scholarly journals A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis.

1997 ◽  
Vol 41 (12) ◽  
pp. 2629-2633 ◽  
Author(s):  
M A Lety ◽  
S Nair ◽  
P Berche ◽  
V Escuyer
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Single Point ◽  
Mycobacterium Leprae ◽  
Single Point Mutation ◽  
Missense Mutations ◽  
Mycobacterial Species ◽  
Mechanisms Of Resistance ◽  
Embb Gene ◽  
Mycobacterial Cell Wall

Ethambutol [EMB; dextro-2,2'-(ethylenediimino)-di-1-butanol] is an effective drug when used in combination with isoniazid for the treatment of tuberculosis. It inhibits the polymerization of arabinan in the arabinogalactan and lipoarabinomannan of the mycobacterial cell wall. Recent studies have shown that arabinosyltransferases could be targets of EMB. These enzymes are encoded by the emb locus that was identified in Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium avium, and Mycobacterium tuberculosis. We demonstrate that a missense mutation in the M. smegmatis embB gene, one of the genes of the emb locus, confers resistance to EMB. The level of resistance is not dependent on the number of copies of the mutated embB gene, indicating that this is a true mechanism of resistance. The mutation is located in a region of the EmbB protein that is highly conserved among the different mycobacterial species. We also identified in this region two other independent mutations that confer EMB resistance. Furthermore, mutations have recently been described in the same region of the EmbB protein from clinical EMB-resistant M. tuberculosis isolates. Together, these data strongly suggest that one of the mechanisms of resistance to EMB consists of missense mutations in a particular region of the EmbB protein that could be directly involved in the interaction with the EMB molecule.

scholarly journals Mycobacterium tuberculosis Lipomannan Induces Apoptosis and Interleukin-12 Production in Macrophages

2004 ◽  
Vol 72 (4) ◽  
pp. 2067-2074 ◽  
Author(s):  
D. N. Dao ◽  
L. Kremer ◽  
Y. Guérardel ◽  
A. Molano ◽  
W. R. Jacobs ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Function Analysis ◽  
Specific Interaction ◽  
Interleukin 12 ◽  
Mycobacterial Species ◽  
Immunostimulatory Activity ◽  
Mycobacterium Chelonae ◽  
Toll Like Receptor 2 ◽  
Mycobacterial Cell Wall

ABSTRACT The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as a virulence factor of Mycobacterium tuberculosis, and modification of the terminal arabinan residues of this compound with mannose caps (producing mannosyl-capped LAM [ManLAM]) in M. tuberculosis or with phosphoinositol caps (producing phosphoinositol-capped LAM [PILAM]) in Mycobacterium smegmatis has been implicated in various functions associated with these lipoglycans. A structure-function analysis was performed by using LAMs and their biosynthetic precursor lipomannans (LMs) isolated from different mycobacterial species on the basis of their capacity to induce the production of interleukin-12 (IL-12) and/or apoptosis of macrophage cell lines. Independent of the mycobacterial species, ManLAMs did not induce IL-12 gene expression or apoptosis of macrophages, whereas PILAMs induced IL-12 secretion and apoptosis. Interestingly, uncapped LAM purified from Mycobacterium chelonae did not induce IL-12 secretion or apoptosis. Furthermore, LMs, independent of their mycobacterial origins, were potent inducers of IL-12 and apoptosis. The precursor of LM, phosphatidyl-myo-inositol dimannoside, had no activity, suggesting that the mannan core of LM was required for the activity of LM. The specific interaction of LM with Toll-like receptor 2 (TLR-2) but not with TLR-4 suggested that these responses were mediated via the TLR-2 signaling pathway. Our experiments revealed an important immunostimulatory activity of the biosynthetic LAM precursor LM. The ratio of LAM to LM in the cell wall of mycobacteria may be an important determinant of virulence, and enzymes that modify LM could provide targets for development of antituberculosis drugs and for derivation of attenuated strains of M. tuberculosis.

scholarly journals Cell Wall Core Galactofuran Synthesis Is Essential for Growth of Mycobacteria

2001 ◽  
Vol 183 (13) ◽  
pp. 3991-3998 ◽  
Author(s):  
Fei Pan ◽  
Mary Jackson ◽  
Yufang Ma ◽  
Michael McNeil
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Separate Experiment ◽  
Temperature Sensitive ◽  
Origin Of Replication ◽  
Knockout Mutant ◽  
Allelic Replacement ◽  
Higher Temperature ◽  
Mycobacterial Cell Wall

