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

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.

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

International Journal of Bacteriology ◽

10.1155/2014/564109 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 1

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.

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Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination

Journal of Bacteriology ◽

10.1128/jb.01005-12 ◽

2012 ◽

Vol 194 (23) ◽

pp. 6398-6409 ◽

Cited By ~ 45

Author(s):

P. Plocinski ◽

N. Arora ◽

K. Sarva ◽

E. Blaszczyk ◽

H. Qin ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Cell Shape ◽

Double Mutant ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Content Type ◽

Ring Assembly ◽

Double Mutant Strain

ABSTRACTBacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein fromMycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog inM. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [14C]N-acetylglucosamine cell wall precursor. TheM. smegmatisMSMEG_0023crgAdouble mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA forcellwall synthesis and cellshape proteinA, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.

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Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00659-21 ◽

2021 ◽

Author(s):

Loes van Schie ◽

Katlyn Borgers ◽

Gitte Michielsen ◽

Evelyn Plets ◽

Marnik Vuylsteke ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Growth Inhibition ◽

Cell Envelope ◽

Mycolic Acid ◽

Wall Layer ◽

Cell Wall Degrading Enzymes ◽

Growth Inhibition Assay ◽

Degrading Enzymes ◽

Hydrophobic Barrier

Background The major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope – a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer – which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives (i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. Methods We inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. Results Our biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80. Conclusions Synergistically acting cell wall degrading enzymes are potently inhibiting Mtb – which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05708-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1797-1809 ◽

Cited By ~ 292

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.

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Mycobacterium Tuberculosis infection and Tissue Factor Expression in Macrophages

Blood ◽

10.1182/blood.v118.21.1198.1198 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1198-1198

Author(s):

