A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus.

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 a 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.

Multiple Mycobacterium abscessus O-acetyltransferases influence glycopeptidolipid structure and colony morphotype.

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.

Efficacy of three sputum specimens for the diagnosis of Mycobacterium avium complex pulmonary disease.

BACKGROUND: In Mycobacterium avium complex pulmonary disease (MAC-PD), diagnosis requires a positive culture from at least two separate expectorated sputum specimens. The optimal number of sputum examinations remains unclear. OBJECTIVE: This study sought to elucidate the diagnostic yield of acid-fast bacilli in MAC-PD using 3 sputum specimens and to clarify the clinical characteristics of patients with MAC-PD diagnosed using 3 sputum specimens. Furthermore, we investigated the correlation between increased number of sputum specimens and diagnostic yield. METHODS: We reviewed the medical records of 139 patients with MAC-PD diagnosed at Toho University Omori Medical Center for whom at least three sputum specimens were examined before treatment from November 2014 through June 2021. Patients were classified into the 3-sputum diagnosed and the non-3 sputum diagnosed groups based on diagnostic procedure; clinical and radiological characteristics were compared. We also assessed diagnostic yield with the increased number of sputum specimens. RESULTS: Diagnostic yield with 3 sputum specimens was 16.5% (23/139). The 3-sputum diagnosed group had a lower body mass index [18.6(17-19.5) vs. 19.5(18-21.5); p = 0.014], and higher chest CT score [9(6.5-13) vs. 6(4-9); p = 0.011] including cavitary lesions (39.1% vs. 19%; p = 0.037) compared with the non-3 sputum diagnosed group. When the number of sputum specimens was increased to 6, the diagnostic yield increased to 23.7% (33/139). CONCLUSION: Diagnostic yield with 3 sputum specimens was 16.5%. Patients diagnosed using 3 sputum specimens had more severe chest CT findings including cavitary lesions. Increasing the number of sputum specimens to 6 improved diagnostic yield by 7.2%.

Glycopeptidolipid Genotype Correlates With the Severity of Mycobacterium abscessus Lung Disease.

BACKGROUND: Smooth and rough colony morphotypes of Mycobacterium abscessus are associated with virulence, but some isolates form both smooth and rough colonies, impeding successful morphotype identification. Reportedly, smooth/rough morphotypes are also related to the glycopeptidolipid (GPL) genotype. However, the accuracy of GPL genotyping to discriminate morphotypes and the relationship between GPL genotype and clinical characteristics of M abscessus lung disease have not been verified. METHODS: A retrospective analysis of colony morphology, GPL genotype, and clinical data from 182 patients with M abscessus lung disease was conducted. RESULTS: Of 194 clinical isolates, 126 (65.0%), 15 (7.7%), and 53 (27.3%) exhibited rough, smooth, and mixed colony morphotypes, respectively. Glycopeptidolipid genotyping indicated that 86.7% (13 of 15) of smooth isolates belonged to the GPL-wild type (WT) group, whereas 98.4% (124 of 126) of rough isolates belonged to the GPL-mutant type (MUT) group. Therefore, GPL genotyping accurately distinguished between smooth and rough morphotypes. Mixed colony morphotypes were also divided into GPL-WT (18.9%) and GPL-MUT (81.1%) groups. Further analysis revealed that patients infected with the GPL-MUT group presented with significantly worse baseline clinical characteristics and exacerbated episodes of lung disease. CONCLUSIONS: Glycopeptidolipid genotyping accurately distinguishes smooth and rough colony morphotypes. Patients infected with the GPL-MUT genotype exhibit worse clinical characteristics and are at a higher risk of exacerbated lung disease.

A description of Mycobacterium chelonae subsp. gwanakae subsp. nov., a rapidly growing mycobacterium with a smooth colony phenotype due to glycopeptidolipids.

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36WT 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 39630T (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 35752T and M. chelonae subsp. bovis KCTC 39630T. 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 MOTT36WT (=KCTC 29127T=JCM 32454T).

Biomarker for nontuberculous mycobacterial pulmonary disease in patients with rheumatoid arthritis: Anti-glycopeptidolipid core antigen immunoglobulin A antibodies.