ABSTRACT The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here thatglf can be knocked out in Mycobacterium smegmatis by allelic replacement only in the presence of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of replication and the M. smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and theglf-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both glf and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.

scholarly journals Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics

2013 ◽  
Vol 57 (9) ◽  
pp. 4267-4275 ◽  
Author(s):  
Yong Heon Lee ◽  
John D. Helmann
Keyword(s):  
Cell Wall ◽  
Single Point ◽  
Single Point Mutation ◽  
Wild Type ◽  
Content Type ◽  
Ribosome Binding ◽  
Peptidoglycan Biosynthesis ◽  
Elevated Expression ◽  
Antibacterial Target ◽  
Bacterial Peptidoglycan

ABSTRACTUndecaprenyl pyrophosphate synthase (UppS) catalyzes the formation of the C55lipid carrier (UPP) that is essential for bacterial peptidoglycan biosynthesis. We selected here a vancomycin (VAN)-resistant derivative ofBacillus subtilisW168 that contains a single-point mutation in the ribosome-binding site of theuppSgene designateduppS1. Genetic reconstruction experiments demonstrate that theuppS1allele is sufficient to confer low-level VAN resistance and causes reduced UppS translation. The decreased level of UppS rendersB. subtilisslightly more susceptible to many late-acting cell wall antibiotics, including β-lactams, but significantly more resistant to fosfomycin andd-cycloserine, antibiotics that interfere with the very early steps of cell wall synthesis. We further show that theuppS1allele leads to slightly elevated expression of the σMregulon, possibly helping to compensate for the stress caused by a decrease in UPP levels. Notably, theuppS1mutation increases resistance to VAN, fosfomycin, andd-cycloserine in wild-type cells, but this effect is greatly reduced or eliminated in asigMmutant background. Our findings suggest that, although UppS is an attractive antibacterial target, incomplete inhibition of UppS function may lead to increased resistance to some cell wall-active antibiotics.

scholarly journals Functional Complementation of the Essential Gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but Not Escherichia coli fabG

2007 ◽  
Vol 189 (10) ◽  
pp. 3721-3728 ◽  
Author(s):  
Tanya Parish ◽  
Gretta Roberts ◽  
Francoise Laval ◽  
Merrill Schaeffer ◽  
Mamadou Daffé ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Essential Gene ◽  
Permeability Barrier ◽  
Type I ◽  
Type Ii ◽  
Mycolic Acids ◽  
Mycobacterial Cell Wall

ABSTRACT Mycolic acids are a key component of the mycobacterial cell wall, providing structure and forming a major permeability barrier. In Mycobacterium tuberculosis mycolic acids are synthesized by type I and type II fatty acid synthases. One of the enzymes of the type II system is encoded by fabG1. We demonstrate here that this gene can be deleted from the M. tuberculosis chromosome only when another functional copy is provided elsewhere, showing that under normal culture conditions fabG1 is essential. FabG1 activity can be replaced by the corresponding enzyme from the closely related species Mycobacterium smegmatis but not by the enzyme from Escherichia coli. M. tuberculosis carrying FabG from M. smegmatis showed no phenotypic changes, and both the mycolic acids and cell wall permeability were unchanged. Thus, M. tuberculosis and M. smegmatis enzymes are interchangeable and do not control the lengths and types of mycolic acids synthesized.

scholarly journals The relA Homolog of Mycobacterium smegmatis Affects Cell Appearance, Viability, and Gene Expression

2005 ◽  
Vol 187 (7) ◽  
pp. 2439-2447 ◽  
Author(s):  
John L. Dahl ◽  
Kriti Arora ◽  
Helena I. Boshoff ◽  
Danelle C. Whiteford ◽  
Sophia A. Pacheco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Metabolic Pathways ◽  
Pathogenic Bacteria ◽  
Regulatory Mechanism ◽  
Stringent Response ◽  
Wall Structure ◽  
Mycobacterial Species

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.

scholarly journals Mutations at embB Codon 306 Are an Important Molecular Indicator of Ethambutol Resistance in Mycobacterium tuberculosis

2008 ◽  
Vol 53 (3) ◽  
pp. 1061-1066 ◽  
Author(s):  
Angela M. Starks ◽  
Aysel Gumusboga ◽  
Bonnie B. Plikaytis ◽  
Thomas M. Shinnick ◽  
James E. Posey
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Allele ◽  
Single Point ◽  
Single Point Mutation ◽  
Missense Mutations ◽  
Wild Type ◽  
Molecular Tests ◽  
Resistant Mutants ◽  
Spontaneous Mutants

