-
Journal of Bacteriology Jan 2023The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium... (Review)
Review
The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis, designated mc155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research. Classical bacterial models, such as Escherichia coli, were inadequate for mycobacteria research because they have low genetic conservation, different physiology, and lack the novel envelope structure that distinguishes the Mycobacterium genus. By contrast, M. smegmatis encodes thousands of conserved mycobacterial gene orthologs and has the same cell architecture and physiology. Dissection and characterization of conserved genes, structures, and processes in genetically tractable M. smegmatis mc155 have since provided previously unattainable insights on these same features in its slow-growing relatives. Notably, tuberculosis (TB) drugs, including the first-line drugs isoniazid and ethambutol, are active against M. smegmatis, but not against E. coli, allowing the identification of their physiological targets. Furthermore, Bedaquiline, the first new TB drug in 40 years, was discovered through an M. smegmatis screen. M. smegmatis has become a model bacterium, not only for M. tuberculosis, but for all other Mycobacterium species and related genera. With a repertoire of bioinformatic and physical resources, including the recently established Mycobacterial Systems Resource, M. smegmatis will continue to accelerate mycobacterial research and advance the field of microbiology.
Topics: Humans; Mycobacterium smegmatis; Escherichia coli; Mycobacterium tuberculosis; Tuberculosis; Isoniazid
PubMed: 36598232
DOI: 10.1128/jb.00337-22 -
MBio May 2021The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent warrant new strategies for TB control. The...
The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent warrant new strategies for TB control. The successful use of a bacteriophage cocktail to treat a infection suggests that phages could play a role in tuberculosis therapy. To assemble a phage cocktail with optimal therapeutic potential for tuberculosis, we have explored mycobacteriophage diversity to identify phages that demonstrate tuberculocidal activity and determined the phage infection profiles for a diverse set of strains spanning the major lineages of human-adapted strains of the complex. Using a combination of genome engineering and bacteriophage genetics, we have assembled a five-phage cocktail that minimizes the emergence of phage resistance and cross-resistance to multiple phages, and which efficiently kills the strains tested. Furthermore, these phages function without antagonizing antibiotic effectiveness, and infect both isoniazid-resistant and -sensitive strains. Tuberculosis kills 1.5 million people each year, and resistance to commonly used antibiotics contributes to treatment failures. The therapeutic potential of bacteriophages against offers prospects for shortening antibiotic regimens, provides new tools for treating multiple drug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB infections, and protects newly developed antibiotics against rapidly emerging resistance to them. Identifying a suitable suite of phages active against diverse isolates circumvents many of the barriers to initiating clinical evaluation of phages as part of the arsenal of antituberculosis therapeutics.
Topics: Antitubercular Agents; Humans; Mycobacteriophages; Mycobacterium smegmatis; Mycobacterium tuberculosis; Phage Therapy; Tuberculosis, Multidrug-Resistant
PubMed: 34016711
DOI: 10.1128/mBio.00973-21 -
PLoS Pathogens Jul 2022Mycobacteriophages-bacteriophages infecting Mycobacterium hosts-contribute substantially to our understanding of viral diversity and evolution, provide resources for... (Review)
Review
Mycobacteriophages-bacteriophages infecting Mycobacterium hosts-contribute substantially to our understanding of viral diversity and evolution, provide resources for advancing Mycobacterium genetics, are the basis of high-impact science education programs, and show considerable therapeutic potential. Over 10,000 individual mycobacteriophages have been isolated by high school and undergraduate students using the model organism Mycobacterium smegmatis mc2155 and 2,100 have been completely sequenced, giving a high-resolution view of the phages that infect a single common host strain. The phage genomes are revealed to be highly diverse and architecturally mosaic and are replete with genes of unknown function. Mycobacteriophages have provided many widely used tools for Mycobacterium genetics including integration-proficient vectors and recombineering systems, as well as systems for efficient delivery of reporter genes, transposons, and allelic exchange substrates. The genomic insights and engineering tools have facilitated exploration of phages for treatment of Mycobacterium infections, although their full therapeutic potential has yet to be realized.
Topics: Bacteriophages; Genome, Viral; Humans; Mycobacteriophages; Mycobacterium; Mycobacterium Infections; Mycobacterium smegmatis
PubMed: 35797343
DOI: 10.1371/journal.ppat.1010602 -
Proceedings of the National Academy of... Mar 2023Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the...
Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found in low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox-sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel, comparable to ubiquinone in other structures and suggesting a conserved quinone binding mechanism.
Topics: Electron Transport Complex I; Ubiquinone; Vitamin K 2; Quinones; Mycobacterium smegmatis
PubMed: 36952383
DOI: 10.1073/pnas.2214949120 -
Microbiology (Reading, England) Oct 2023Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a...
Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. has two recognized morphotypes, smooth and rough. Interestingly, exists in only a rough form. The shift from smooth to rough in both and was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among and variants. Deletion of the global regulator shifted the intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.
Topics: Humans; Mycobacterium abscessus; Mycobacterium smegmatis; Mycobacterium Infections, Nontuberculous; Mycobacterium
PubMed: 37862100
DOI: 10.1099/mic.0.001402 -
BioRxiv : the Preprint Server For... Mar 2023The growth and division of mycobacteria, which include several clinically relevant pathogens, deviate significantly from that of canonical bacterial models. Despite...
