Journal articles on the topic 'Mycobacterium smegmati'
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Deng, Guoying, Fei Zhang, Shufeng Yang, Jian Kang, Shanshan Sha, and Yufang Ma. "Mycobacterium tuberculosis Rv0431 expressed in Mycobacterium smegmati s, a potentially mannosylated protein, mediated the immune evasion of RAW 264.7 macrophages." Microbial Pathogenesis 100 (November 2016): 285–92. http://dx.doi.org/10.1016/j.micpath.2016.10.013.
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Pandey, N., K. Singh, F. Ahmad, and R. Sharma. "Characterization of biofilm formation by Mycobacterium smegmatis during different environmental stress conditions: An in-vitro study." Journal of Environmental Biology 43, no. 6 (November 15, 2022): 771–78. http://dx.doi.org/10.22438/jeb/43/6/mrn-4081.
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Aim: In-vitro characterisation of biofilm produced by Mycobacterium smegmatis (M. smegmatis), a surrogate model for biofilm production by Mycobacteria, and to evaluate the impact of different environmental stress on mycobacterial growth and biofilm formation. Methodology: M. smegmatis biofilms were studied using tissue culture plate and tube adherence methods. Confocal Laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to study the 3D structure and surface morphology, respectively. Additionally, the effect of different environmental stress, such as the absence of essential ions, exposure to harsh environmental conditions, such as acidic environment or oxidative stress, on mycobacterial biofilm formation and mycobacterial growth was assessed. Results: All the exposures, except for carbon supplemented media had a detrimental effect on the number of viable counts and on biofilm formation by mycobacteria (p<0.001). Growth in low pH and oxidative stress was found to be maximum showing reduction by 98% when compared with control. Interpretation: Our findings present various environmental conditions that profoundly affect biofilm formation and thus, may find practical implications in future as effective mycobacterial control strategies having attributes of mycobacterial growth as well as biofilm inhibition. Key words: Biofilm, Environmental stress, Multi-drug resistance, Mycobacterium
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Burguière, Adeline, Paul G. Hitchen, Lynn G. Dover, Anne Dell, and Gurdyal S. Besra. "Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole." Microbiology 151, no. 6 (June 1, 2005): 2087–95. http://dx.doi.org/10.1099/mic.0.27938-0.
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The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium–intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus.
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Cai, Xiaoying, Lei Liu, Chunhong Qiu, Chongzheng Wen, Yao He, Yanxiang Cui, Siyu Li, et al. "Identification and architecture of a putative secretion tube across mycobacterial outer envelope." Science Advances 7, no. 34 (August 2021): eabg5656. http://dx.doi.org/10.1126/sciadv.abg5656.
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Tuberculosis-causing mycobacteria have thick cell-wall and capsule layers that are formed from complex structures. Protein secretion across these barriers depends on a specialized protein secretion system, but none has been reported. We show that Mycobacterium tuberculosis Rv3705c and its homologous MSMEG_6251 in Mycobacterium smegmatis are tube-forming proteins in the mycobacterial envelope (TiME). Crystallographic and cryo-EM structures of these two proteins show that both proteins form rotationally symmetric rings. Two layers of TiME rings pack together in a tail-to-tail manner into a ring-shaped complex, which, in turn, stacks together to form tubes. M. smegmatis TiME was detected mainly in the cell wall and capsule. Knocking out the TiME gene markedly decreased the amount of secreted protein in the M. smegmatis culture medium, and expression of this gene in knocked-out strain partially restored the level of secreted protein. Our structure and functional data thus suggest that TiME forms a protein transport tube across the mycobacterial outer envelope.
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Poupin, P., J. J. Godon, E. Zumstein, and N. Truffaut. "Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450." Canadian Journal of Microbiology 45, no. 3 (March 1, 1999): 209–16. http://dx.doi.org/10.1139/w99-002.
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Nine bacterial strains that grew on morpholine and pyrrolidine as sole carbon, nitrogen, and energy sources were isolated from three different environments with no known morpholine contamination. One of these strains could also degrade piperidine. These bacteria were identified as Mycobacterium strains. A phylogenetic analysis based on the partial 16S rDNA sequences indicated that the isolated strains clustered within the fast growing group of mycobacteria. When the above-mentioned cyclic amines were used as growth substrates, the synthesis of a soluble cytochrome P450 was induced in all these bacteria. Other laboratory strains, Mycobacterium fortuitum and Mycobacterium smegmatis mc2155, were tested for their abilities to degrade morpholine. Neither of them degraded morpholine but could use pyrrolidine and piperidine. The growth of M. fortuitum and M. smegmatis mc2155 on these compounds involved a soluble cytochrome P450, suggesting that mycobacterial strains are naturally able to use pyrrolidine and have developed a similar enzymatic pathway to metabolize this amine.Key words: mycobacteria, morpholine, piperidine, pyrrolidine, cytochrome P450.
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Harth, Günter, Saša Masleša-Galić, and Marcus A. Horwitz. "A two-plasmid system for stable, selective-pressure-independent expression of multiple extracellular proteins in mycobacteria." Microbiology 150, no. 7 (July 1, 2004): 2143–51. http://dx.doi.org/10.1099/mic.0.27113-0.
