation of niosomes to determine the best option for niosome functionalizing for precise delivery to the intracellular pathogen Mycobacterium abscessus. Silver nanoparticles (AgNPs) were synthesized using gallic acid as an antibacterial agent. Then, niosomes were prepared and characterized, following the encapsulation of AgNPs functionalized with a single-chain antibody screened against the cell wall glycopeptidolipid of Mycobacterium abscessus. For a precise delivery of the cargo into macrophages, the niosomes were also functionalized with the polysaccharide fucoidan, taken specifically by the scavenger receptor class A expressed on the surface of macrophages. Results of the study showed a steady decrease in the intracellular pathogen load after 48 h post-infection. In conclusion, this system could be developed into a platform to target other types of intracellular pathogens and as an option for antimicrobial therapy. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus causes severe lung infections in cystic fibrosis patients and exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in the S-to-R transition but the underlying molecular mechanisms of this transition remain incompletely understood. Herein, we characterized MAB_4111c in relation to GPL synthesis and investigated the effects of MAB_4111c deletion in M. abscessus pathogenicity. An enzymatic assay indicated that MAB_4111c, also designated Tle for Talose epimerase, is converting dTDP-L-Rhamnose into dTDP-6-deoxy-L-Talose. A tle deletion mutant was constructed in the S variant of M. abscessus and relative areas of Rhamnose and 6-deoxy-Talose and their methylated forms expressed as ratios of total monosaccharides, showed an altered GPL profile lacking 6-deoxy-Talose. Thus, Tle provides dTDP-6-deoxy-L-Talose, subsequently used by the glycosyltransferase Gtf1 to transfer 6-deoxy-Talose to the GPL backbone. Strikingly, the tle mutant exhibited an R morphotype, showed impaired sliding motility and biofilm formation, and these phenotypes were rescued upon functional complementation. Moreover, deletion of tle in M. abscessus results in increased pathogenicity and killing in zebrafish embryos. Together, our results underscore the importance of the dTDP-L-Rhamnose 4-epimerase activity in GPL biosynthesis and in influencing M. abscessus virulence., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this review we will focus on unique aspects of Mycobacterium abscessus (MABS) which we feel earn it the designation of “shapeshifter of the mycobacterial world.” We will review its emergence as a distinct species, the recognition and description of MABS subspecies which are only now being clearly defined in terms of pathogenicity, its ability to exist in different forms favoring a saprophytic lifestyle or one more suitable to invasion of mammalian hosts, as well as current challenges in terms of antimicrobial therapy and future directions for research. One can see in the various phases of MABS, a species transitioning from a free living saprophyte to a host-adapted pathogen.

Mycobacterium abscessus is a rapidly-growing species causing a diverse panel of clinical manifestations, ranging from cutaneous infections to severe respiratory disease. Its unique cell wall, contributing largely to drug resistance and to pathogenicity, comprises a vast panoply of complex lipids, among which the glycopeptidolipids (GPLs) have been the focus of intense research. These lipids fulfill various important functions, from sliding motility or biofilm formation to interaction with host cells and intramacrophage trafficking. Being highly immunogenic, the induction of a strong humoral response is likely to select for rough low-GPL producers. These, in contrast to the smooth high-GPL producers, display aggregative properties, which strongly impacts upon intracellular survival. A propensity to grow as extracellular cords allows these low-GPL producing bacilli to escape the innate immune defenses. Transitioning from high-GPL to low-GPL producers implicates mutations within genes involved in biosynthesis or transport of GPL. This leads to induction of an intense pro-inflammatory response and robust and lethal infections in animal models, explaining the presence of rough isolates in patients with decreased pulmonary functions. Herein, we will discuss how, thanks to the generation of defined GPL mutants and the development of appropriate cellular and animal models to study pathogenesis, GPL contribute to M. abscessus biology and physiopathology.

Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective.