Hema Kothari ◽

Ramakrishna Vankayalapati ◽

Padmaja Paidipally ◽

L. Vijaya Mohan Rao ◽

Usha R Pendurthi

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Tissue Factor ◽

Conflicts Of Interest ◽

Human Monocyte ◽

Mycolic Acid ◽

Coagulation Cascade ◽

Mycobacterium Tuberculosis Infection ◽

Aberrant Expression ◽

Mycobacterial Cell Wall

Abstract Abstract 1198 Tissue factor (TF), the primary initiator of the coagulation cascade, is normally absent from cells that regularly come in contact with blood such as endothelial cells and monocytes yet could be induced in these cells in a variety of pathological conditions. The aberrant expression of TF by cells of the monocyte/macrophage lineage is thought to be a major contributor to thrombotic disorders and inflammation. Macrophages play a central role in the innate immune response essential for host defense against pathogenic infections. Tuberculosis (TB), a fatal disease caused by Mycobacterium tuberculosis (Mtb), affects nearly one third of the world's population. A number of studies have reported the presence of thrombotic complications, particularly disseminated intravascular coagulation (DIC) in TB patients. However, it is unclear how Mtb infection causes DIC or other thrombotic disorders as mycobacteria are not known to produce endotoxins or exotoxins that otherwise initiate the clotting cascade. In the present study, we have investigated whether Mtb infection induces TF expression in macrophages and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. Exposure of human monocyte-derived macrophages (MDM) to live virulent Mtb H37Rv (at 1 to 5 bacteria/macrophage) or γ-Mtb H37Rv (10 μg/ml) markedly increased TF expression in MDM (5 to 20-fold increase compared to untreated MDM). TF expression in macrophages in response to Mtb is predominantly higher in CD14hi (CD14+) macrophages. Although CD14loCD16hi (CD16+) macrophages also expressed TF in response to Mtb, the level of TF induction in these cells was much lower. γ-Mtb induced TF expression in macrophages much more robustly than E.coli-derived LPS. The γ-Mtb-mediated induction of TF expression in macrophages peaked around 9 h and was sustained throughout 48 h, which markedly varies from the kinetics of LPS-induced TF expression, which peaks between 3–6 h and then reduced down to basal level by 18–24 h. To identify the potential macrophage receptor(s) responsible for the induction of TF by Mtb, MDM were pre-treated with antibodies against CD14 and Toll-like receptors (TLR) before they were exposed to γ-Mtb. Although CD14 antibodies markedly inhibited LPS-induced TF activity, they only suppressed Mtb-induced TF activity minimally. Pretreatment of MDMs with TLR2 or TLR4 antibodies had no significant effect on γ-Mtb-induced TF expression. However, combining the CD14 antibodies together with TLR2 and TLR4 antibodies showed nearly a 50% reduction in γ-Mtb-induced TF activity. These data indicate that a cooperative action of multiple receptors and signaling pathways may be responsible for the robust and sustained induction of TF expression. In order to identify the specific component(s) of γ-Mtb that are responsible for TF induction, we have treated MDMs with various subcellular fractions or purified components derived from Mtb H37Rv. The whole cell lysate of Mtb, cell wall, cell membrane and culture filterate proteins induced TF activity to varying degrees. Of all the purified components tested, the mycobacterial cell wall core component mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) elicited induction of TF protein and activity in the order of mAGP > PIM6 > LM. It is interesting to note that the treatment of MDMs with individual components, mycolic acid, arabinogalactan and peptidoglycan of mAGP complex did not induce TF expression, indicating that the structure of mAGP may be required for recognition by pattern recognition receptors on macrophages. In summary, our data show that Mtb induces TF expression in macrophages, probably through a novel mechanism. Although Mtb-induced TF expression in monocytes/macrohages may lead to systemic thrombotic disorders through the release of TF containing microparticles, it may be the localized and sustained expression of TF on macrophages that internalize the infectious bacteria at the site of infection in lung is responsible for granuloma formation in tuberculosis patients that prevents the spread of the bacteria. Disclosures: No relevant conflicts of interest to declare.

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Synthesis of a homologous series of galactofuranose-containing mycobacterial arabinogalactan fragments

Canadian Journal of Chemistry ◽

10.1139/cjc-2016-0416 ◽

2016 ◽

Vol 94 (11) ◽

pp. 976-988 ◽

Cited By ~ 2

Author(s):

Maju Joe ◽

Todd L. Lowary

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Chemical Synthesis ◽

Mycolic Acid ◽

Wall Structure ◽

Complex Cell ◽

Human Pathogen ◽

Structural Element ◽

Target Molecules ◽

Mycobacterial Cell Wall

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, produce a complex cell wall structure made of carbohydrates and lipids. The major structural element of the mycobacterial cell wall is a glycoconjugate called the mycolic acid – arabinogalactan – peptidoglycan (mAGP) complex. Inhibition of mAGP biosynthesis is a proven strategy for developing anti-mycobacterial drugs, and thus, understanding the pathways and enzymes involved in the assembly of this molecule is of interest. In this paper, we describe the chemical synthesis of a panel of nine oligosaccharide fragments (4–12) of the galactan domain of the mAGP complex designed as biosynthetic probes. These structures, ranging in size from a hexasaccharide to a tetradecasaccharide, are potential substrates for two biosynthetic enzymes, GlfT2 and AftA, and represent the largest mycobacterial galactan fragments synthesized to date. The route developed was iterative and provided multimilligram quantities of the target molecules 4–12 in good overall yield.

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An essential mycolate remodeling program for mycobacterial adaptation in host cells

10.1101/354431 ◽

2018 ◽

Cited By ~ 1

Author(s):

Eliza J.R. Peterson ◽

Rebeca Bailo ◽

Alissa C. Rothchild ◽

Mario Arrieta-Ortiz ◽

Amardeep Kaur ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

New Technology ◽

Mycolic Acid ◽

Host Cells ◽

Macrophage Infection ◽

Cell Wall Remodeling ◽

Mycobacterial Cell Wall

AbstractThe success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.One Sentence SummaryNovel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages

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