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Recombinant mycobacteria expressing Mycobacterium tuberculosis extracellular proteins are leading candidates for new vaccines against tuberculosis and other mycobacterial diseases, and important tools both in antimycobacterial drug development and basic research in mycobacterial pathogenesis. Recombinant mycobacteria that stably overexpress and secrete major extracellular proteins of M. tuberculosis in native form on plasmids pSMT3 and pNBV1 were previously constructed by the authors. To enhance the versatility of this plasmid-based approach for mycobacterial protein expression, the Escherichia coli/mycobacteria shuttle plasmid pGB9 was modified to accommodate mycobacterial genes expressed from their endogenous promoters. Previous studies showed that the modified plasmid, designated pGB9.2, derived from the cryptic Mycobacterium fortuitum plasmid pMF1, was present at a low copy number in both E. coli and mycobacteria, and expression of recombinant M. tuberculosis proteins was found to be at levels paralleling its copy number, that is, approximating their endogenous levels. Plasmid pGB9.2 was compatible with the shuttle vectors pSMT3 and pNBV1 and in combination with them it simultaneously expressed the M. tuberculosis 30 kDa extracellular protein FbpB. Plasmid pGB9.2 was stably maintained in the absence of selective pressure in three mycobacterial species: Mycobacterium bovis BCG, M. tuberculosis and M. smegmatis. Plasmid pGB9.2 was found to be self-transmissible between both fast- and slow-growing mycobacteria, but not from mycobacteria to E. coli or between E. coli strains. The combination of two compatible plasmids in one BCG strain allows expression of recombinant mycobacterial proteins at different levels, a potentially important factor in optimizing vaccine potency.
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Converse, Scott E., and Jeffery S. Cox. "A Protein Secretion Pathway Critical for Mycobacterium tuberculosis Virulence Is Conserved and Functional in Mycobacterium smegmatis." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1238–45. http://dx.doi.org/10.1128/jb.187.4.1238-1245.2005.
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ABSTRACT The Snm protein secretion system is a critical determinant of Mycobacterium tuberculosis virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte Mycobacterium smegmatis. We show that the Snm system is operational in M. smegmatis and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in M. smegmatis requires genes that are homologous to those required for secretion in M. tuberculosis. Using a gene knockout strategy in M. smegmatis, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between M. smegmatis and M. tuberculosis, the M. smegmatis Snm system can secrete the M. tuberculosis ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species. M. smegmatis, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.
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Korycka-Machala, Malgorzata, Ewelina Rychta, Anna Brzostek, Heather R. Sayer, Anna Rumijowska-Galewicz, Richard P. Bowater, and Jarosław Dziadek. "Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics." Antimicrobial Agents and Chemotherapy 51, no. 8 (June 4, 2007): 2888–97. http://dx.doi.org/10.1128/aac.00254-07.
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ABSTRACT Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.
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Cayer, Marie-Pierre, Marc Veillette, Pascal Pageau, Richard Hamelin, Marie-Josée Bergeron, Anne Mériaux, Yvon Cormier, and Caroline Duchaine. "Identification of mycobacteria in peat moss processing plants: application of molecular biology approaches." Canadian Journal of Microbiology 53, no. 1 (January 1, 2007): 92–99. http://dx.doi.org/10.1139/w06-105.
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Peat moss processing plant workers are exposed to high concentrations of bioaerosols. Although mycobacteria have been cultured from peat moss, no study has examined the workers' exposure to mycobacterial bioaerosols. We evaluated the presence of mycobacteria in air samples from peat moss processing plants using molecular biology approaches (cloning-sequencing and polymerase chain reaction (PCR)) and the workers exposure using immunoglobulin G (IgG) complexes to mycobacteria. In addition, species detected in air samples and in peat moss were compared. Two peat moss processing plants were chosen among 14 previously studied. A total of 49 clones were sequenced. Real-time PCR was also performed on the same air samples to evaluate the airborne concentration of mycobacteria and estimate exposure levels. Several Mycobacterium species were present in the air samples (M. malmoense, M. smegmatis, M. graceum, M. bohemicum, and M. interjectum). Mycobacterium avium was recovered by culture in peat moss but not in the air using the molecular approach. Total airborne Mycobacterium concentration was estimated at 8.2 × 108/m3. Workers had IgG against the mycobacterial mix and M. avium, suggesting significant exposure. The findings from air samples, supported by IgG measurements, demonstrate that peat moss processing plant workers are exposed to mycobacteria in addition to other biological agents.Key words: exposure, peat moss, airborne mycobacteria.
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El-Etr, Sahar H., Ling Yan, and Jeffrey D. Cirillo. "Fish Monocytes as a Model for Mycobacterial Host-Pathogen Interactions." Infection and Immunity 69, no. 12 (December 1, 2001): 7310–17. http://dx.doi.org/10.1128/iai.69.12.7310-7317.2001.
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ABSTRACT Mycobacterium marinum, a relatively rapid-growing fish and human pathogen, has become an important model for the investigation of mycobacterial pathogenesis. M. marinumis closely related to the Mycobacterium tuberculosiscomplex and causes a disease in fish and amphibians with pathology similar to tuberculosis. We have developed an in vitro model for the study of M. marinum virulence mechanisms using the carp monocytic cell line CLC (carp leukocyte culture). We found that fish monocytes can differentiate between pathogenic and nonpathogenic mycobacterial species. Interestingly, M. marinum enters fish monocytes at a 40- to 60-fold-higher rate thanMycobacterium smegmatis. In addition, M. marinum survives and replicates in fish monocytes whileM. smegmatis is killed. We also found that M. marinum inhibits lysosomal fusion in fish monocytes, indicating that these cells may be used to dissect the mechanisms of intracellular trafficking in mycobacteria. We conclude from these observations that monocytic cells from fish, a natural host for M. marinum, provide an extremely valuable model for the identification and characterization of mycobacterial virulence determinants in the laboratory.