Mycobacterium abscessus is a rapidly-growing species causing a diverse panel of clinical manifestations, ranging from cutaneous infections to severe respiratory disease. Its unique cell wall, contributing largely to drug resistance and to pathogenicity, comprises a vast panoply of complex lipids, among which the glycopeptidolipids (GPLs) have been the focus of intense research. These lipids fulfill various important functions, from sliding motility or biofilm formation to interaction with host cells and intramacrophage trafficking. Being highly immunogenic, the induction of a strong humoral response is likely to select for rough low-GPL producers. These, in contrast to the smooth high-GPL producers, display aggregative properties, which strongly impacts upon intracellular survival. A propensity to grow as extracellular cords allows these low-GPL producing bacilli to escape the innate immune defenses. Transitioning from high-GPL to low-GPL producers implicates mutations within genes involved in biosynthesis or transport of GPL. This leads to induction of an intense pro-inflammatory response and robust and lethal infections in animal models, explaining the presence of rough isolates in patients with decreased pulmonary functions. Herein, we will discuss how, thanks to the generation of defined GPL mutants and the development of appropriate cellular and animal models to study pathogenesis, GPL contribute to M. abscessus biology and physiopathology. [ABSTRACT FROM AUTHOR]

Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus is a rapidly growing mycobacterium (RGM) causing serious infections especially among cystic fibrosis patients. Extremely limited therapeutic options against M. abscessus and a rise in infections with this mycobacterium require novel chemotherapies and a better understanding of how the bacterium causes infection. Different from most RGM, M. abscessus can survive inside macrophages and persist for long durations in infected tissues. We recently delineated differences in the infective programs followed by smooth (S) and rough (R) variants of M. abscessus. Unexpectedly, we found that the S variant behaves like pathogenic slow growing mycobacteria, through maintaining a block on the phagosome maturation process and by inducing phagosome-cytosol communications. On the other hand, R variant infection triggers autophagy and apoptosis, reminiscent of the way that macrophages control RGM. However, the R variant has an exquisite capacity to form extracellular cords, allowing these bacteria to rapidly divide and evade phagocytosis. Therefore, new chemotherapeutic interventions against M. abscessus need to efficiently deal with both the reservoir of intracellular bacilli and the extracellular cords. In this context, we recently identified two chemical entities that were very effective against both M. abscessus populations. Although being structurally unrelated these two chemotypes inhibit the activity of the essential mycolic acid transporter, MmpL3. In this Perspective, we aimed to highlight recent insights into how M. abscessus interacts with phagocytic cells and how the inhibition of mycolic acid transport in this pathogenic RGM could be an efficient means to control both intracellular and extracellular populations of the bacterium.

Mycobacterium abscessus is a rapidly growing mycobacterium (RGM) causing serious infections especially among cystic fibrosis patients. Extremely limited therapeutic options against M. abscessus and a rise in infections with this mycobacterium require novel chemotherapies and a better understanding of how the bacterium causes infection. Different from most RGM, M. abscessus can survive inside macrophages and persist for long durations in infected tissues. We recently delineated differences in the infective programs followed by smooth (S) and rough (R) variants of M. abscessus. Unexpectedly, we found that the S variant behaves like pathogenic slow growing mycobacteria, through maintaining a block on the phagosome maturation process and by inducing phagosome-cytosol communications. On the other hand, R variant infection triggers autophagy and apoptosis, reminiscent of the way that macrophages control RGM. However, the R variant has an exquisite capacity to form extracellular cords, allowing these bacteria to rapidly divide and evade phagocytosis. Therefore, new chemotherapeutic interventions against M. abscessus need to efficiently deal with both the reservoir of intracellular bacilli and the extracellular cords. In this context, we recently identified two chemical entities that were very effective against both M. abscessus populations. Although being structurally unrelated these two chemotypes inhibit the activity of the essential mycolic acid transporter, MmpL3. In this Perspective, we aimed to highlight recent insights into how M. abscessus interacts with phagocytic cells and how the inhibition of mycolic acid transport in this pathogenic RGM could be an efficient means to control both intracellular and extracellular populations of the bacterium. [ABSTRACT FROM AUTHOR]

These studies focus on the evolving pathogenic sequence of I M. abscessus i lung infection when the low pathogenic colonizing lung airway phenotype is transitioning to its more virulent, proinflammatory phenotype. One important aspect of I M. abscessus i pathogenesis is colonization of abnormal lung airways with glycopeptidolipid (GPL) - expressing smooth colony variants that are able to form biofilms. Keywords: Mycobacterium abscessus; cystic fibrosis; porin; biofilm; glycopeptidolipid EN Mycobacterium abscessus cystic fibrosis porin biofilm glycopeptidolipid 1 2 2 06/14/22 20220610 NES 220610 I Mycobacterium abscessus i is a non-tuberculous mycobacteria that is an important cause of progressive human pulmonary infection. [Extracted from the article]