OBJECTIVE: Nontuberculous mycobacterial (NTM) pulmonary disease is occasionally associated with rheumatoid arthritis (RA), influencing the therapeutic strategy of RA. Since chronic lung diseases are frequently associated with RA, the diagnosis of NTM pulmonary disease is quite difficult in RA patients. Recently, a serological diagnostic test detecting serum immunoglobulin A against the glycopeptidolipid (GPL) core antigen was developed. We investigated the serum levels of anti-GPL antibodies in RA patients to determine the usefulness for detecting NTM pulmonary disease. METHODS: Anti-GPL antibodies were detected in the sera from RA patients with or without NTM pulmonary disease. RESULTS: The positivity of anti-GPL antibodies in RA patients with NTM pulmonary disease was higher than in RA without (p = 1.76 × 10-14, odds ratio 70.29, 95% confidence interval [CI] 22.28-221.83). Anti-GPL Ab titers were increased in RA with NTM pulmonary disease (mean titer ± standard deviation [U/ml], RA with NTM pulmonary disease: 4.1 ± 7.0, RA without NTM pulmonary disease: 0.4 ± 1.6, p = 1.51 × 10-10). The area under the curve (AUC) value of the receiver operating characteristic (ROC) curve for anti-GPL antibodies was 0.917 (95%CI 0.860-0.974, p = 3.32 × 10-47). CONCLUSIONS: Serum anti-GPL antibodies are useful for detecting NTM pulmonary disease in RA patients.

Clinical efficacy of anti-glycopeptidolipid-core IgA test for diagnosing Mycobacterium avium complex infection in lung.

BACKGROUND AND OBJECTIVE: It is difficult to verify the bacteriological diagnosis of Mycobacterium avium complex (MAC) infection. The anti-glycopeptidolipid (GPL)-core IgA antibody test was recently developed as a diagnostic method for MAC pulmonary disease. Only a few studies evaluate its clinical efficacy. We conducted retrospective evaluations of clinical characteristics of patients suspected of MAC infection to explore the usefulness of the anti-GPL-core IgA antibody test. METHODS: We retrospectively evaluated 296 patients who were suspected to have MAC infection and underwent anti-GPL-core IgA antibody test between March 2013 and July 2014 in Jikei University hospital. RESULTS: A total of 29 patients were diagnosed with 'definite MAC' based on the American Thoracic Society (ATS) criteria with multiple identifications of MAC. On the other hand, 106 patients were diagnosed with other pulmonary diseases than MAC. The sensitivity and specificity of anti-GPL-core IgA antibody test for MAC diagnosis were 58.6% and 98.1%, respectively. The definite MAC group showed no significant differences in strains, treatment history or number of segments involved. The duration of MAC disease in the positive-antibody group was significantly longer than in the negative-antibody group (P = 0.046). A significant increase in the false-negative rate was observed in patients with malignant disease (P = 0.029). CONCLUSIONS: The anti-GPL-core IgA antibody test demonstrated high sensitivity and specificity for the diagnosis of MAC infection especially in patients without malignant diseases.

Glycopeptidolipid Defects Leading to Rough Morphotypes of Mycobacterium abscessus Do Not Confer Clinical Antibiotic Resistance.

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_4099cZeoR) mutant and compare it to a fully isogenic parent strain (ATCC 19977) bearing wild-type zeocin resistance (WTZeoR). Antibiotic susceptibility profiles of Tn_4099cZeoR and WTZeoR 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_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.

Utility of serum immunoglobulin A antibody against glycopeptidolipid core antigen in the diagnosis and management of hypersensitivity pneumonitis associated with Mycobacterium avium complex: A case report.

Measurement of the levels of serum immunoglobulin A antibody against glycopeptidolipid (GPL) core antigen, a cell surface antigen found in Mycobacterium avium complex (MAC), has been reported to be useful in the diagnosis and management of pulmonary MAC infection. However, evidence on its utility in hypersensitivity pneumonitis (HP) associated with MAC (i.e., "hot-tub lung") is limited. We herein report a case of HP associated with MAC in which the GPL core antibody levels were serially measured from diagnosis to treatment and thereafter. A 61-year-old man was suspected to have non-fibrotic HP based on the clinical course, laboratory findings, imaging pattern, bronchoalveolar lavage (BAL) lymphocytosis, and histopathological findings. Based on the history of whirlpool bath use, inhalation of aerosolized MAC was suspected as the cause of HP. The GPL core antibody level, measured using an enzyme-linked immunosorbent assay kit, was elevated, suggesting an immunological sensitization to MAC. A provocation test using the patient's whirlpool bath was positive. An identical MAC strain was isolated from the BAL fluid and bathtub. Accordingly, the patient was diagnosed with HP caused by the inhalation of aerosolized MAC from the whirlpool bath. The patient recovered after steroid treatment and discontinuation of the whirlpool bath. The GPL core antibody levels decreased with disease improvement. In conclusion, GPL core antibody levels could be elevated in HP associated with MAC and decrease with disease improvement. Thus, measurement of the GPL core antibody level may be useful for the diagnosis and management of HP associated with MAC.