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Bashyam, Murali D., and Anil K. Tyagi. "Identification and Analysis of “Extended −10” Promoters from Mycobacteria." Journal of Bacteriology 180, no. 9 (May 1, 1998): 2568–73. http://dx.doi.org/10.1128/jb.180.9.2568-2573.1998.
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ABSTRACT Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their Escherichia coli counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their E. colicounterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a Mycobacterium tuberculosispromoter and a Mycobacterium smegmatis promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and E. coli suggests that functioning of the TGN motif in promoters of these two species is similar.
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Nguyen, Liem, Satheesh Chinnapapagari, and Charles J. Thompson. "FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis." Journal of Bacteriology 187, no. 19 (October 1, 2005): 6603–11. http://dx.doi.org/10.1128/jb.187.19.6603-6611.2005.
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ABSTRACT Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating α,α′-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.
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Cougoule, Céline, Patricia Constant, Gilles Etienne, Mamadou Daffé, and Isabelle Maridonneau-Parini. "Lack of Fusion of Azurophil Granules with Phagosomes during Phagocytosis of Mycobacterium smegmatis by Human Neutrophils Is Not Actively Controlled by the Bacterium." Infection and Immunity 70, no. 3 (March 2002): 1591–98. http://dx.doi.org/10.1128/iai.70.3.1591-1598.2002.
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ABSTRACT Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as β-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of β-glucuronidase, indicating that M. smegmatis does not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatis opsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatis does not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.
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Malik, Richard, Carolyn O'Brien, and Janet Fyfe. "Infections of cats attributable to slow growing or ‘non-culturable’ mycobacteria." Microbiology Australia 30, no. 2 (2009): 92. http://dx.doi.org/10.1071/ma09092.
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Cats are susceptible to a range of different mycobacterial infections. Tuberculosis has not been seen in domestic species living in Australia (including the cat) for decades. Mycobacterial infections most commonly develop in cats subsequent to penetrating injuries (typically inflicted by other cats) that become contaminated with soil or dirt. Most of these infections are caused by rapidly growing mycobacteria, especially Mycobacterium smegmatis and related species, although occasionally other species such as Mycobacterium avium and Mycobacterium ulcerans are involved. In this report we briefly review infections caused by some novel mycobacterial species, which are either impossible or very difficult to grow in vitro using the usual range of liquid and solid media available in reference laboratories. Our understanding of these infections, sometimes referred to as ?feline leprosy-like syndromes?, has increased greatly since the application of molecular techniques and the systematic investigation of affected cats.
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NIGOU, Jérôme, and Gurdyal S. BESRA. "Cytidine diphosphate-diacylglycerol synthesis in Mycobacterium smegmatis." Biochemical Journal 367, no. 1 (October 1, 2002): 157–62. http://dx.doi.org/10.1042/bj20020370.
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Recent studies have demonstrated that, during infection of macrophages by mycobacteria, phospholipids (PLs) are released from the mycobacterial cell wall within infected macrophages and transported out of this compartment into intracellular vesicles. The release of these PLs may have functions that influence the outcome of mycobacterial infections. Despite their important role, little is known about the biosynthesis of PLs in mycobacteria. In all organisms, PL biosynthesis begins with acylation of sn-glycerol 3-phosphate to form phosphatidic acid (PA), which is then converted to the central liponucleotide intermediate, cytidine diphosphate-diacylglycerol (CDP-DAG) via the CDP-DAG synthase (CDS). The present work examines CDS activity in Mycobacterium smegmatis extracts, with regard to subcellular localization, pH dependence, bivalent and univalent cation requirement, substrate specificity and regulation by nucleotides. We show that CDS activity, which is mainly found within the cytoplasmic membrane, is Mg2+-dependent and activated by K+ ions. Among PAs containing saturated fatty acids, dipalmitoyl-PA is the preferred substrate [Km = 0.23±0.03mM for Triton X-100 (v/v)/PA in the ratio 5:1]. Moreover, CDS activity is inhibited by the reaction products PPi (IC50 = 1.5mM), CDP-DAG (IC50 = 0.3mM) and the nucleotides ATP, UTP and GTP. This study contributes to the delineation of PL biosynthesis in mycobacteria.
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Do, Thi Thuy, Jerónimo Rodríguez-Beltran, Esmeralda Cebrián-Sastre, Alexandro Rodríguez-Rojas, Alfredo Castañeda-García, and Jesús Blázquez. "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis." Antibiotics 11, no. 4 (April 12, 2022): 509. http://dx.doi.org/10.3390/antibiotics11040509.
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Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.
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Gibbons, Henry S., Frank Wolschendorf, Michelle Abshire, Michael Niederweis, and Miriam Braunstein. "Identification of Two Mycobacterium smegmatis Lipoproteins Exported by a SecA2-Dependent Pathway." Journal of Bacteriology 189, no. 14 (May 11, 2007): 5090–100. http://dx.doi.org/10.1128/jb.00163-07.
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ABSTRACT The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and ΔsecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a ΔsecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.
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Pagán-Ramos, E., J. Song, M. McFalone, M. H. Mudd, and V. Deretic. "Oxidative Stress Response and Characterization of theoxyR-ahpC and furA-katG Loci inMycobacterium marinum." Journal of Bacteriology 180, no. 18 (September 15, 1998): 4856–64. http://dx.doi.org/10.1128/jb.180.18.4856-4864.1998.
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ABSTRACT Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and likeMycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate geneahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to theoxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region ofahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katGexpression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream ofkatG in M. marinum. The furA-katGlinkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC andkatG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.
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Guo, Ming, Zhonghe Sun, and Ying Zhang. "Mycobacterium smegmatis Has Two Pyrazinamidase Enzymes, PncA and PzaA." Journal of Bacteriology 182, no. 13 (July 1, 2000): 3881–84. http://dx.doi.org/10.1128/jb.182.13.3881-3884.2000.
