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

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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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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The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence

Infection and Immunity ◽

10.1128/iai.00131-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 31

Author(s):

Catherine C. Wright ◽

Fong Fu Hsu ◽

Eusondia Arnett ◽

Jennifer L. Dunaj ◽

Patrick M. Davidson ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Mycolic Acid ◽

Wax Ester ◽

Wild Type ◽

Content Type ◽

Immune Pressure ◽

Oxygen Starvation ◽

Mycobacterial Cell Wall

ABSTRACT The mycobacterial cell wall is crucial to the host-pathogen interface, because it provides a barrier against antibiotics and the host immune response. In addition, cell wall lipids are mycobacterial virulence factors. The mycobacterial membrane protein large (MmpL) proteins are cell wall lipid transporters that are important for basic mycobacterial physiology and Mycobacterium tuberculosis pathogenesis. MmpL3 and MmpL11 are conserved across pathogenic and nonpathogenic mycobacteria, a feature consistent with an important role in the basic physiology of the bacterium. MmpL3 is essential and transports trehalose monomycolate to the mycobacterial surface. In this report, we characterize the role of MmpL11 in M. tuberculosis. M. tuberculosis mmpL11 mutants have altered biofilms associated with lower levels of mycolic acid wax ester and long-chain triacylglycerols than those for wild-type bacteria. While the growth rate of the mmpL11 mutant is similar to that of wild-type M. tuberculosis in macrophages, the mutant exhibits impaired survival in an in vitro granuloma model. Finally, we show that the survival or recovery of the mmpL11 mutant is impaired when it is incubated under conditions of nutrient and oxygen starvation. Our results suggest that MmpL11 and its cell wall lipid substrates are important for survival in the context of adaptive immune pressure and for nonreplicating persistence, both of which are critically important aspects of M. tuberculosis pathogenicity.

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Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Current Molecular Pharmacology ◽

10.2174/1874467211666181025141114 ◽

2019 ◽

Vol 12 (1) ◽

pp. 27-49 ◽

Cited By ~ 6

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.

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Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol

Journal of Bacteriology ◽

10.1128/jb.00488-09 ◽

2009 ◽

Vol 191 (21) ◽

pp. 6584-6591 ◽

Cited By ~ 97

Author(s):

Anna Brzostek ◽

Jakub Pawelczyk ◽

Anna Rumijowska-Galewicz ◽

Bozena Dziadek ◽

Jaroslaw Dziadek

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Carbon Source ◽

Sole Carbon Source ◽

Cytoplasmic Membrane ◽

Degradation Pathway ◽

Cholesterol Accumulation ◽

Targeted Disruption ◽

Future Studies

ABSTRACT It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.

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Synergistic Lethality of a Binary Inhibitor ofMycobacterium tuberculosisKasA

mBio ◽

10.1128/mbio.02101-17 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 14

Author(s):

Pradeep Kumar ◽

Glenn C. Capodagli ◽

Divya Awasthi ◽

Riju Shrestha ◽

Karishma Maharaja ◽

...

Keyword(s):

Crystal Structure ◽

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Substrate Binding ◽

Content Type ◽

X Ray ◽

Structure Activity Relationships ◽

Structure Activity ◽

Transcriptional Pattern

ABSTRACTWe report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) inMycobacterium tuberculosis. Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented thein vitroactivity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model ofM. tuberculosisinfection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCECell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors ofMycobacterium tuberculosisKasA—a key component for biosynthesis of the mycolic acid layer of the bacterium’s cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

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