Mycobacterium abscessus causes severe lung infections. Clinical isolates can have either smooth (S) or rough (R) colony morphotypes; of these, S but not R variants have abundant cell wall glycopeptidolipids (GPL) consisting of a peptidolipid core substituted by a 6-deoxy-α-L-talose (6-dTal) and rhamnose residues. Deletion of gtf1, encoding the 6-dTal transferase, results in the S-to-R transition, mycobacterial cord formation, and increased virulence, underscoring the importance of 6-dTal in infection outcomes. However, since 6-dTal is di-O-acetylated, it is unclear whether the gtf1 mutant phenotypes are related to the loss of the 6-dTal or the result of the absence of acetylation. Here, we addressed whether M. abscessus atf1 and atf2, encoding two putative O-acetyltransferases located within the gpl biosynthetic locus, transfer acetyl groups to 6-dTal. We found deletion of atf1 and/or atf2 did not drastically alter the GPL acetylation profile, suggesting there are additional enzymes with redundant functions. We subsequently identified two paralogs of atf1 and atf2, MAB_1725c and MAB_3448. While deletion of MAB_1725c and MAB_3448 had no effect on GPL acetylation, the triple atf1-atf2-MAB_1725c mutant did not synthetize fully acetylated GPL, and the quadruple mutant was totally devoid of acetylated GPL. Moreover, both triple and quadruple mutants accumulated hyper-methylated GPL. Finally, we show deletion of atf genes resulted in subtle changes in colony morphology but had no effect on M. abscessus internalization by macrophages. Overall, these findings reveal the existence of functionally redundant O-acetyltransferases and suggest that O-acetylation influences the glycan moiety of GPL by deflecting biosynthetic flux in M. abscessus., Competing Interests: Conflict of interest The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication. G. H. receives support through a collaborative research agreement with Janssen Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. Within chronically infected patients, M. abscessus isolates undergo molecular changes leading to increased virulence and antibiotic resistance. Specifically, mutations in glycopeptidolipid (GPL) synthesis genes, leading to the rough phenotype, are associated with invasive, nonremitting infections and a severe clinical course. It has been unclear whether GPL defects confer antibiotic resistance independently of other molecular changes. We used transposon technology to isolate a rough (GPL-defective; Tn MABS&#95;4099c ZeoR ) mutant and compare it to a fully isogenic parent strain (ATCC 19977) bearing wild-type zeocin resistance (WT ZeoR ). Antibiotic susceptibility profiles of Tn&#95;4099c ZeoR and WT ZeoR were tested and compared using the Sensititre RAPMYCOI antimicrobial susceptibility test plate. MICs were evaluated within clinically relevant values according to the Clinical and Laboratory Standards Institute (CLSI) standards. We found that M. abscessus with rough colony morphotype (Tn&#95;4009c) had comparable antibiotic susceptibility to its smooth isogenic WT counterpart. Small differences (a 1:2 dilution) in MICs were found for imipenem, cefoxitin, and tigecycline, yet those small differences did not change the clinical susceptibility report for these antibiotics, as they fell within the same CLSI cutoffs for resistance. While small alternations in susceptibility to imipenem, cefoxitin, and tigecycline were noted, we conclude that the GPL mutations in M. abscessus did not confer clinically significant antibiotic resistance. Increased antibiotic resistance in the clinical setting may occur in an unrelated and parallel manner to GPL mutations. IMPORTANCE Mycobacterium abscessus chronically infects patients with preexisting lung diseases, leading to progressive deterioration in pulmonary function. The common perception among clinicians is that the rough phenotype is associated with progressive disease and severe clinical course, manifested as a widespread inflammatory response and resistance to antibacterials. However, as clinical isolates accumulate hundreds of mutations over the prolonged course of infection, it is unclear whether the rough phenotype per se is responsible for the antibiotic resistance seen in late-stage infections, or whether the resistance is related to other genetic changes in the bacteria. Previous studies mostly compared rough and smooth clinical isolates. Here, for the first time, we compared WT smooth bacteria to a specific rough, GPL-associated, otherwise-isogenic mutant. We determined that the rough morphotype had essentially identical antibiotic susceptibilities as the parent strain. The mechanistic basis for the antibiotic resistance observed in rough clinical isolates is therefore most probably related to other genetic determinants.