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ABSTRACT The Mycobacterium smegmatis pncA gene, encoding nicotinamidase/pyrazinamidase, was identified. While it was similar to counterparts from other mycobacteria, the M. smegmatis PncA had little homology to the other M. smegmatispyrazinamidase/nicotinamidase, encoded by the pzaA gene. Transformation of Mycobacterium bovis strain BCG withM. smegmatis pncA or pzaA conferred susceptibility to pyrazinamide.
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Menendez, M. C., M. J. Garcia, M. C. Navarro, J. A. Gonzalez-y-Merchand, S. Rivera-Gutierrez, L. Garcia-Sanchez, and R. A. Cox. "Characterization of an rRNA Operon (rrnB) of Mycobacterium fortuitum and Other Mycobacterial Species: Implications for the Classification of Mycobacteria." Journal of Bacteriology 184, no. 4 (February 15, 2002): 1078–88. http://dx.doi.org/10.1128/jb.184.4.1078-1088.2002.
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ABSTRACT Mycobacteria are thought to have either one or two rRNA operons per genome. All mycobacteria investigated to date have an operon, designated rrnA, located downstream from the murA gene. We report that Mycobacteriun fortuitum has a second rrn operon, designated rrnB, which is located downstream from the tyrS gene; tyrS is very close to the 3" end of a gene (3-mag) coding for 3-methylpurine-DNA-glycosylase. The second rrn operon of Mycobacterium smegmatis was shown to have a similar organization, namely, 5" 3-mag-tyrS-rrnB 3". The rrnB operon of M. fortuitum was found to have a single dedicated promoter. During exponential growth in a rich medium, the rrnB and rrnA operons were the major and minor contributors, respectively, to pre-rRNA synthesis. Genomic DNA was isolated from eight other fast-growing mycobacterial species. Samples were investigated by Southern blot analysis using probes for murA, tyrS, and 16S rRNA sequences. The results revealed that both rrnA and rrnB operons were present in each species. The results form the basis for a proposed new scheme for the classification of mycobacteria. The approach, which is phylogenetic in concept, is based on particular properties of the rrn operons of a cell, namely, the number per genome and a feature of 16S rRNA gene sequences.
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Chauhan, Priyanka, Santhe Amber van der Meulen, João Miguel Simões Caetano, Hojjat Ghasemi Goojani, Dennis Botman, Rob van Spanning, Holger Lill, and Dirk Bald. "Response of Mycobacterium smegmatis to the Cytochrome bcc Inhibitor Q203." International Journal of Molecular Sciences 23, no. 18 (September 7, 2022): 10331. http://dx.doi.org/10.3390/ijms231810331.
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For the design of next-generation tuberculosis chemotherapy, insight into bacterial defence against drugs is required. Currently, targeting respiration has attracted strong attention for combatting drug-resistant mycobacteria. Q203 (telacebec), an inhibitor of the cytochrome bcc complex in the mycobacterial respiratory chain, is currently evaluated in phase-2 clinical trials. Q203 has bacteriostatic activity against M. tuberculosis, which can be converted to bactericidal activity by concurrently inhibiting an alternative branch of the mycobacterial respiratory chain, cytochrome bd. In contrast, non-tuberculous mycobacteria, such as Mycobacterium smegmatis, show only very little sensitivity to Q203. In this report, we investigated factors that M. smegmatis employs to adapt to Q203 in the presence or absence of a functional cytochrome bd, especially regarding its terminal oxidases. In the presence of a functional cytochrome bd, M. smegmatis responds to Q203 by increasing the expression of cytochrome bcc as well as of cytochrome bd, whereas a M. smegmatisbd-KO strain adapted to Q203 by increasing the expression of cytochrome bcc. Interestingly, single-cell studies revealed cell-to-cell variability in drug adaptation. We also investigated the role of a putative second cytochrome bd isoform postulated for M. smegmatis. Although this putative isoform showed differential expression in response to Q203 in the M. smegmatisbd-KO strain, it did not display functional features similar to the characterised cytochrome bd variant.
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Wolschendorf, Frank, Maysa Mahfoud, and Michael Niederweis. "Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis." Journal of Bacteriology 189, no. 6 (January 5, 2007): 2435–42. http://dx.doi.org/10.1128/jb.01600-06.
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ABSTRACT Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.
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Saviola, Beatrice, Samuel C. Woolwine, and William R. Bishai. "Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology." Infection and Immunity 71, no. 3 (March 2003): 1379–88. http://dx.doi.org/10.1128/iai.71.3.1379-1388.2003.
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ABSTRACT A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.
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Sharbati-Tehrani, Soroush, Joachim Stephan, Gudrun Holland, Bernd Appel, Michael Niederweis, and Astrid Lewin. "Porins limit the intracellular persistence of Mycobacterium smegmatis." Microbiology 151, no. 7 (July 1, 2005): 2403–10. http://dx.doi.org/10.1099/mic.0.27969-0.
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The genus Mycobacterium comprises highly pathogenic as well as opportunistic or apathogenic species exhibiting a great variability with respect to their ability to persist or multiply within monocytic host cells. The impact of the permeability of the mycobacterial outer membrane on intracellular persistence was studied. For this purpose, a Mycobacterium smegmatis mutant with a deletion of the major porin gene mspA and a second mutant lacking mspA and the homologous porin gene mspC were used. Deletion of mspA together with mspC significantly enhanced intracellular persistence in murine bone marrow macrophages, the mouse macrophage cell line J774A.1 and Acanthamoeba castellanii. Complementation of mspA in the porin mutant strains resulted in restoration of the wild-type phenotype with respect to intracellular persistence. This is the first report to show that the deletion of porins of mycobacteria results in improved persistence in eukaryotic cells, demonstrating that the intracellular persistence of M. smegmatis depends upon the permeability of the outer membrane.