ABSTRACT Ethambutol resistance in clinical Mycobacterium tuberculosis isolates is associated primarily with missense mutations in the embB gene. However, recent reports have described the presence of embB mutations, especially those at embB codon 306, in isolates susceptible to ethambutol. To clarify the role of embB mutations in ethambutol resistance, we sequenced the ethambutol resistance-determining region in spontaneous ethambutol-resistant mutants. In our study, 66% of spontaneous mutants contained a single point mutation in embB, with 55% of these occurring at embB 306. The MIC of ethambutol for spontaneous mutants was increased two- to eightfold relative to the pansusceptible M. tuberculosis strains from which the mutants were generated. To further characterize the role of embB 306 mutations, we directly introduced mutant alleles, embB(M306V) or embB(M306I), into pansusceptible M. tuberculosis strains and conversely reverted mutant alleles in spontaneous ethambutol-resistant mutants back to those of the wild type via allelic exchange using specialized linkage transduction. We determined that the MIC of ethambutol was reduced fourfold for three of the four spontaneous ethambutol-resistant embB 306 mutants when the mutant allele was replaced with the wild-type embB allele. The MIC for one of the spontaneous mutants genetically reverted to wild-type embB was reduced by only twofold. When the wild-type embB allele was converted to the mutant allele embB(M306V), the ethambutol MIC was increased fourfold, and when the allele was changed to M306I, the ethambutol MIC increased twofold. Our data indicate that embB 306 mutations are sufficient to confer ethambutol resistance, and detection of these mutations should be considered in the development of rapid molecular tests.

scholarly journals Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Filipa Gil ◽  
Anna E. Grzegorzewicz ◽  
Maria João Catalão ◽  
João Vital ◽  
Michael R. McNeil ◽  
...  
Keyword(s):  
Cell Wall ◽  
Outer Membrane ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Head Group ◽  
Lipolytic Enzyme ◽  
Ester Bond ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan–peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6′-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

scholarly journals FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis

2005 ◽  
Vol 187 (19) ◽  
pp. 6603-6611 ◽  
Author(s):  
Liem Nguyen ◽  
Satheesh Chinnapapagari ◽  
Charles J. Thompson
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Wall Structure ◽  
Single Mutation ◽  
Mycolic Acids ◽  
Trehalose Dimycolate ◽  
Colonial Morphology ◽  
Mycobacterial Cell Wall

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.

scholarly journals The conserved translation factor LepA is required for optimal synthesis of a porin family in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Skye R.S. Fishbein ◽  
Francesca G. Tomasi ◽  
Ian D. Wolf ◽  
Charles L. Dulberger ◽  
Albert Wang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Antibiotic Susceptibility ◽  
Drug Targets ◽  
Drug Tolerance ◽  
Drug Susceptibility ◽  
Cell Integrity ◽  
Translation Factor ◽  
Associated Proteins ◽  
Mycobacterial Cell Wall

The recalcitrance of mycobacteria to antibiotic therapy is in part due to its ability to build proteins into a multi-layer cell wall. Proper synthesis of both cell wall constituents and associated proteins is crucial to maintaining cell integrity, and intimately tied to antibiotic susceptibility. How mycobacteria properly synthesize the membrane-associated proteome, however, remains poorly understood. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. LepA is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cellular processes is not clear. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to strains with lepA. We find that LepA is important for the steady-state abundance of a number of membrane-associated proteins, including an outer membrane porin, MspA, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane which leads to the drug tolerance phenotype of the lepA mutant. In mycobacteria, the translation factor LepA modulates mycobacterial membrane homeostasis, which in turn affects antibiotic tolerance. Importance The mycobacterial cell wall is a promising target for new antibiotics due to the abundance of important membrane-associated proteins. Defining mechanisms of synthesis of the membrane proteome will be critical to uncovering and validating drug targets. We found that LepA, a universally conserved translation factor, controls the synthesis of a number of major membrane proteins in M. smegmatis. LepA primarily controls synthesis of the major porin MspA. Loss of LepA results in decreased permeability through the loss of this porin, including permeability to antibiotics like rifampin and vancomycin. In mycobacteria, regulation from the ribosome is critical for the maintenance of membrane homeostasis and, importantly, antibiotic susceptibility.

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