Background:Mycobacterium avium complex (MAC) pulmonary disease (PD) is often difficult and complicated to diagnose or to discriminate from follicular bronchitis, bronchiectasis, or other conditions associated with rheumatoid arthritis (RA) lung in the clinical setting. Objective: We investigated whether a serologic test for anti-glycopeptidolipid (GPL) antibody was useful for distinguishing MAC-PD from RA lung in diagnosis. Methods: Serum IgA antibody to MAC-specific GPL core antigen was measured by an enzyme immunoassay. Antibody levels were measured in sera from 14 RA patients with MAC-PD (RA + MAC), 20 RA patients with bronchial or bronchiolar lesions without MAC-PD (RA w/o MAC), 20 RA patients without pulmonary lesions (RA only), and 25 healthy volunteers (HV). Results: The levels of serum anti-GPL antibodies were higher in the RA + MAC group than in the RA w/o MAC, RA-only, and HV groups (2.87 ± 2.83 vs. 0.50 ± 0.45, 0.31 ± 0.24, and 0.38 ± 0.10 U/ml, respectively; p < 0.001). With the cutoff point in receiver-operating characteristic analysis set at 0.7 U/ml, the serologic test differentiated RA + MAC from RA w/o MAC with a sensitivity of 100% and specificity of 90%. Conclusions: This serologic test for anti-GPL antibody is useful for diagnosing MAC-PD in RA. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e- reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e- reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium avium complex (MAC) is a group of non-tuberculous mycobacteria that cause tuberculosis-like diseases in humans. Unlike Mycobacterium tuberculosis, MAC expresses high levels of glycopeptidolipids (GPLs) containing a well-defined tetrapeptide-amino alcohol core, composed of d-phenylalanine, d-allo-threonine, d-alanine, and l-alaninol, that is modified with a fatty acid and sugar residues. Surprisingly, however, a careful scrutiny of the mass spectrum of MAC GPLs revealed the presence of ions that could not readily accountable for the known GPL structure. The magnitude of the ions was increased prominently when GPLs were isolated from the valine-supplemented culture, and the ions representing the authentic GPL species were diminished, suggesting the possibility that the basic structure of the peptide backbone might be altered in response to the exogenously added valine. Indeed, further mass spectrometry (MS)/MS and gas chromatography–MS analysis indicated a substitution of d-valine for the N-terminal d-phenylalanine of the tetrapeptide core, and the presence of d-valine and the absence of d-phenylalanine was confirmed by high-performance liquid chromatography, using the derivatized amino acid residues that were released from the tetrapeptide. Finally, specific antibodies to the purified valine-containing GPL species were detected in the serum of a MAC-infected guinea pig. Therefore, these results identify a new molecular species of MAC GPLs with immunogenic potential. [Copyright &y& Elsevier]

Summary: Mycobacterium smegmatis is a rapidly growing, non-pathogenic mycobacterium, and M. smegmatis strain mc2155 in particular has been used as a tool for molecular analysis of mycobacteria because of its high rate of transformation. We examined another strain, M. smegmatis J15cs, which has the advantage of surviving for six days in murine macrophages. The J15cs strain produces a rough dry colony, and we hypothesized that the long survival of the J15cs strain was correlated with its cell wall components. Therefore, the lipid compositions of these two strains were compared. The subclasses and carbon species of the mycolic acids were very similar, and the major glycolipids and phospholipids were expressed in both strains. However, apolar glycopeptidolipids were deleted only in the J15cs strain. The presence of apolar glycopeptidolipids gives the cell wall a different structure. Moreover, the apolar glycopeptidolipids were recognized by macrophages via toll-like receptor 2, but not 4. We concluded that the absence of apolar glycopeptidolipids is a definitive feature of the J15cs strain, and affects its morphology and survival in host cells. [Copyright &y& Elsevier]

The cell surface of mycobacteria is quite rich in lipids. Glycopeptidolipids, surface-exposed lipids that typify some mycobacterial species, have been associated with a phenotypic switch between rough and smooth colony morphotypes. This conversion in Mycobacterium smegmatis is correlated with the absence/presence of glycopeptidolipids on the cell surface and is due to insertion sequence mobility. Here, we show that the occurrence of a high amount of glycopeptidolipids in the smooth variant leads to lower invasion abilities and lower internalization by macrophages. We further show that the high production of glycopeptidolipids on the cell surface can confer a selective advantage to the smooth variant when grown in vitro. This higher fitness under the laboratory condition might explain the selection of smooth variants in several independent laboratories. The implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Glycopeptidolipids (GPLs) comprise a major surface glycolipid of Mycobacterium avium complex (MAC), and their unique oligosaccharide extensions are known to define MAC serotypes. Beside the mature form of “serotype-specific” GPLs (ssGPLs), those that share the backbone structure but lack the oligosaccharide extensions exist as abundantly in all MAC serotypes, but the presumption was that antibody responses might not be directed to these “serotype-nonspecific” GPLs (nsGPLs) due to the lack of the sugar chain epitope. Here, we show that IgG responses to nsGPLs indeed occur in MAC-infected guinea pigs. The pool of anti-nsGPL antibodies was distinct from that of anti-ssGPL antibodies in terms of requirements for the oligosaccharide and acetylation for their target recognition. Because nsGPLs are shared in virtually all MAC strains, but totally absent in Mycobacterium tuberculosis, this study suggests that detecting serum anti-nsGPL antibodies can potentially be useful for differential diagnosis of MAC infection and tuberculosis. [Copyright &y& Elsevier]