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Dahl, John L., Kriti Arora, Helena I. Boshoff, Danelle C. Whiteford, Sophia A. Pacheco, Olaus J. Walsh, Dalia Lau-Bonilla, William B. Davis, and Anthony G. Garza. "The relA Homolog of Mycobacterium smegmatis Affects Cell Appearance, Viability, and Gene Expression." Journal of Bacteriology 187, no. 7 (April 1, 2005): 2439–47. http://dx.doi.org/10.1128/jb.187.7.2439-2447.2005.
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ABSTRACT The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of relMsm . The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.
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Arthur, Patrick K., Vincent Amarh, Precious Cramer, Gloria B. Arkaifie, Ethel J. S. Blessie, Mohammed-Sherrif Fuseini, Isaac Carilo, Rebecca Yeboah, Leonard Asare, and Brian D. Robertson. "Characterization of Two New Multidrug-Resistant Strains of Mycobacterium smegmatis: Tools for Routine In Vitro Screening of Novel Anti-Mycobacterial Agents." Antibiotics 8, no. 1 (January 2, 2019): 4. http://dx.doi.org/10.3390/antibiotics8010004.
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Mycobacterium tuberculosis is a pathogen of global public health concern. This threat is exacerbated by the emergence of multidrug-resistant and extremely-drug-resistant strains of the pathogen. We have obtained two distinct clones of multidrug-resistant Mycobacterium smegmatis after gradual exposure of Mycobacterium smegmatis mc2 155 to increasing concentrations of erythromycin. The resulting resistant strains of Mycobacterium smegmatis exhibited robust viability in the presence of high concentrations of erythromycin and were found to be resistant to a wide range of other antimicrobials. They also displayed a unique growth phenotype in comparison to the parental drug-susceptible Mycobacterium smegmatis mc2 155, and a distinct colony morphology in the presence of cholesterol. We propose that these two multidrug-resistant clones of Mycobacterium smegmatis could be used as model organisms at the inceptive phase of routine in vitro screening of novel antimicrobial agents targeted against multidrug-resistant Mycobacterial tuberculosis.
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Cheadle, Eleanor J., Dearbhaile O'Donnell, Peter J. Selby, and Andrew M. Jackson. "Closely Related Mycobacterial Strains Demonstrate Contrasting Levels of Efficacy as Antitumor Vaccines and Are Processed for Major Histocompatibility Complex Class I Presentation by Multiple Routes in Dendritic Cells." Infection and Immunity 73, no. 2 (February 2005): 784–94. http://dx.doi.org/10.1128/iai.73.2.784-794.2005.
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ABSTRACT Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growing Mycobacterium smegmatis and the slow-growing M. bovis M. bovis BCG, were engineered to express a model tumor antigen, the Kb-restricted dominant cytotoxic T-lymphocyte epitope OVA257-264. Recombinant M. bovis BCG but not recombinant M. smegmatis conferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinant M. smegmatis infection led to a greater degree of dendritic cell maturation than recombinant M. bovis BCG infection. Antigen from recombinant M. smegmatis was processed and presented as OVA257-264 on Kb molecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinant M. bovis BCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growing M. bovis BCG but not that vectored by fast-growing, readily destroyed M. smegmatis is processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development.
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Dziadek, Jaroslaw, Stacey A. Rutherford, Murty V. Madiraju, Mark A. L. Atkinson, and Malini Rajagopalan. "Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene." Microbiology 149, no. 6 (June 1, 2003): 1593–603. http://dx.doi.org/10.1099/mic.0.26023-0.
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To understand the role of Mycobacterium smegmatis ftsZ (ftsZsmeg ) in the cell division process, the ftsZ gene was characterized at the genetic level. This study shows that ftsZsmeg is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy. Expression of ftsZsmeg in M. smegmatis from a constitutively active mycobacterial promoter resulted in lethality whereas that from a chemically inducible acetamidase (ami) promoter led to FtsZ accumulation, filamentation and cell lysis. To further understand the roles of ftsZ in cell division a conditionally complementing ftsZsmeg mutant strain was constructed in which ftsZ expression is controlled by acetamide. Growth in the presence of 0·2 % acetamide increased FtsZ levels approximately 1·4-fold, but did not decrease viability or change cell length. Withdrawal of acetamide reduced FtsZ levels, decreased viability, increased cell length and eventually lysed the cells. Finally, it is shown that ftsZsmeg function in M. smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZ tb can independently initiate the formation of Z-rings and catalyse the septation process. It is concluded that optimal levels of M. smegmatis FtsZ are required to sustain cell division and that the cell division initiation mechanisms are similar in mycobacteria.
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Park, Sae W., Eun H. Hwang, Hyuck Park, Jeong A. Kim, Jinho Heo, Key H. Lee, Taeksun Song, et al. "Growth of Mycobacteria on Carbon Monoxide and Methanol." Journal of Bacteriology 185, no. 1 (January 1, 2003): 142–47. http://dx.doi.org/10.1128/jb.185.1.142-147.2003.
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ABSTRACT Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.
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Marmiesse, Magali, Priscille Brodin, Carmen Buchrieser, Christina Gutierrez, Nathalie Simoes, Veronique Vincent, Philippe Glaser, Stewart T. Cole, and Roland Brosch. "Macro-array and bioinformatic analyses reveal mycobacterial ‘core’ genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex." Microbiology 150, no. 2 (February 1, 2004): 483–96. http://dx.doi.org/10.1099/mic.0.26662-0.
