scholarly journals The Assessment of Viability of M. Tuberculosis after Exposure to CPC Using Different Methods

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Gomathi Sekar ◽  
R. Lakshmi ◽  
N. Selvakumar
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Metabolic Activity ◽  
Acid Content ◽  
Reference Strain ◽  
Vital Staining ◽  
Mycolic Acid ◽  
Viable Count ◽  
Mycolic Acids ◽  
Minimal Reduction

Settings. National Institute for Research in Tuberculosis, Chennai. Objective. To assess the proportion of metabolically active cells of Mycobacterium tuberculosis after exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content in M. tuberculosis after exposure to CPC was estimated using HPLC. Methods. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis H37Rv were used for FDA-EB, viable count, and HPLC. Results. FDA/EB consistently stained 70–90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65–70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability of M. tuberculosis was maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells of M. tuberculosis were treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall of M. tuberculosis.

scholarly journals Carbon Source-Induced Modifications in the Mycolic Acid Content and Cell Wall Permeability of Rhodococcus erythropolis E1

2003 ◽  
Vol 69 (12) ◽  
pp. 7019-7027 ◽  
Author(s):  
Ivana Sokolovská ◽  
Raoul Rozenberg ◽  
Christophe Riez ◽  
Paul G. Rouxhet ◽  
Spiros N. Agathos ◽  
...  
Keyword(s):  
Cell Wall ◽  
Carbon Source ◽  
Acid Content ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Mycolic Acids ◽  
Carbon Chains ◽  
Linear Alkanes ◽  
Wall Permeability ◽  
Cell Wall Permeability

ABSTRACT The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.

scholarly journals Two Accessory Proteins Govern MmpL3 Mycolic Acid Transport in Mycobacteria

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Allison Fay ◽  
Nadine Czudnochowski ◽  
Jeremy M. Rock ◽  
Jeffrey R. Johnson ◽  
Nevan J. Krogan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Complex Structure ◽  
Mycolic Acid ◽  
Accessory Proteins ◽  
Acid Transport ◽  
Content Type ◽  
Mycolic Acids ◽  
Antibiotic Target

ABSTRACT Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named “TMM transport factor A”, TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis. IMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.

scholarly journals SQ109 Targets MmpL3, a Membrane Transporter of Trehalose Monomycolate Involved in Mycolic Acid Donation to the Cell Wall Core of Mycobacterium tuberculosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1797-1809 ◽  
Author(s):  
Kapil Tahlan ◽  
Regina Wilson ◽  
David B. Kastrinsky ◽  
Kriti Arora ◽  
Vinod Nair ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycolic Acid ◽  
Spontaneous Generation ◽  
Content Type ◽  
Mycolic Acids ◽  
Cell Wall Assembly ◽  
Resistant Mutants ◽  
Trehalose Monomycolate

ABSTRACTSQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core ofMycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates.In vitroassays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essentialmmpL3gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals Redundant Function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis Cyclopropanation of Oxygenated Mycolates

2010 ◽  
Vol 192 (14) ◽  
pp. 3661-3668 ◽  
Author(s):  
Daniel Barkan ◽  
Vivek Rao ◽  
George D. Sukenick ◽  
Michael S. Glickman
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Genetic Analysis ◽  
Biosynthetic Pathway ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Acid Modification ◽  
Mycolic Acids ◽  
Powerful Approach ◽  
Long Chain

ABSTRACT The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

2013 ◽  
Vol 51 (5) ◽  
pp. 619-626 ◽  
Author(s):  
Sally A. Cantrell ◽  
Michael D. Leavell ◽  
Olivera Marjanovic ◽  
Anthony T. Iavarone ◽  
Julie A. Leary ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Mycolic Acid ◽  
Acid Accumulation

In vitro effect of ursolic acid on the inhibition of Mycobacterium tuberculosis and its cell wall mycolic acid

2015 ◽  
Vol 33 ◽  
pp. 17-24 ◽  
Author(s):  
Md. Anirban Jyoti ◽  
Tamanna Zerin ◽  
Tae-Hyun Kim ◽  
Tae-Seon Hwang ◽  
Woong Sik Jang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Ursolic Acid ◽  
Mycolic Acid ◽  
Vitro Effect

scholarly journals Synthesis and Biological Aspects of Mycolic Acids: An Important Target AgainstMycobacterium tuberculosis

2008 ◽  
Vol 8 ◽  
pp. 720-751 ◽  
Author(s):  
Marcus Vinícius Nora de Souza ◽  
Marcelle de Lima Ferreira ◽  
Alessandra Campbell Pinheiro ◽  
Maurício Frota Saraiva ◽  
Mauro Vieira de Almeida ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Walls ◽  
Important Class ◽  
Cellular Targets ◽  
Mycolic Acids ◽  
Important Target ◽  
Novel Drugs ◽  
Biological Aspects ◽  
Mycobacterial Cell Wall

Mycolic acids are an important class of compounds, basically found in the cell walls of a group of bacteria known as mycolata taxon, exemplified by the most famous bacteria of this group, theMycobacterium tuberculosis(M. tb.), the agent responsible for the disease known as tuberculosis (TB). Mycolic acids are important for the survival of M. tb. For example, they are able to help fight against hydrophobic drugs and dehydration, and also allow this bacterium to be more effective in the host's immune system by growing inside macrophages. Due to the importance of the mycolic acids for maintenance of the integrity of the mycobacterial cell wall, these compounds become attractive cellular targets for the development of novel drugs against TB. In this context, the aim of this article is to highlight the importance of mycolic acids in drug discovery.

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.

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