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Molecular Microbiology Sep 2003Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering... (Review)
Review
Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant to many antibiotics. Pore proteins spanning the outer membrane mediate the diffusion of hydrophilic nutrients. Mycobacterium tuberculosis possesses at least two porins in addition to the low activity channel protein OmpATb. OmpATb is essential for adaptation of M. tuberculosis to low pH and survival in macrophages and mice. The channel activity of OmpATb is likely to play a major role in the defence of M. tuberculosis against acidification within the phagosome of macrophages. MspA is the main porin of Mycobacterium smegmatis. It forms a tetrameric complex with a single central pore of 10 nm length and a cone-like structure. This structure differs clearly from that of the trimeric porins of Gram-negative bacteria, which form one 4 nm long pore per monomer. The 45-fold lower number of porins compared to Gram-negative bacteria and the exceptional length of the pores are two major determinants of the low permeability of the outer membrane of M. smegmatis for hydrophilic solutes. The importance of the synergism between slow transport through the porins and drug efflux or inactivation for the development of drugs against M. tuberculosis is discussed.
Topics: Animals; Bacterial Proteins; Cell Membrane Permeability; Hydrogen-Ion Concentration; Macrophages; Mice; Mycobacterium; Mycobacterium smegmatis; Mycobacterium tuberculosis; Porins; Protein Conformation
PubMed: 12940978
DOI: 10.1046/j.1365-2958.2003.03662.x -
American Journal of Respiratory Cell... Mar 2020
Topics: Humans; Mycobacterium; Mycobacterium Infections, Nontuberculous; Nontuberculous Mycobacteria; Phospholipids
PubMed: 31580721
DOI: 10.1165/rcmb.2019-0324ED -
Nature Communications May 2024Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through...
Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.
Topics: Proteolysis; Mycobacterium smegmatis; Bacterial Proteins; Anti-Bacterial Agents; Mycobacterium tuberculosis; Humans; Microbial Sensitivity Tests; Machine Learning
PubMed: 38744895
DOI: 10.1038/s41467-024-48506-8 -
Scientific Reports Dec 2019Nontuberculous mycobacteria, NTM, are of growing concern and among these members of the Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clades can...
Nontuberculous mycobacteria, NTM, are of growing concern and among these members of the Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clades can cause infections in humans and they are resistant to first-line anti-tuberculosis drugs. They can be isolated from different ecological niches such as soil, tap water and ground water. Mycobacteria, such as Mmuc and Mneo, are classified as rapid growing mycobacteria, RGM, while the most familiar, Mycobacterium tuberculosis, belongs to the slow growing mycobacteria, SGM. Modern "omics" approaches have provided new insights into our understanding of the biology and evolution of this group of bacteria. Here we present comparative genomics data for seventeen NTM of which sixteen belong to the Mmuc- and Mneo-clades. Focusing on virulence genes, including genes encoding sigma/anti-sigma factors, serine threonine protein kinases (STPK), type VII (ESX genes) secretion systems and mammalian cell entry (Mce) factors we provide insight into their presence as well as phylogenetic relationship in the case of the sigma/anti-sigma factors and STPKs. Our data further suggest that these NTM lack ESX-5 and Mce2 genes, which are known to affect virulence. In this context, Mmuc- and Mneo-clade members lack several of the genes in the glycopeptidolipid (GLP) locus, which have roles in colony morphotype appearance and virulence. For the M. mucogenicum type strain, Mmuc, we provide RNASeq data focusing on mRNA levels for sigma factors, STPK, ESX proteins and Mce proteins. These data are discussed and compared to in particular the SGM and fish pathogen Mycobacterium marinum. Finally, we provide insight into as to why members of the Mmuc- and Mneo-clades show resistance to rifampin and isoniazid, and why Mmuc forms a rough colony morphotype.
Topics: Bacterial Proteins; Drug Resistance, Bacterial; Genomics; Humans; Isoniazid; Mycobacteriaceae; Mycobacterium Infections, Nontuberculous; Rifampin; Virulence Factors
PubMed: 31848383
DOI: 10.1038/s41598-019-55464-5 -
Antimicrobial Agents and Chemotherapy Feb 2021Infections caused by nontuberculous mycobacteria (NTM) are increasing globally. complex (MAC) and complex are the most frequently encountered NTM, and oral treatment...
Infections caused by nontuberculous mycobacteria (NTM) are increasing globally. complex (MAC) and complex are the most frequently encountered NTM, and oral treatment options are extremely limited for these pathogens, especially for the complex. In this study, the potency of omadacycline, a new tetracycline derivative, was tested against 111 isolates of NTM. MIC testing was performed as recommended by the Clinical and Laboratory Standards Institute against 70 isolates of rapidly growing mycobacteria (RGM), of which >90% were tetracycline resistant. These included subsp. (20 isolates), subsp. (3), (15 isolates), (7 isolates), the group, including six doxycycline-resistant isolates (12 isolates), and the group, including four doxycycline-resistant isolates (10 isolates). Forty-one isolates of slowly growing mycobacteria (SGM), including 16 isolates of MAC, were also tested. Omadacycline was active against all RGM species, with MIC ranges of 0.004 to 0.25 and 0.06 to 1 μg/ml for 80% and 100% inhibition, respectively. For subsp. , MICs were 0.06 and 0.12 μg/ml with 80% and 100% inhibition, respectively. There was considerable trailing of the omadacycline endpoint with the RGM. MICs of tigecycline exhibited no trailing and were generally within 1 to 2 dilutions of the 100% inhibition omadacycline MICs. While there was no trailing observed in SGM, omadacycline MICs were higher (MIC range, 8 to >16 μg/ml; = 41), as previously noted with tigecycline. This study supports further research of omadacycline, including clinical trials, for the treatment of RGM infections, especially .