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To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter ‘core’ genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the ‘core’ genes conserved among M. tuberculosis H37Rv and M. leprae are also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented.
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Tran, Huyen Thi, Julia Solnier, Eva-Maria Pferschy-Wenzig, Olaf Kunert, Liam Martin, Sanjib Bhakta, Loi Huynh, Tri Minh Le, Rudolf Bauer, and Franz Bucar. "Antimicrobial and Efflux Pump Inhibitory Activity of Carvotacetones from Sphaeranthus africanus Against Mycobacteria." Antibiotics 9, no. 7 (July 8, 2020): 390. http://dx.doi.org/10.3390/antibiotics9070390.
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Carvotacetones (1–7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.
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Sharbati, Soroush, Faustine Ravon, Ralf Einspanier, and Jennifer zur Bruegge. "Mycobacterium smegmatis But Not Mycobacterium avium subsp. hominissuis Causes Increased Expression of the Long Non-Coding RNA MEG3 in THP-1-Derived Human Macrophages and Associated Decrease of TGF-β." Microorganisms 7, no. 3 (February 27, 2019): 63. http://dx.doi.org/10.3390/microorganisms7030063.
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Pathogenic mycobacteria are able to persist intracellularly in macrophages, whereas non-pathogenic mycobacteria are effectively combated and eliminated after their phagocytosis. It is known that TGF-β plays an important role in this context. Infection with pathogenic mycobacteria such as Mycobacterium tuberculosis or M. avium leads to production of active TGF-β, which blocks the ability of IFN-γ and TNF-α to inhibit intracellular replication. On the other hand, it is known that the long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) is involved in the regulation of TGF-β. In this study, we show how the infection of THP-1-derived human macrophages with the saprophytic M. smegmatis but not with the facultatively pathogenic M. avium subsp. hominissuis leads to increased MEG3 expression. This is associated with the downregulation of DNA methyltransferases (DNMT) 1 and 3b, which are known to regulate MEG3 expression via promoter hypermethylation. Consequently, we observe a significant downregulation of TGF-β in M. smegmatis-infected macrophages but not in M. avium subsp. hominissuis pointing to lncRNAs as novel mediators of host cell response during mycobacterial infections.
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Udou, Takezo. "Extracellular hemolytic activity in rapidly growing mycobacteria." Canadian Journal of Microbiology 40, no. 4 (April 1, 1994): 318–21. http://dx.doi.org/10.1139/m94-052.
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Little is known about virulence factors associated with rapidly growing mycobacteria. We evaluated 42 clinical isolates of Mycobacterium fortuitum and Mycobacterium chelonae and 4 reference strains of Mycobacterium smegmatis for the production of hemolysin (or hemolytic substance) as a possible contributor to the pathogenesis of disease caused by these organisms. All the strains tested possessed extracellular hemolytic activity that was stable after heating and proteinase treatment, and the active substance had a molecular weight less than 10 000. The activity accumulated in culture medium during the late exponential to mid stationary phase of growth. Hemolysis in vitro was relatively slow; incubation for 10 h at 35 °C was required to obtain maximal activity. Some specificity of the hemolysis with regard to red blood cells from different animals was observed.Key words: rapidly growing mycobacteria, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium smegmatis, hemolytic activity.
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Šimunović, Katarina, Julia Solnier, Fabian Alperth, Olaf Kunert, Sonja Smole Smole Možina, and Franz Bucar. "Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins." Antibiotics 10, no. 9 (September 5, 2021): 1075. http://dx.doi.org/10.3390/antibiotics10091075.
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Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, Peucedanum ostruthium extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in Mycobacterium smegmatis. P. ostruthium extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in M. smegmatis. The plant P. ostruthuim has a complex effect on M. smegmatis, including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes P. ostruthium and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.
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Nash, Kevin A. "Intrinsic Macrolide Resistance in Mycobacterium smegmatis Is Conferred by a Novel erm Gene, erm(38)." Antimicrobial Agents and Chemotherapy 47, no. 10 (October 2003): 3053–60. http://dx.doi.org/10.1128/aac.47.10.3053-3060.2003.
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ABSTRACT High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc2155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 μg/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.
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Fernandes, Norvin D., Qi-long Wu, Dequan Kong, Xiaoling Puyang, Sumeet Garg, and Robert N. Husson. "A Mycobacterial Extracytoplasmic Sigma Factor Involved in Survival following Heat Shock and Oxidative Stress." Journal of Bacteriology 181, no. 14 (July 15, 1999): 4266–74. http://dx.doi.org/10.1128/jb.181.14.4266-4274.1999.
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ABSTRACT Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor, sigH, was cloned fromMycobacterium tuberculosis and Mycobacterium smegmatis. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified inM. smegmatis and one of two identified in M. bovis BCG were found to have −35 promoter sequences that match the ECF-dependent −35 promoter consensus. Expression from these promoters was strongly induced by 50°C heat shock. In comparison to the wild type, an M. smegmatis sigH mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an M. smegmatis sigH sigE double mutant was found to be markedly decreased following 53°C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the sigH operon is required for complementation of the sigH stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.
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McDonough, Justin A., Kari E. Hacker, Anthony R. Flores, Martin S. Pavelka, and Miriam Braunstein. "The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial β-Lactamases." Journal of Bacteriology 187, no. 22 (November 15, 2005): 7667–79. http://dx.doi.org/10.1128/jb.187.22.7667-7679.2005.
