Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae

<p>Abstract</p> <p>Background</p> <p>The outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism <it>Mycobacterium smegmatis</it>, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb.</p> <p>Results</p> <p>We have recently sequenced the complete genomes of two fast-growers causing human infections, <it>Mycobacterium abscessus </it>(CIP 104536T) and <it>M. chelonae </it>(CIP 104535T). We show here that these two species contain genes corresponding to all those of the <it>M. smegmatis </it>"GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in <it>M. chelonae </it>and <it>M. abscessus</it>. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (<it>atf1 </it>and <it>atf2</it>) in <it>M. abscessus </it>and <it>M. chelonae </it>has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (<it>M. chelonae</it>, 900 kb; <it>M. abscessus</it>, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins.</p> <p>Conclusion</p> <p>Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in <it>M. smegmatis </it>represents the ancestral form and that evolution has scattered various pieces throughout the genome in <it>M. abscessus </it>and <it>M. chelonae</it>.</p

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

Background: Recently, 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.Results: We 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.Conclusions: Our 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.OAIID:oai:osos.snu.ac.kr:snu2013-01/102/0000006653/7SEQ:7PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:0000006653ADJUST_YN:YEMP_ID:A077651DEPT_CD:806CITE_RATE:4.397FILENAME:rough colony morphology of mycobacterium.pdfDEPT_NM:의과학과SCOPUS_YN:YCONFIRM:

Evaluation of the role of sigma B in Mycobacterium smegmatis

The alternate sigma factor, sigB, is known to play a crucial role in maintaining the stationary phase in mycobacteria. In this communication, we have studied the proteomics of Mycobacterium smegmatis mc2155 and its two derivatives, one of which has a disrupted sigB gene and the other, PMVSigB, which contains a multicopy plasmid containing sigB. We have identified by two-dimensional gel analyses, several proteins that are over-expressed in PMVSigB compared to mc2155. These proteins are either stress proteins or participate actively in different metabolic pathways of the organisms. On the other hand, when sigB deleted mycobacteria were grown until the stationary phase and its two-dimensional protein profile was compared to that of mc2155, few DNA binding proteins were found to be up-regulated. We have shown recently that upon over-expressing sigB, the cell surface glycopeptidolipids of M. smegmatis are hyperglycosylated, a situation similar to what was observed for nutritionally starved bacteria. Gene expression profile through quantitative PCR presented here identified a Rhamnosyltransferase responsible for this hyperglycosylation

Discovery and biosynthesis of the redox cofactor mycofactocin

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), can survive as an intracellular pathogen residing in a granulomatous lesion in the lungs. Current TB treatments comprise a long course of several antibiotics with various side effects. Incomplete treatment is leading to antibiotic resistance, a growing threat worldwide. It has been recognized that both antibiotic inactivation and intracellular survival is often mediated by organic cofactors. Furthermore, novel therapeutic compounds in use are prodrugs that require cofactors for in vivo conversion into an active pharmaceutical entity. Mycobacterial cofactors are an up-and-coming field of research for developing novel therapeutic compounds. As such, novel cofactors offer an exciting opportunity to discover novel aspects in the pathogen's physiology and pathology as well as inspiring novel avenues for TB management. Mycofactocin (MFT) is a novel redox cofactor, part of the family of natural products termed ribosomally produced and post-translationally modified peptides and a universal genomic feature of the Mycobacterium genus. Its discovery was spurred from the bioinformatic observation of a gene locus in the vicinity of a radical SAM maturase, containing a small peptide precursor, a chaperone, a peptidase, and a glycosyltransferase, alongside putative MFT-dependent redox enzymes and other genes encoding proteins of unknown function. In-vitro investigation of the biosynthesis pathway showed the formation of the redox-active precursor premycofactocin. In addition, in-vivo studies with the model organism Mycolicibacterium smegmatis have associated MFT to the metabolism of primary alcohols for assimilation as a carbon source. Critical questions remained, such as whether the in vitro biosynthetic model is valid in vivo, the function of the putative glycosyltransferase, the enzymology of MFT-dependent oxidoreductases and dehydrogenases, and the role of MFT in mycobacterial pathogenesis

Antibiotic Resistance Characteristics of Mycobacterium\textit{Mycobacterium} Abscessus\textit{Abscessus} Rely on Specific Outer Membrane Porins and Surface-Associated Glycopeptidolipids