Glycopeptidolipid glycosylation controls surface properties and pathogenicity in Mycobacterium abscessus.

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.

The diagnostic yield and characteristics of bronchoalveolar lavage in suspected nontuberculous mycobacterial pulmonary disease.

Background: Bronchoalveolar lavage (BAL) has widely been used to manage respiratory diseases including respiratory infections. The aim of this study was to evaluate the diagnostic yield of BAL for detecting nontuberculous Mycobacterium (NTM). Methods: We retrospectively reviewed the records of 54 patients who underwent bronchoscopy due to suspected NTM pulmonary disease. Positive culture results of respiratory specimens were defined as NTM pulmonary disease. For BAL, two or three aliquots of 50 mL (total 100 or 150 mL) of sterile normal saline were instilled through bronchoscope. Results: NTM was detected in 31 of 54 (57.4%) patients. The detection rates were not different between the patients who underwent bronchoscopy with BAL (24 of 39, 61.5%) and those without (7 of 15, 46.7%) (P = 0.437). BAL fluid was mostly neutrophil dominant in both positive and negative NTM culture groups. In the subgroup analysis of 33 patients who underwent both the BAL and anti- glycopeptidolipid (GPL)-core immunoglobulin A (IgA) antibody measurements, 12 of 19 (63.2%) positive Mycobacterium avium complex (MAC) culture patients and 8 of 14 (57.1%) negative MAC culture patients were positive for anti-GPL-core IgA antibody (seropositive) (P = 0.991). There was no severe complication related to BAL. Conclusions: The diagnostic yield of BAL with ≥100 mL sterile saline was not superior to that of bronchial wash or sputum aspiration in patients with suspected NTM pulmonary disease. Patients with seropositive but negative culture results for MAC suggest pseudonegative for pulmonary MAC disease.

Combination of anti-glycopeptidolipid-core IgA antibody and clinical features for diagnosing potential nontuberculous mycobacterium pulmonary disease in routine practice.

BACKGROUND: The anti-Mycobacterium avium complex (MAC) antibody test measures levels of IgA antibody against the glycopeptidolipid (GPL) core in the bacterial cell walls and is a useful clinical indicator of nontuberculous mycobacterium pulmonary disease (NTM-PD). However, it is not currently possible to diagnose the disease using anti-MAC antibodies alone. OBJECTIVES: The study aim was to assess the efficacy of the combination of anti-MAC antibodies and clinical findings for diagnosing potential NTM-PD. METHODS: This cross-sectional study included 938 patients tested using the anti-MAC antibody. NTM-PD was diagnosed by multiple positive cultures of the same species in sputum samples. Multivariate logistic regression models were used to identify the clinical factors related to NTM-PD. RESULTS: Overall, 19.6% (184/938) of participants were diagnosed with NTM-PD. In multivariate analysis, positive anti-MAC antibodies, low body mass index, absence of malignancy, and cavity-forming lung lesions were significantly associated with NTM-PD at diagnosis. The positive rates of the anti-MAC antibody test were 79.4% (135/170) for MAC and 55.6% (5/9) for Mycobacterium abscessus complex, respectively. CONCLUSIONS: Bronchoscopic examinations should be performed especially in certain types of individuals from whom sputum samples cannot be obtained. Anti-MAC antibodies are also positive in patients other than those harboring MAC, but the rate may be low because of the different components in GPLs.

Long-Term Prognosis and Antimycobacterial Glycolipid Antibody as Biomarker in Mycobacterium avium-intracellulare Complex Pulmonary Disease.