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ABSTRACT The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of ΔtatA and ΔtatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active β-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active ′BlaC. Thus, ′BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.
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Su, Chih-Chia, Philip A. Klenotic, Meng Cui, Meinan Lyu, Christopher E. Morgan, and Edward W. Yu. "Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport." PLOS Biology 19, no. 8 (August 12, 2021): e3001370. http://dx.doi.org/10.1371/journal.pbio.3001370.
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The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.
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Acharya, Narottam, Pradeep Kumar, and Umesh Varshney. "Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases." Microbiology 149, no. 7 (July 1, 2003): 1647–58. http://dx.doi.org/10.1099/mic.0.26228-0.
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Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the β1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E. coli (Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the β1-edge of Ugi) and with mutants of EcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.
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Vieira, Otilia V., Rene E. Harrison, Cameron C. Scott, Harald Stenmark, David Alexander, Jun Liu, Jean Gruenberg, Alan D. Schreiber, and Sergio Grinstein. "Acquisition of Hrs, an Essential Component of Phagosomal Maturation, Is Impaired by Mycobacteria." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4593–604. http://dx.doi.org/10.1128/mcb.24.10.4593-4604.2004.
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ABSTRACT Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.
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Radhakrishnan, Anjana, Christopher M. Furze, Mohd Syed Ahangar, and Elizabeth Fullam. "A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis." RSC Advances 8, no. 58 (2018): 33087–95. http://dx.doi.org/10.1039/c8ra06237d.
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Ezquerra-Aznárez, José Manuel, Giulia Degiacomi, Henrich Gašparovič, Giovanni Stelitano, Josè Camilla Sammartino, Jana Korduláková, Paolo Governa, et al. "The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme." International Journal of Molecular Sciences 23, no. 2 (January 11, 2022): 771. http://dx.doi.org/10.3390/ijms23020771.
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Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-β-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.
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Agustí, Gemma, Oihane Astola, Elisabeth Rodríguez-Güell, Esther Julián, and Marina Luquin. "Surface Spreading Motility Shown by a Group of Phylogenetically Related, Rapidly Growing Pigmented Mycobacteria Suggests that Motility Is a Common Property of Mycobacterial Species but Is Restricted to Smooth Colonies." Journal of Bacteriology 190, no. 20 (August 8, 2008): 6894–902. http://dx.doi.org/10.1128/jb.00572-08.
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ABSTRACT Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.
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Meyers, Paul R., William R. Bourn, Lafras M. Steyn, Paul D. van Helden, Albert D. Beyers, and Gordon D. Brown. "Novel Method for Rapid Measurement of Growth of Mycobacteria in Detergent-Free Media." Journal of Clinical Microbiology 36, no. 9 (1998): 2752–54. http://dx.doi.org/10.1128/jcm.36.9.2752-2754.1998.
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We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, andMycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.
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Mo, Yongkai, Natalie M. Quanquin, William H. Vecino, Uma Devi Ranganathan, Lydia Tesfa, William Bourn, Keith M. Derbyshire, Norman L. Letvin, William R. Jacobs, and Glenn J. Fennelly. "Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization." Infection and Immunity 75, no. 10 (July 30, 2007): 4804–16. http://dx.doi.org/10.1128/iai.01877-06.
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ABSTRACT Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120h E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120h E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.
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Liu, Hanrui, Xuwen Gui, Shixing Chen, Weizhe Fu, Xiang Li, Tingyuan Xiao, Jie Hou, and Tao Jiang. "Structural Variability of Lipoarabinomannan Modulates Innate Immune Responses within Infected Alveolar Epithelial Cells." Cells 11, no. 3 (January 21, 2022): 361. http://dx.doi.org/10.3390/cells11030361.
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Mycobacterium tuberculosis (M. tb) is an intracellular pathogen persisting in phagosomes that has the ability to escape host immune surveillance causing tuberculosis (TB). Lipoarabinomannan (LAM), as a glycolipid, is one of the complex outermost components of the mycobacterial cell envelope and plays a critical role in modulating host responses during M. tb infection. Different species within the Mycobacterium genus exhibit distinct LAM structures and elicit diverse innate immune responses. However, little is known about the mechanisms. In this study, we first constructed a LAM-truncated mutant with fewer arabinofuranose (Araf) residues named M. sm-ΔM_6387 (Mycobacterium smegmatis arabinosyltransferase EmbC gene knockout strain). It exhibited some prominent cell wall defects, including tardiness of mycobacterial migration, loss of acid-fast staining, and increased cell wall permeability. Within alveolar epithelial cells (A549) infected by M. sm-ΔM_6387, the uptake rate was lower, phagosomes with bacterial degradation appeared, and microtubule-associated protein light chain 3 (LC3) recruitment was enhanced compared to wild type Mycobacterium smegmatis (M. smegmatis). We further confirmed that the variability in the removal capability of M. sm-ΔM_6387 resulted from host cell responses rather than the changes in the mycobacterial cell envelope. Moreover, we found that M. sm-ΔM_6387 or its glycolipid extracts significantly induced expression changes in some genes related to innate immune responses, including Toll-like receptor 2 (TLR2), class A scavenger receptor (SR-A), Rubicon, LC3, tumor necrosis factor alpha (TNF-α), Bcl-2, and Bax. Therefore, our studies suggest that nonpathogenic M. smegmatis can deposit LC3 on phagosomal membranes, and the decrease in the quantity of Araf residues for LAM molecules not only impacts mycobacterial cell wall integrity but also enhances host defense responses against the intracellular pathogens and decreases phagocytosis of host cells.
