The diversity of clinical Mycobacterium abscessus isolates in morphology, glycopeptidolipids and infection rates in a macrophage model.

Introduction. Mycobacterium abscessus (MABS) is a pathogenic bacterium that can cause severe lung infections, particularly in individuals with cystic fibrosis. MABS colonies can exhibit either a smooth (S) or rough (R) morphotype, influenced by the presence or absence of glycopeptidolipids (GPLs) on their surface, respectively. Despite the clinical significance of these morphotypes, the relationship between GPL levels, morphotype and the pathogenesis of MABS infections remains poorly understood.Gap statement. The mechanisms and implications of GPL production and morphotypes in clinical MABS infections are unclear. There is a gap in understanding their correlation with infectivity and pathogenicity, particularly in patients with underlying lung disease.Aim. This study aimed to investigate the correlation between MABS morphology, GPL and infectivity by analysing strains from cystic fibrosis patients' sputum samples.Methodology. MABS was isolated from patient sputum samples and categorized by morphotype, GPL profile and replication rate in macrophages. A high-content ex vivo infection model using THP-1 cells assessed the infectivity of both clinical and laboratory strains.Results. Our findings revealed that around 50 % of isolates displayed mixed morphologies. GPL analysis confirmed a consistent relationship between GPL content and morphotype that was only found in smooth isolates. Across morphotype groups, no differences were observed in vitro, yet clinical R strains were observed to replicate at higher levels in the THP-1 infection model. Moreover, the proportion of infected macrophages was notably higher among clinical R strains compared to their S counterparts at 72 h post-infection. Clinical variants also infected THP-1 cells at significantly higher rates compared to laboratory strains, highlighting the limited translatability of lab strain infection data to clinical contexts.Conclusion. Our study confirmed the general correlation between morphotype and GPL levels in smooth strains yet unveiled more variability within morphotype groups than previously recognized, particularly during intracellular infection. As the R morphotype is the highest clinical concern, these findings contribute to the expanding knowledge base surrounding MABS infections, offering insights that can steer diagnostic methodologies and treatment approaches.

Stress-induced non-replicating Mycobacterium smegmatis incorporates exogenous fatty acids into glycopeptidolipids.

Nontuberculous mycobacteria (NTM) such as Mycobacterium smegmatis accumulate high levels of glycopeptidolipids (GPLs) on their outer surface. The biosynthesis of GPLs is critically linked to biofilm formation by NTM which also includes opportunistic pathogens such as Mycobacterium abscessus. Although GPLs have been investigated in many earlier studies, the biosynthesis of GPLs using exogenous fatty acids in M. smegmatis subjected to stresses encountered by mycobacteria during infection of the human body has not been studied. Therefore, we subjected M. smegmatis to different combinations of the three stresses of hypoxia, acidic pH and nutrient starvation and report here that the metabolic incorporation of radiolabeled long-chain fatty acids into alkali-stable GPLs was significantly increased under these stress conditions. Endogenously synthesized fatty acids were not preferred for GPL biosynthesis by M. smegmatis subjected to the triple stress combination. Our observations indicate that GPLs may play important roles in cell surface modifications associated with the non-replicating state of M. smegmatis. Our experimental model reported here would be useful in the further study of GPL biosynthesis from exogenous fatty acid sources in M. smegmatis subjected to hypoxia, nutrient starvation and acidic stress conditions and help in the screening of candidate drugs that target this biochemical pathway in pathogenic NTM.

Growth medium and nitric oxide alter Mycobacterium abscessus morphotype and virulence.

Mycobacterium abscessus complex (MABC) infections cause significant morbidity and mortality among patients with chronic lung disease, like cystic fibrosis. MABC exists in smooth (S) and rough (R) morphotypes, but triggers of morphotype switching and associated pathogenicity or antimicrobial susceptibility are poorly understood. We demonstrate that M. abscessus subspecies abscessus (Mab), massiliense (Mms), and bolletii (Mbl) cultured in Middlebrook (MB) broth exhibit S morphotype, whereas the bacteria grown in Luria Bertani (LB) broth adopt the R morphotype, characterized by low glycopeptidolipid (GPL) expression. The components of broth that mediate this selection are complex, with albumin supplementation promoting growth of S morphotype, but not sufficient for complete selection. Consistent with the findings of other groups, R forms of Mab, Mms and Mbl selected by LB broth were internalized in RAW 264.7 macrophages with higher efficiency than S. Intracellular survival of broth-selected organisms was variable and was higher for S Mab, but lower for S Mms and Mbl. It is proposed that growth in R morphotype is induced during stress conditions, such as nutrient poor environments or during inflammation. One key component of inflammation is release of nitric oxide. We demonstrated that a nitric oxide donor (DETA-NONOate) appears to induce growth in an R morphotype, as indicated by reduced GPL expression of Mab. Mab treated with DETA-NONOate also enhanced susceptibility to azithromycin at sub-MIC concentrations. In conclusion, morphotype and macrophage intracellular bacterial load of MABC subspecies can be manipulated by growing the bacteria in different culture conditions. Nitric oxide may also drive morphotype selection and enhanced azithromycin activity against Mab and macrophage killing.

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen.

Mycobacterium avium: an overview.

Mycobacterium avium is an environmental microorganism found in soil and water sources worldwide. It is the most prevalent species of nontuberculous mycobacteria that causes infectious diseases, especially in immunocompromised individuals. This review discusses and highlights key topics about M. avium, such as epidemiology, pathogenicity, glycopeptidolipids, laboratory identification, genotyping, antimicrobial therapy and antimicrobial resistance. Additionally, the main comorbidities associated with M. avium infection are discussed.

Two dd-Carboxypeptidases from Mycobacterium smegmatis Affect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids.

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (between d-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The dd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used by ld-transpeptidases as substrates to form 3-3 cross-links. Therefore, dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of dd-CPases in mycobacteria is relatively unexplored. In this study, we deleted two dd-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single dd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz, a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to ß-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the dd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCE The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including Mycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.