“Mycobacterium abscessus is an opportunistic pathogen, ubiquitous in the environment, that often causes infections in humans with compromised natural defences such as patients with cystic fibrosis or other chronic lung diseases. A current taxonomic classification suggests separation of M. abscessus into three distinct subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Although a saprophyte in water and soil, following lung infection M. abscessus can swiftly grow and survive intra-cellularly within macrophages as well as in extra-cellular caseous lesions and airway mucus. Several factors contribute to the success of this rapidly growing mycobacterium. A plethora of intrinsic resistance mechanisms renders almost all clinically used antibiotics ineffective against M. abscessus. In addition, the presence of a highly dynamic open pan-genome in M. abscessus might explain the ease with which the bacterium evolves and adapts to a wide-spectrum of stressful environmental conditions encountered in diverse habitats. Importantly, the respiratory habitat of M. abscessus brings it in close proximity to highly virulent pathogens (for example, Pseudomonas aeruginosa in cystic fibrosis lung) which can serve as donors of novel drug resistance or virulence genes” (Luthra, S. et al.). “Mechanisms underpinning intrinsic drug resistance of M. abscessus are multi-fold and fall into two main groups: first, the presence of a highly impermeable cell envelope and/or multi-drug efflux pumps might reduce the effective concentration of antibiotics within the bacterial cells; second, the genome of M. abscessus encodes several putative enzymes which can inactivate antibiotics by modification and/or degradation or lower the affinity of the drug for its target by modifying the target. For long, molecular investigations aimed at elucidating antibiotic resistance mechanisms of M. abscessus were limited, however, significant progress has been made in recent years owing to the development of efficient tools for genetic manipulation of this bacterium” (Luthra, S. et al.). We examined, in this study, the role of limiting factors [i.e. β-barrel proteins that function as porins and surface-exposed glycopeptidolipids (GPL)] in regulating β-lactam transport across the mycomembrane and the impact of changes in cell envelope composition with or without the presence of a specific mechanism involved in β-lactam removal (i.e. deactivation by periplasmic β-lactamase) on the antibacterial susceptibility of M. abscessus toward a representative set of compounds from three different β-lactam subclasses (i.e. cephalosporins, carbapenems and a penem). This was accomplished through a series of gene knockouts (obtained via two-step homologous recombination) and by the determination of minimal inhibitory concentrations of antibiotics for all M. abscessus strains. Here, we reveal that in M. abscessus, β-lactam susceptibility is linked to the presence of the general porin MapA and that the chromosomally encoded β-lactamase BlaMab can confer a higher level of β-lactam resistance in a mapA mutant than in wildtype M. abscessus. Additionally, the results suggest that the GPL environment can affect the levels of susceptibility to drugs that mainly diffuse across the membrane barrier through non-specific porins. This work has yielded new insights into the types and numbers of factors that influence susceptibility to β-lactam antibiotics in M. abscessus and how sets of genes act in concert, rather than in isolation, to elicit bacterial resistance to antibiotics. Through a better understanding of how the modification of cell envelope permeability elicits bacterial resistance, we might be able to develop new, effective means to overcome the ‘impermeability’ resistance strategy in an effort to fight M. abscessus infections. Reference Reproduced from Luthra, S. et al. The role of antibiotic-target-modifying and antibiotic-modifying enzymes in Mycobacterium abscessus drug resistance. Front. Microbiol. 9, 1–13 (2018) (see chapter 2)

An investigation into the molecular genetics of cell wall biosynthesis in Mycobacterium leprae