Clinical characteristics and outcomes of multidrug chemotherapy have been used as the main prognostic factors for Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD) over the last decade; however, no useful prognostic biomarkers have been reported. The aim is to ascertain whether the serum antibody titers could include useful prognostic predictors of MAC-PD. Ninety-four patients with MAC-PD were enrolled and regularly followed up with for more than 5 years or until death. Cox proportional hazard regression and receiver operating characteristic (ROC) curve analyses were used to identify predictors of mortality in this prospective observational study. According to treatment outcomes, 85 patients completed follow-up and were classified into four groups. Seventeen patients (20%) died during follow-up (median, 10.1 years; interquartile range, 8.1 to 12.4 years). All 11 patients with MAC-PD-specific death were included in the 14 patients of the group nonresponsive to the multidrug chemotherapy. They had significantly higher anti-Mycobacterium glycolipid (MBGL) antibody titers than those in the other groups and a significantly (P < 0.0001) poorer survival prognosis. The anti-MBGL antibody titers also served as a negative prognostic factor. A cutoff score of 7, which was calculated by clinical poor prognostic characteristics and anti-MBGL antibody titers, differentiated the nonresponse group and the other groups at baseline (sensitivity, specificity, and area under the curve: 92.9%, 81.7%, and 0.95, respectively). In conclusion, anti-MBGL antibody titers were useful to assess the refractory MAC-PD. The predictions of treatment outcome and mortality become more accurate by using anti-MBGL antibody and clinical poor prognostic characteristics together. IMPORTANCE The natural history of MAC-PD is challenging to predict in immunocompetent patients at diagnosis, and the current multidrug chemotherapy options are not strong enough to eliminate mycobacteria from the lungs. Therefore, the diagnosis of MAC-PD does not necessarily lead to the decision to start chemotherapy. We have also observed refractory patients in clinical practice, who were resistant to multiple-drug chemotherapy and showed persistent excretion of MAC bacilli and progressive worsening of chest radiographic findings until death. We have reported that the measurements of anti-MBGL antibody titers helped assess refractory MAC-PD in this study. Furthermore, the predictions of treatment outcome and mortality become more accurate by using the anti-MBGL antibody in addition to clinical poor prognostic characteristics, which were older age, lower body mass index, the positive results of a smear test for acid-fast bacteria (AFB), and presence of cavitary disease.

The Diagnosis of Nontuberculous Mycobacterial Pulmonary Disease by Single Bacterial Isolation Plus Anti-GPL-Core IgA Antibody.

Although serum anti-glycopeptidolipid (GPL)-core IgA antibody is a highly specific test for infection with Mycobacterium avium complex (MAC), Mycobacterium abscessus, and its subspecies abscessus, subsp. massiliense, and subsp. bolletii (MAB), its use for the definitive diagnosis of MAC pulmonary disease (PD) and MAB-PD are unknown. To clarify the diagnostic accuracy of the anti-GPL-core IgA antibody test among patients with radiologically suspected MAC-PD or MAB-PD who already have a single positive sputum culture test. The first isolations of MAC and MAB from patients with radiologically suspected MAC-PD or MAB-PD at the Osaka Toneyama Medical Center between January 2006 and December 2020 were collected. Patients were enrolled when their serum anti-GPL-core IgA antibody was measured during the 3 months before and after the first isolation. We retrospectively compared the results of anti-GPL-core IgA antibody testing with the final diagnoses based on the current guidelines. We included 976 patients for analysis. The serum anti-GPL-core IgA antibody was positive in 699 patients (71.6%). The positive predictive value of anti-GPL-core IgA antibody for the diagnosis of MAC-PD or MAB-PD was 97.4%. The median time required for the second positive culture after the first isolation was 51 days (interquartile range 12 to 196 days). The positive serum anti-GPL-core IgA antibody test allowed an early and definitive diagnosis of MAC-PD or MAB-PD in those who already had a single positive sputum culture test. IMPORTANCE To satisfy the microbiologic criteria of the current diagnostic guideline for nontuberculous mycobacterial pulmonary disease (PD), at least two positive sputum cultures of the same species of mycobacteria from sputum are required to avoid the casual isolation of mycobacteria. This study showed that the positivity of a serum anti-glycopeptidolipid (GPL)-core IgA antibody test has an excellent diagnostic ability among patients with radiologically suspected Mycobacterium avium complex (MAC)-PD or Mycobacterium abscessus (MAB)-PD who already had a single positive sputum culture test. The usage of single culture isolation plus anti-GPL-core IgA antibody as another diagnostic criterion has a time, cost, and effort-saving effect. Furthermore, it will facilitate the diagnosis of MAC-PD or MAB-PD in the early stage of disease because serum anti-GPL-core IgA antibody becomes high in these patients. Therefore, we proposed adding single culture isolation plus anti-GPL-core IgA antibody as "combined microbiological and serological criteria" to the diagnostic guidelines for MAC-PD and MAB-PD.