y. nishiuchi, s. kitada and r. maekura. 2004.The antigenic glycopeptidolipids (GPLs) fromMycobacterium avium–intracellularecomplex (MAC) are grouped into 28 serovars on the basis of the variable oligosaccharide sequences and the core structures. To facilitate the identification of MAC serovars by employing liquid chromatography/mass spectrometry (LC/MS), the diversity in fatty acyl moieties and the number of acetyl groups of GPLs should be characterized.Employing a small-scale preparation method, sufficient quantities of intact GPLs could be obtained from several colonies of MAC within 4 h. Tandem mass spectrometry of GPLs showed the presence of common fragment ion atm/z1048 in the main molecular species of all reference strains. It revealed that the acyl moieties had similar diversity among all serovars. Furthermore, intact GPLs had mainly one or two acetyl groups. This allowed us to determine the masses of each serovar based on intact GPLs and to classify 16 isolates from patients by LC/MS.The present serotyping method using LC/MS analysis improved the precision of measurements and shortened the procedure time compared with conventional thin-layer chromatography or the seroagglutination test method.This proposed method proves useful for identifying serovars of MAC for epidemiological and pathogenic research purposes. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subsp. paratuberculosis (MAP) is a member of the Mycobacterium avium complex (MAC) and causes the inflammatory bowel disease, Johne’s disease, in livestock. MAP has also been implicated as the causative agent of a similar disease, Crohn’s disease, in humans. One of three major genetic differences between MAP and non-pathogenic MAC is the 6,496-bp GS element. Based on the output from freely available protein sequence and structural bioinformatics tools, and the close homology of GS genes with the ser2 region of the closely related Mycobacterium avium subsp. avium (MAA), we predict that GS encoded enzymes are involved in the biosynthesis of GDP-fucose, and the addition to, and modification of fucose on, the oligosaccharide moiety of GPL. GPL is a major constituent of the cell wall of the MAC and has immunomodulatory properties. Therefore, the enzymes involved in its synthesis may provide novel drug targets against MAP and other pathogenic MAC members.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

An effective synthesis of the pentasaccharide hapten from the glycopeptidolipid antigen of Mycobacterium avium serovar 12 in the protected p-nitrophenyl glycoside, using a 3+2 block synthesis strategy, is described. [Copyright &y& Elsevier]

Mycobacterium abscessus is an emerging and difficult-to-manage mycobacterial species that exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in transition from S to R and severe lung disease. A structure-activity relationship study was undertaken to decipher the role of GPL glycosylation in morphotype transition and pathogenesis. Deletion of gtf3 uncovered the prominent role of the extra rhamnose in enhancing mannose receptor-mediated internalization of M. abscessus by macrophages. In contrast, the absence of the 6-deoxy-talose and the first rhamnose in mutants lacking gtf1 and gtf2, respectively, affected M abscessus phagocytosis but also resulted in the S-to-R transition. Strikingly, gtf1 and gtf2 mutants displayed a strong propensity to form cords and abscesses in zebrafish, leading to robust and lethal infection. Together, these results underscore the importance and differential contribution of GPL monosaccharides in promoting virulence and infection outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Among the fast-growing mycobacteria, members of the Mycobacterium fortuitum complex are the most-commonly cited opportunistic human pathogens, notably in post-surgical infections. Previous studies showed that this complex was composed of four well-identified species and a group of isolates that did not correspond to recognized species, which has been referred to as the third biovariant complex. The occurrence and chemical structure of the glycolipid antigens of six strains that belong to this latter group were examined in the present study. Based on the TLC profiles, resistance to alkali and seroreactivities of their glycolipids, the examined strains were classified into three groups: one group was devoid of species-specific glycolipid and the two other groups contained alkali-stable or alkali-labile glycoconjugates. The structures of the major glycolipid antigens of the latter two groups were elucidated by fast-atom-bombardment MS, one-dimensional and two-dimensional NMR spectroscopy and conventional chemical analyses. The alkali-stable glycolipids were structurally identical to the C-mycoside-type glycopeptidolipids characterized in the taxonomically related species Mycobacterium peregrinum. The major alkali-labile glycolipid was identified as β-Glcp-1 → 6)-α-Glcp2Acyl-(1 → 1)-α-GLcp3,4,6Acyl3. The acyl substituents consisted on one acetyl group and three fatty acyl residues composed mainly of tetradecanoyl residues, but significant amounts of 2-methylhexadecanoyl and 2-methyloctadecanoyl substituents were also present. The heterogeneity of the glycolipid content of members of the third biovariant complex of M. fortuitum demonstrated in the present study confirms the heterogeneity of the complex. In addition, the occurrence of a species-specific glycolipid in some strains supports the hypothesis that some strains of this complex of M. fortuitum may belong to a new mycobacterial species. [ABSTRACT FROM AUTHOR]