Topics: Anti-Bacterial Agents; Humans; Microbial Sensitivity Tests; Mycobacteriaceae; Mycobacterium Infections, Nontuberculous; Nontuberculous Mycobacteria; Tetracyclines
PubMed: 33288634
DOI: 10.1128/AAC.01947-20 -
International Journal of... 2023The introduction of a method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF mass spectrometry) into the practice of...
BACKGROUND
The introduction of a method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF mass spectrometry) into the practice of laboratories significantly increased the identification of acid-resistant bacteria (ARB).
METHODS
Seventy-four nontuberculous mycobacteria (NTM) cultures identified by deoxyribonucleic acid (DNA) hybridization, polymerase chain reaction, Sanger sequencing, and MALDI-ToF mass spectrometry.
RESULTS
Analysis of the identification results obtained by the methods of DNA hybridization and Sanger sequencing showed a complete match only for 67.6% of samples of the total number of cultures included in the study. The partial match of the identification results was 68.9%. When comparing the results of the identification of 74 samples obtained by MALDI-ToF mass spectrometry to the results obtained by sequencing, full match of identification of Mycobacterium chimaera/Mycobacterium intracelullare, Mycobacterium porcinum/Mycobacterium peregrinum and Mycobacterium tuberculosis complex was found for 90.5% of the samples; the partial match of the results - for 4.1%.. DNA hybridization as a method for identifying NTM showed acceptable sensitivity and specificity; however, for mass spectrometry, a significantly higher sensitivity with comparable specificity was determined.
CONCLUSIONS
Mass spectrometry is an important element in the modern system of species identification of microorganisms. The optimization of sample preparation protocols and assessment of the impact on the identification of new techniques of cultivation of microorganisms can significantly improve the quality of identification of microorganisms from the ARB group. In this case, accurate species identification and the development of algorithms for its application will improve the diagnosis of diseases caused by ARB.
Topics: Humans; Angiotensin Receptor Antagonists; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Angiotensin-Converting Enzyme Inhibitors; Nontuberculous Mycobacteria; DNA
PubMed: 37338472
DOI: 10.4103/ijmy.ijmy_68_23 -
MBio May 2019Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many assimilates cholesterol during chronic...
Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many assimilates cholesterol during chronic infection, and its growth in the presence of cholesterol requires most of the MFT biosynthesis genes (, , , , , and ), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in , , and We found that the growth deficit of deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, was indispensable for ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in During ethanol treatment, mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems. Tuberculosis is caused by , and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.
Topics: Biological Factors; Biosynthetic Pathways; Cholesterol; Ethanol; Gene Deletion; Mycobacterium marinum; Mycobacterium smegmatis; Mycobacterium tuberculosis; Peptides
PubMed: 31113891
DOI: 10.1128/mBio.00190-19 -
International Immunopharmacology Feb 2023Macrophage is believed to play a vital role in the fight against Mycobacterium tuberculosis (M.tb) infection by activating autophagy. Recently, receptor-interacting...
Macrophage is believed to play a vital role in the fight against Mycobacterium tuberculosis (M.tb) infection by activating autophagy. Recently, receptor-interacting protein kinase-3 (RIP3), an essential kinase for necroptotic cell death signaling, has been demonstrated to be involved in autophagy. However, RIP3's role in fighting against M.tb infection remains elusive. Here we show that a substantial increase in inflammatory cell infiltration and higher bacterial burden are observed in the lungs of RIP3 mice with Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection. Meanwhile, RIP3 ameliorates lung injury and promote autophagy via induce autophagosome and autophagolysosome formation which indicate that RIP3 is indispensable for host clearance of BCG via autophagy. Mechanically, RIP3 enhances p62 binding to ubiquitylated proteins and LC3 by interacting with p62, and RHIM domain is required for RIP3-p62 interaction. Hence, our results conclusively show that RIP3 impedes M.tb survival and promotes p62-mediated autophagy. The findings provide further insight into understanding the mechanism of M.tb immune escape and pathogenesis of tuberculosis.
Topics: Mice; Animals; Mycobacterium tuberculosis; BCG Vaccine; Tuberculosis; Autophagy; Mycobacterium bovis
PubMed: 36638666
DOI: 10.1016/j.intimp.2023.109696 -
Nature Communications Mar 2024Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be...
Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.
Topics: Mycobacterium smegmatis; Mycobacterium tuberculosis; Proteolysis; Dimerization; Drug Discovery
PubMed: 38443338
DOI: 10.1038/s41467-024-46218-7 -
The European Respiratory Journal Mar 2017
Topics: Humans; Lung Diseases; Mycobacterium avium; Mycobacterium avium Complex; Mycobacterium avium-intracellulare Infection; Sputum
PubMed: 28275180
DOI: 10.1183/13993003.00110-2017