The growth and division of mycobacteria, which include several clinically relevant pathogens, deviate significantly from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. In addition to being structurally distinct, the molecular components of the mycobacterial envelope are also evolutionarily unique, including the phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM). LM and LAM modulate host immunity during infection, but their role outside of intracellular survival remains poorly understood, despite their widespread conservation among non-pathogenic and opportunistically pathogenic mycobacteria. Previously, and mutants producing structurally altered LM and LAM were shown to grow slowly under certain conditions and to be more sensitive to antibiotics, suggesting that mycobacterial lipoglycans may support cellular integrity or growth. To test this, we constructed multiple biosynthetic lipoglycan mutants of and determined the effect of each mutation on cell wall biosynthesis, envelope integrity, and division. We found that mutants deficient in LAM, but not LM, fail to maintain cell wall integrity in a medium-dependent manner, with envelope deformations specifically associated with septa and new poles. Conversely, a mutant producing abnormally large LAM formed multiseptated cells in way distinct from that observed in a septal hydrolase mutant. These results show that LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell envelope integrity and septal placement.
PubMed: 36993273
DOI: 10.1101/2023.03.26.534150 -
Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass.Current Protocols Jun 2022Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The...
Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac PIM and Ac PIM , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac PIM and 1.8 mg of Ac PIM . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.
Topics: Biomass; Chromatography, Thin Layer; Mannosides; Mycobacterium smegmatis; Mycobacterium tuberculosis; Phosphatidylinositols
PubMed: 35758621
DOI: 10.1002/cpz1.458 -
Transcription Apr 2020Recent biophysical studies of mycobacterial transcription have shed new light on this fundamental process in a group of bacteria that includes deadly pathogens such as... (Review)
Review
Recent biophysical studies of mycobacterial transcription have shed new light on this fundamental process in a group of bacteria that includes deadly pathogens such as ( ( (), as well as the nonpathogenic (). Most of the research has focused on , the causative agent of tuberculosis (TB), which remains one of the top ten causes of death globally. The enzyme RNA polymerase (RNAP) is responsible for all bacterial transcription and is a target for one of the crucial antibiotics used for TB treatment, rifampicin (Rif). Here, we summarize recent biophysical studies of mycobacterial RNAP that have advanced our understanding of the basic process of transcription, have revealed novel paradigms for regulation, and thus have provided critical information required for developing new antibiotics against this deadly disease.
Topics: Mycobacterium; RNA-Dependent RNA Polymerase; Transcription, Genetic; Tuberculosis
PubMed: 31880185
DOI: 10.1080/21541264.2019.1707612 -
Biotechnology Advances Oct 2022After several decades during which proteases and after lipases took the biotransformation world scene as the predominant biocatalysts, a new, promising enzyme was... (Review)
Review
After several decades during which proteases and after lipases took the biotransformation world scene as the predominant biocatalysts, a new, promising enzyme was discovered and characterized. The acyltransferase from Mycobacterium smegmatis (MsAcT) has in fact an extraordinary activity for a wide array of reactions, such as trans-esterification, amidation, trans-amidation and perhydrolysis, both in water and solvent media, giving rise to a series of interesting compounds including APIs (i.e., active pharmaceutical ingredients), natural flavors and fragrances, monomers for polymer synthesis, and peracids employed as disinfectants or antimicrobials. Although the most used acylating agent has been ethyl acetate (EtOAc), depending on the reaction type also acetamide, dimethyl carbonate and a variety of other esters, have been reported. The best yields were reached using very reactive donors such as vinyl or isopropenyl esters (almost complete conversion in rapid reaction times and water media for condensation reactions). In this review article the most innovative scientific advances on MsAcT, its mechanism and engineering are summarized, putting a particular focus on the different kind of processes (batch and flow) that it is possible to carry out using this enzyme as free or immobilized form. In conclusion, the author personal view on the unexplored reaction possibilities using MsAcT is reported as a window on the future of the topic.
Topics: Acyltransferases; Biocatalysis; Enzymes, Immobilized; Esterification; Esters; Mycobacterium smegmatis; Water
PubMed: 35609801
DOI: 10.1016/j.biotechadv.2022.107985 -
Human Vaccines & Immunotherapeutics Dec 2023The immune escape mutations of SARS-CoV-2 variants emerged frequently, posing a new challenge to weaken the protective efficacy of current vaccines. Thus, the...
The immune escape mutations of SARS-CoV-2 variants emerged frequently, posing a new challenge to weaken the protective efficacy of current vaccines. Thus, the development of novel SARS-CoV-2 vaccines is of great significance for future epidemic prevention and control. We herein reported constructing the attenuated () as a bacterial surface display system to carry the spike (S) and nucleocapsid (N) of SARS-CoV-2. To mimic the native localization on the surface of viral particles, the S or N antigen was fused with truncated PE_PGRS33 protein, which is a transportation component onto the cell wall of (). The sub-cellular fraction analysis demonstrated that S or N protein was exactly expressed onto the surface (cell wall) of the recombinant . After the immunization of the -based COVID-19 vaccine candidate in mice, S or N antigen-specific T cell immune responses were effectively elicited, and the subsets of central memory CD4+ T cells and CD8+ T cells were significantly induced. Further analysis showed that there were some potential cross-reactive CTL epitopes between SARS-CoV-2 and . Overall, our data provided insights that -based bacterial surface display system could be a suitable vector for developing T cell-based vaccines against SARS-CoV-2 and other infectious diseases.
Topics: Mice; Humans; Animals; Mycobacterium smegmatis; COVID-19 Vaccines; COVID-19; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 36785935
DOI: 10.1080/21645515.2023.2171233