Ultrastructure of the Mycobacterium avium subsp. hominissuis Biofilm.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial pathogens responsible for chronic lung disease in humans. It is widely distributed in biofilms in natural and living environments. It is considered to be transmitted from the environment. Despite its importance in public health, the ultrastructure of the MAH biofilm remains largely unknown. The ultrastructure of a MAH-containing multispecies biofilm that formed naturally in a bathtub inlet was herein reported along with those of monoculture biofilms developed from microcolonies and pellicles formed in the laboratory. Scanning electron microscopy revealed an essentially multilayered bathtub biofilm that was packed with cocci and short and long rods connected by an extracellular matrix (ECM). Scattered mycobacterium-like rod-shaped cells were observed around biofilm chunks. The MAH monoculture biofilms that developed from microcolonies in vitro exhibited an assembly of flat layers covered with thin film-like ECM membranes. Numerous small bacterial cells (0.76±0.19| |µm in length) were observed, but not embedded in ECM. A glycopeptidolipid-deficient strain did not develop the layered ECM membrane architecture, suggesting its essential role in the development of biofilms. The pellicle biofilm also consisted of flat layered cells covered with an ECM membrane and small cells. MAH alone generated a flat layered biofilm covered with an ECM membrane. This unique structure may be suitable for resistance to water flow and disinfectants and the exclusion of fast-growing competitors, and small cells in biofilms may contribute to the formation and transmission of bioaerosols.

O-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of Mycobacterium abscessus and Macrophage Invasion.

Mycobacterium abscessus, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the fmt gene encoding a putative O-methyltransferase was deleted in the M. abscessus S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the Δfmt mutant, and this defect was rescued upon complementation with a functional fmt gene. In contrast, the introduction of fmt derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that fmt encodes an O-methyltransferase. Unexpectedly, phenotypic analyses showed that Δfmt was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by Δfmt was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt O-methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of M. abscessus as well as in the early steps of the interaction between the bacilli and macrophages.

Serum immunoglobulin a antibodies to glycopeptidolipid core antigen for Mycobacteroides abscessus complex lung disease.

Background: Mycobacteroides abscessus complex (MABC) exhibits smooth morphotypes, expressing glycopeptidolipid (GPL), and rough morphotypes, expressing diminished GPL, on the MABC cell wall. Few reports have focused on the relationship between anti-GPL-core immunoglobulin A (IgA) antibody and colony morphology in MABC lung disease. Methods: This study aimed to test GPL core antigen in patients with MABC lung disease to investigate the relationship between coinfection/contamination in other nontuberculous mycobacteria species and colony morphology variant in MABC isolates. Patients with MABC lung disease and contamination diagnosed between 2012 and 2017 at our hospital were enrolled retrospectively. Results: Of the assessed patients, 43 patients with MABC lung disease and 13 with MABC contamination were included. There was a significant difference in anti-GPL-core IgA antibody levels between them (P = 0.02). Forty-three patients with MABC lung disease were divided into two groups as positive and negative antibodies groups. A significant increase in the positive anti-GPL-core IgA antibody was observed in coexistence with both Mycobacterium avium complex (MAC) (P = 0.02) and the isolate of the smooth variant (P = 0.03) in MABC. Conclusions: Anti-GPL-core IgA antibodies in patients with MABC are greatly influenced by MAC coexistence, and colony morphology variant of the MABC isolate.

Mycobacterium avium Complex Pleuritis with Elevated Anti-glycopeptidolipid-core IgA Antibody Levels in Pleural Effusion.

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.

A paucity of knowledge regarding nontuberculous mycobacterial lipids compared to the tubercle bacillus.

All mycobacteria, including nontuberculous mycobacteria (NTM), synthesize an array of lipids including phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM). While absent from Mycobacterium tuberculosis (M. tb), glycopeptidolipids (GPL) are critical to the biology of NTM. M. tb and some NTM also synthesize trehalose-containing glycolipids and phenolic glycolipids (PGL), key membrane constituents with essential roles in metabolism. While lipids facilitate immune evasion, they also induce host immunity against tuberculosis. However, much less is known about the significance of NTM-derived PIM, LM, LAM, GPL, trehalose-containing glycolipids, and PGL as virulence factors, warranting further investigation. While culling the scientific literature on NTM lipids, it's evident that such studies were relatively few in number with the overwhelming majority of prior work dedicated to understanding lipids from the saprophyte Mycobacterium smegmatis. The identification and functional analysis of immune reactive NTM-derived lipids remain challenging, but such work is likely to yield a greater understanding of the pathogenesis of NTM lung disease. In this review, we juxtapose the vast literature of what is currently known regarding M. tb lipids to the lesser number of studies for comparable NTM lipids. But because GPL is the most widely recognized NTM lipid, we highlight its role in disease pathogenesis.

A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants.

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.