The mycobacterial cell envelope is a complex structure the components of which have been implicated in the survival, virulence, permeability and resistance to antibiotics of Mycobacterium. The elucidation of the synthesis of the cell envelope would improve the understanding of the structure and its influential properties and could lead to the identification of drug target sites. The isolation of genes encoding proteins involved in the biosynthesis of the cell envelope structures and their mutants would enable the biosynthetic pathways and functions of individual structures to be determined. The objective of this project was to isolate genes encoding cell envelope structures in Mycobacterium leprae, using Mycobacterium smegmatis as model organism. The aim was to isolate an M. smegmatis strain with a mutant cell envelope and to complement the mutation using a genomic library of M. leprae. M. smegmatis mc2155 was successfully mutagenised using /V-methyl-N-Nitro-fV- nitrosoguanidine (NTG), generating 0.1-0.2% auxotrophic mutants. A bank of 2,000 NTG- treated M. smegmatis strains were screened for alterations in phenotypes that may have reflected a change in the cell envelope i.e. mycobacteriophage resistance, temperature sensitivity and increased resistance and sensitivity to antibiotics. M. smegmatis strains with increased resistance to ofloxacin and ciprofloxacin (15) were isolated along with strains more sensitive to penicillin G(5) and pyrazinamide (1). The pyrazinamide sensitive mutant, Pyramidll was further characterised and found to be 20% more sensitive to pyrazinamide than the M. smegmatis mc2155 wild type strain. Pyramidll was found to be less hydrophobic than the wild type strain, variably more sensitive to penicillin G and to exhibit a smooth colony morphology. The cell wall components of Pyramid II were analysed but no gross differences were observed in comparison to the wild type M. smegmatis strain. Pyramidll is believed to contain a mutation effecting its permeability to pyrazinamide. Pyramidll was transformed with an M. leprae genomic pYUB18 shuttle vector library and complementing clones isolated (6%). A complementing cosmid, 57, found to map to cosmid B1308 in the ordered M. leprae library, was used to create a sub-library in pMV206 from which a 3.5kb fragment of complementing M. leprae DNA was isolated and found to contain three complete putative coding regions, possibly involved in osmoregulation

Contenido de glicopeptidolípidos, motilidad y formación de biopelículas en aislados clínicos de Mycobacterium colombiense

Mycobacterium colombiense es una micobacteria perteneciente al Complejo Mycobacterium avium (MAC), aislada a partir de pacientes infectados con el virus de inmunodeficiencia humana (VIH) de la ciudad de Bogotá. M. colombiense ha sido relacionada con la producción de linfadenitis e infecciones pulmonares en pacientes inmunodeprimidos y VIH positivos en Colombia, España y Francia [1][2]. Hasta la fecha, las bases genéticas y morfológicas involucradas en el comportamiento fenotípico de las distintas cepas de M. colombiense son completamente desconocidas. En el presente trabajo se ha demostrado por primera vez que M. colombiense es una micobacteria motil y con capacidad de formar biopelículas (BP) restringida a cepas con morfología de colonia lisa. Hemos observado que las cepas de M. colombiense de morfología lisa contienen en su pared celular glicolípidos con un comportamiento cromatográfico similar a los glicopeptidolípidos (GPLs) de M. avium. Una variante natural rugosa de M. colombiense (57B) mostró ausencia de motilidad, una reducida capacidad de formar BP y sintetizar GPLs en las condiciones experimentales utilizadas en el presente estudio. Utilizando diferentes análisis bioinformáticos, se ubicó una agrupación de genes que podrían estar involucrados en la biosíntesis de GPLs en la cepa de referencia M. colombiense CECT 3035, cuya secuencia genómica se reportó recientemente. Por último, experimentos de qPCR, sugieren que la transcripción de genes posiblemente involucrados en actividades O-metiltransferasa y glicosiltransferasa podrían estar relacionados en la producción de GPLs de las distintas cepas de M. colombiense cultivadas en condiciones planctónicas y de motilidad.Abstract. Mycobacterium colombiense is a novel member of Mycobacterium avium Complex (MAC) that produces respiratory and disseminated infections especially in immunosuppressed patients [1] [2]. At present, the morphologic and genetic bases involved in the phenotypic features of M. colombiense strains are completely unknown. In this study, we show for the first time that M. colombiense strains have the ability to biofilm formation and displayed motility restricted to strains with smooth morphology. In addition, Thin-layer chromatography analysis showed that strains displaying smooth morphology contain glycolipids that migrated as glycopeptidolipids (GPLs). A natural rough variant, M. colombiense 57B, displayed absence of motility and impaired capacity of both, biofilm formation and production of lipids with chromatographic behavior similar to GPLs. According to bioinformatic analyses, a gene cluster of GPLs biosynthesis for the recently sequenced M. colombiense CECT 3035 strain is proposed. Real-time PCR experiments suggested that transcription of genes possibly involved in methyl and glycosyl transfer activity could be correlated to diversity of glycolipids associated to GPLs in M. colombiense strains.Maestrí