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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 -
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 -
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 -
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 -
Nano Letters Oct 2023Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of...
Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.
Topics: Nanopores; Nucleosides; Reproducibility of Results; Porins; Mycobacterium smegmatis
PubMed: 37818841
DOI: 10.1021/acs.nanolett.3c02872 -
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 -
Frontiers in Cellular and Infection... 2023Mycobacteriophages are viruses that infect members of genus . Because of the rise in antibiotic resistance in mycobacterial diseases such as tuberculosis,...
Mycobacteriophages are viruses that infect members of genus . Because of the rise in antibiotic resistance in mycobacterial diseases such as tuberculosis, mycobacteriophages have received renewed attention as alternative therapeutic agents. Mycobacteriophages are highly diverse, and, on the basis of their genome sequences, they are grouped into 30 clusters and 10 singletons. In this article, we have described the isolation and characterization of a novel mycobacteriophage Kashi-VT1 (KVT1) infecting mc 155 () and isolated from Varanasi, India. KVT1 is a cluster K1 temperate phage that belongs to family as visualized in transmission electron microscopy. The phage genome is 61,010 base pairs with 66.5% Guanine/Cytosine (GC) content, encoding 101 putative open reading frames. The KVT1 genome encodes an immunity repressor, a tyrosine integrase, and an excise protein, which are the characteristics of temperate phages. It also contains genes encoding holin, lysin A, and lysin B involved in host cell lysis. The one-step growth curve demonstrated that KVT1 has a latency time of 90 min and an average burst size of 101 phage particles per infected cell. It can withstand a temperature of up to 45°C and has a maximum viability between pH 8 and 9. Some mycobacteriophages from cluster K are known to infect the pathogenic (); hence, KVT1 holds potential for the phage therapy against tuberculosis, and it can also be engineered to convert into an exclusively lytic phage.
Topics: Humans; Mycobacteriophages; Genome, Viral; Mycobacterium tuberculosis; Mycobacterium smegmatis; Tuberculosis; Bacteriophages
PubMed: 37545854
DOI: 10.3389/fcimb.2023.1173894 -
Journal of Thoracic Disease Aug 2023As the first line of defense, epithelial cells play a vital role in the initiation and control of both innate and adaptive immunity, which participate in the development...
BACKGROUND
As the first line of defense, epithelial cells play a vital role in the initiation and control of both innate and adaptive immunity, which participate in the development of disease. Despite its therapeutic significance, little is understood about the specific interaction between pathogenic microorganisms and lung epithelial cells.
METHODS
In this study, we performed a head-to-head comparison of the virulence and infection mechanisms of () and (), which represent Gram-negative/positive respiratory pathogens, respectively, in lung epithelial cell models for the first time.
RESULTS
Through scanning electron microscopy combined with bacterial infection experiments, we confirmed the ability of and strains to form biofilm and cord factor out of the cell wall. has stronger adhesion and intracellular retention ability, while is more likely to induce acute infection. These pathogens could stay and proliferate in lung epithelial cells and stimulate the secretion of specific cytokines and chemokines through a gene transcription regulator. infection can promote crosstalk among epithelial cells and other immune cells in the lung from a very early stage by prompting the secretion of pro-inflammatory cytokines. Meanwhile, there were significant correlations between infection and higher levels of interleukin-15 (IL-15), interleukin-1Rα (IL-1Rα), fibroblast growth factor (FGF) basic, and granulocyte colony-stimulating factor (G-CSF). At the same time, infection also led to changes in the expression of cytoskeletal proteins in epithelial cells.
CONCLUSIONS
Our results emphasized the immunoprotection and immunomodulation of lung epithelial cells against exogenous pathogenic microorganisms, indicating that different pathogens damaged the host through different strategies and induced varying innate immune responses. At the same time, they provided important clues and key immune factors for dealing with complicated pulmonary infections.
PubMed: 37691650
DOI: 10.21037/jtd-23-493 -
Cell Reports Dec 2021Although prokaryotic organisms lack traditional organelles, they must still organize cellular structures in space and time, challenges that different species solve...
Although prokaryotic organisms lack traditional organelles, they must still organize cellular structures in space and time, challenges that different species solve differently. To systematically define the subcellular architecture of mycobacteria, we perform high-throughput imaging of a library of fluorescently tagged proteins expressed in Mycobacterium smegmatis and develop a customized computational pipeline, MOMIA and GEMATRIA, to analyze these data. Our results establish a spatial organization network of over 700 conserved mycobacterial proteins and reveal a coherent localization pattern for many proteins of known function, including those in translation, energy metabolism, cell growth and division, as well as proteins of unknown function. Furthermore, our pipeline exploits morphologic proxies to enable a pseudo-temporal approximation of protein localization and identifies previously uncharacterized cell-cycle-dependent dynamics of essential mycobacterial proteins. Collectively, these data provide a systems perspective on the subcellular organization of mycobacteria and provide tools for the analysis of bacteria with non-standard growth characteristics.
Topics: Bacterial Proteins; Cell Cycle; Molecular Imaging; Mycobacterium smegmatis; Organelles; Protein Transport; Spatio-Temporal Analysis
PubMed: 34965429
DOI: 10.1016/j.celrep.2021.110154 -
Microbiology (Reading, England) Feb 2021Multi-subunit SMC complexes are required to perform essential functions, such as chromosome compaction, segregation and DNA repair, from bacteria to humans. Prokaryotic...
Multi-subunit SMC complexes are required to perform essential functions, such as chromosome compaction, segregation and DNA repair, from bacteria to humans. Prokaryotic SMC proteins form complexes with two non-SMC subunits, ScpA and ScpB, to condense the chromosome. The mutants of both and genes in have been shown to display characteristic phenotypes such as growth defects and increased frequency of anucleate cells. Here, we studied the function of the Smc-ScpAB complex from . We observed no significant growth difference between the null mutant and wild-type under both standard and stress conditions. Furthermore, we characterized the Smc-ScpAB holocomplex from . The MsSMC consists of the dimerization hinge and ATPase head domains connected by long coiled-coils. The MsSMC interacts with two non-SMC proteins, ScpA and ScpB, and the resulting holocomplex binds to different DNA substrates independent of ATP. The Smc-ScpAB complex showed DNA-stimulated ATPase activity in the presence of ssDNA. A cytological profiling assay revealed that upon overexpression the Smc-ScpAB ternary complex compacts the decondensed nucleoid of rifampicin-treated wild-type and null mutant of . Together, our study suggests that has a functional Smc-ScpAB complex capable of DNA binding and condensation. Based on our observations, we speculate that the presence of alternative SMCs such as MksB or other SMC homologues might have rescued the mutant phenotype in .
Topics: Adenosine Triphosphatases; Bacterial Proteins; Cell Cycle Proteins; Chromosomes, Bacterial; DNA, Bacterial; DNA-Binding Proteins; Multiprotein Complexes; Mutation; Mycobacterium smegmatis; Protein Binding; Protein Domains; Protein Multimerization
PubMed: 33350902
DOI: 10.1099/mic.0.001011