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Fields, Christopher J., and Robert L. Switzer. "Regulation of pyr Gene Expression in Mycobacterium smegmatis by PyrR-Dependent Translational Repression." Journal of Bacteriology 189, no. 17 (June 29, 2007): 6236–45. http://dx.doi.org/10.1128/jb.00803-07.
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ABSTRACT Regulation of pyrimidine biosynthetic (pyr) genes by a transcription attenuation mechanism that is mediated by the PyrR mRNA-binding regulatory protein has been demonstrated for numerous gram-positive bacteria. Mycobacterial genomes specify pyrR genes and contain obvious PyrR-binding sequences in the initially transcribed regions of their pyr operons, but transcription antiterminator and attenuation terminator sequences are absent from their pyr 5′ leader regions. This work demonstrates that repression of pyr operon expression in Mycobacterium smegmatis by exogenous uracil requires the pyrR gene and the pyr leader RNA sequence for binding of PyrR. Plasmids containing the M. smegmatis pyr promoter-leader region translationally fused to lacZ also displayed pyrR-dependent repression, but transcriptional fusions of the same sequences to a lacZ gene that retained the lacZ ribosome-binding site were not regulated by PyrR plus uracil. We propose that PyrR regulates pyr expression in M. smegmatis, other mycobacteria, and probably in numerous other bacteria by a translational repression mechanism in which nucleotide-regulated binding of PyrR occludes the first ribosome-binding site of the pyr operon.
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Hosseiniporgham, Sepideh, and Leonardo A. Sechi. "A Review on Mycobacteriophages: From Classification to Applications." Pathogens 11, no. 7 (July 7, 2022): 777. http://dx.doi.org/10.3390/pathogens11070777.
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Mycobacterial infections are a group of life-threatening conditions triggered by fast- or slow-growing mycobacteria. Some mycobacteria, such as Mycobacterium tuberculosis, promote the deaths of millions of lives throughout the world annually. The control of mycobacterial infections is influenced by the challenges faced in the diagnosis of these bacteria and the capability of these pathogens to develop resistance against common antibiotics. Detection of mycobacterial infections is always demanding due to the intracellular nature of these pathogens that, along with the lipid-enriched structure of the cell wall, complicates the access to the internal contents of mycobacterial cells. Moreover, recent studies depicted that more than 20% of M. tuberculosis (Mtb) infections are multi-drug resistant (MDR), and only 50% of positive MDR-Mtb cases are responsive to standard treatments. Similarly, the susceptibility of nontuberculosis mycobacteria (NTM) to first-line tuberculosis antibiotics has also declined in recent years. Exploiting mycobacteriophages as viruses that infect mycobacteria has significantly accelerated the diagnosis and treatment of mycobacterial infections. This is because mycobacteriophages, regardless of their cycle type (temperate/lytic), can tackle barriers in the mycobacterial cell wall and make the infected bacteria replicate phage DNA along with their DNA. Although the infectivity of the majority of discovered mycobacteriophages has been evaluated in non-pathogenic M. smegmatis, more research is still ongoing to find mycobacteriophages specific to pathogenic mycobacteria, such as phage DS6A, which has been shown to be able to infect members of the M. tuberculosis complex. Accordingly, this review aimed to introduce some potential mycobacteriophages in the research, specifically those that are infective to the three troublesome mycobacteria, M. tuberculosis, M. avium subsp. paratuberculosis (MAP), and M. abscessus, highlighting their theranostic applications in medicine.
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Cascioferro, Alessandro, Francesca Boldrin, Agnese Serafini, Roberta Provvedi, Giorgio Pal�, and Riccardo Manganelli. "Xer Site-Specific Recombination, an Efficient Tool To Introduce Unmarked Deletions into Mycobacteria." Applied and Environmental Microbiology 76, no. 15 (June 11, 2010): 5312–16. http://dx.doi.org/10.1128/aem.00382-10.
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ABSTRACT Genetic manipulation of mycobacteria still represents a serious challenge due to the lack of tools and selection markers. In this report, we describe the development of an intrinsically unstable excisable cassette for introduction of unmarked mutations in both Mycobacterium smegmatis and Mycobacterium tuberculosis.
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Nguyen, Kiet T., Kristina Piastro, and Keith M. Derbyshire. "LpqM, a Mycobacterial Lipoprotein-Metalloproteinase, Is Required for Conjugal DNA Transfer in Mycobacterium smegmatis." Journal of Bacteriology 191, no. 8 (February 20, 2009): 2721–27. http://dx.doi.org/10.1128/jb.00024-09.
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ABSTRACT We have previously described a novel conjugal DNA transfer process that occurs in Mycobacterium smegmatis. To identify donor genes required for transfer, we have performed a transposon mutagenesis screen; we report here that LpqM, a putative lipoprotein-metalloproteinase, is essential for efficient DNA transfer. Bioinformatic analyses predict that LpqM contains a signal peptide necessary for the protein's targeting to the cell envelope and a metal ion binding motif, the likely catalytic site for protease activity. Using targeted mutagenesis, we demonstrate that each of these motifs is necessary for DNA transfer and that LpqM is located in the cell envelope. The requirement for transfer is specific to the donor strain; an lpqM knockout mutant in the recipient is still proficient in transfer assays. The activity of LpqM is conserved among mycobacteria; homologues from both Mycobacterium tuberculosis and Mycobacterium avium can complement lpqM donor mutants, suggesting that the homologues recognize and process similar proteins. Lipoproteins constitute a significant proportion of the mycobacterial cell wall, but despite their abundance, very few have been assigned an activity. We discuss the potential role of LpqM in DNA transfer and the implications of the conservation of LpqM activity in M. tuberculosis.