Pleuritis caused by nontuberculous mycobacteria is uncommon and difficult to diagnose. We herein report a case of Mycobacterium avium complex (MAC) pleuritis with elevated anti-glycopeptidolipid (GPL)-core IgA antibody levels in the pleural effusion. A 73-year-old woman with MAC pulmonary disease presented with massive left pleural effusion. A pleural biopsy by video-assisted thoracoscopic surgery was performed, revealing many noncaseating epithelioid cell granulomas. MAC was not identified by culture of the pleural effusion or specimens, but the anti-GPL-core IgA antibody level was markedly elevated in the pleural effusion. Measurement of anti-GPL-core IgA levels in the pleural fluid may be useful for diagnosing MAC pleuritis.

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36W T and MOTT68W, were isolated from the sputa of three independent Korean patients co-infected with Mycobacterium yongonense Type II strains. The 16S rRNA gene sequences of all three strains were unique, which were closest to that of Mycobacterium chelonae subsp. bovis KCTC 39630 T (99.9 % similarity). Multilocus sequence typing analysis targeting 10 housekeeping genes including hsp65 and rpoB revealed the distinct phylogenetic location of these strains, which were clustered with M. chelonae subsp. chelonae ATCC 35752 T and M. chelonae subsp. bovis KCTC 39630 T . Phylogenetic analysis based on whole genome sequences revealed a 95.89 % average nucleotide identity (ANI) value with M. chelonae subsp. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, phenotypic characteristics such as a smooth colony morphology and growth inhibition at 37 °C, distinct MALDI-TOF MS profiles of extracted total lipids due to surface glycopeptidolipids, and distinct drug susceptibility profiles further supported the taxonomic characterization of these strains as representing a novel subspecies of Mycobacterium chelonae. Mycobacterium chelonae subsp. gwanakae subsp. nov. is proposed and the type strain is MOTT36W T (=KCTC 29127 T =JCM 32454 T ).

We evaluated serial changes in the levels of serum immunoglobulin A (IgA) antibody to the glycopeptidolipid (GPL) core antigen during antibiotic treatment in 57 patients with Mycobacterium avium complex (MAC) lung disease at baseline (T0) and after 3 months (T3) and 6 months (T6) of treatment. The median patient age was 59 years, and 37 (65%) patients were women. The etiologic organisms included M. avium in 32 (56%) patients and M. intracellulare in 25 (44%) patients. Seven (12%) patients had the fibrocavitary form of the disease on computed tomography. After 12 months of treatment, 42 (74%) patients had a favorable response to treatment, whereas 15 (26%) patients had an unfavorable response to treatment defined as the absence of sputum culture conversion within 12 months of treatment. The initial median serum anti-GPL IgA levels in the 57 patients was 3.50 U/mL, and the antibody levels at T0 (median 3.50 U/mL), T3 (median 2.71 U/mL), and T6 (median 2.61 U/mL) were significantly decreased following treatment (P ＜ 0.001). The results of the multivariate analysis indicated that an initially elevated anti-GPL IgA level (＞ 3.50 U/mL) was associated with an unfavorable response (P = 0.049). Our data suggest that elevated anti-GPL IgA levels may reflect disease activity and may help predict treatment response in patients with MAC lung disease.