Gemfibrozil Inhibits Legionella pneumophila and Mycobacterium tuberculosis Enoyl Coenzyme A Reductases and Blocks Intracellular Growth of These Bacteria in Macrophages

ABSTRACT We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.

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Novel T9 loop conformation of filamenting temperature-sensitive mutant Z from Mycobacterium tuberculosis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19004618 ◽

2019 ◽

Vol 75 (5) ◽

pp. 359-367

Author(s):

E. O. Lazo ◽

J. Jakoncic ◽

S. RoyChowdhury ◽

D. Awasthi ◽

I. Ojima

Keyword(s):

Mycobacterium Tuberculosis ◽

Binding Pocket ◽

Multidrug Resistant ◽

Temperature Sensitive ◽

Bacterial Cell Division ◽

Sensitive Mutant ◽

Temperature Sensitive Mutant ◽

Subunit Interface ◽

Z Ring ◽

Drug Resistant Strains

As of 2017, tuberculosis had infected 1.7 billion people (23% of the population of the world) and caused ten million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multidrug resistant. Thus, the identification of novel druggable targets is essential to combat the proliferation of these drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ) is a key protein involved in cytokinesis, an important process for Mtb proliferation and viability. FtsZ is required for bacterial cell division because it polymerizes into a structure called the Z-ring, which recruits accessory division proteins to the septum. Here, the crystal structure of the MtbFtsZ protein has been determined to 3.46 Å resolution and is described as a dimer of trimers, with an inter-subunit interface between protomers AB and DE. In this work, a novel conformation of MtbFtsZ is revealed involving the T9 loop and the nucleotide-binding pocket of protomers BC and EF.

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ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000441640 ◽

2015 ◽

Vol 25 (6) ◽

pp. 394-402 ◽

Cited By ~ 1

Author(s):

Taylor L. Fischer ◽

Robert J. White ◽

Katherine F.K. Mares ◽

Devin E. Molnau ◽

Justin J. Donato

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Wild Type ◽

E Coli ◽

Enoyl Acp Reductase ◽

Selection For

Background/Aims: We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. Methods: ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. Results: Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. Conclusion: ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

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The Three RelE Homologs of Mycobacterium tuberculosis Have Individual, Drug-Specific Effects on Bacterial Antibiotic Tolerance

Journal of Bacteriology ◽

10.1128/jb.01285-09 ◽

2010 ◽

Vol 192 (5) ◽

pp. 1279-1291 ◽

Cited By ~ 81

Author(s):

Ramandeep Singh ◽

Clifton E. Barry ◽

Helena I. M. Boshoff

Keyword(s):

Mycobacterium Tuberculosis ◽

Survival Rates ◽

Specific Effect ◽

Multidrug Resistant ◽

Cross Resistance ◽

Bacterial Survival ◽

E Coli ◽

The Face

ABSTRACT In Escherichia coli, expression of the RelE and HipA toxins in the absence of their cognate antitoxins has been associated with generating multidrug-tolerant “persisters.” Here we show that unlike persisters of E. coli, persisters of Mycobacterium tuberculosis selected with one drug do not acquire cross-resistance to other classes of drugs. M. tuberculosis has three homologs of RelE arranged in operons with their apparent antitoxins. Each toxin individually arrests growth of both M. tuberculosis and E. coli, an effect that is neutralized by coexpression of the cognate antitoxin. Overexpression or deletion of each of the RelE toxins had a toxin- and drug-specific effect on the proportion of bacilli surviving antibiotic killing. All three toxins were upregulated in vivo, but none of the deletions affected survival during murine infection. RelE2 overexpression increased bacterial survival rates in the presence of rifampin in vitro, while deletion significantly decreased survival rates. Strikingly, deletion of this toxin had no discernible effect on the level of persisters seen in rifampin-treated mice. Our results suggest that, in vivo, RelE-generated persisters are unlikely to play a significant role in the generation of bacilli that survive in the face of multidrug therapy or in the generation of multidrug-resistant M. tuberculosis.

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Membrane Vesicles Shed by Legionella pneumophila Inhibit Fusion of Phagosomes with Lysosomes

Infection and Immunity ◽

10.1128/iai.01382-05 ◽

2006 ◽

Vol 74 (6) ◽

pp. 3285-3295 ◽

Cited By ~ 68

Author(s):

Esteban Fernandez-Moreira ◽

Juergen H. Helbig ◽

Michele S. Swanson

Keyword(s):

Legionella Pneumophila ◽

Surface Composition ◽

Membrane Vesicles ◽

Type Iv Secretion ◽

Phagosome Maturation ◽

Developmentally Regulated ◽

E Coli ◽

Type Iv ◽

Mouse Macrophages ◽

Legionnaires Disease

ABSTRACT When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only ∼25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas ∼75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered ∼72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.

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Sinorhizobium meliloti Functionally Replaces 3-Oxoacyl-Acyl Carrier Protein Reductase (FabG) by Overexpressing NodG During Fatty Acid Synthesis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-15-0148-r ◽

2016 ◽

Vol 29 (6) ◽

pp. 458-467 ◽

Cited By ~ 5

Author(s):

Ya-Hui Mao ◽

Feng Li ◽

Jin-Cheng Ma ◽

Zhe Hu ◽

Hai-Hong Wang

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Sinorhizobium Meliloti ◽

Acyl Carrier Protein ◽

Sequence Similarity ◽

Acid Synthesis ◽

In Vitro Assays ◽

Temperature Sensitive ◽

E Coli

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

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Differential Bacterial Predation by Free-Living Amoebae May Result in Blooms of Legionella in Drinking Water Systems

Microorganisms ◽

10.3390/microorganisms9010174 ◽

2021 ◽

Vol 9 (1) ◽

pp. 174 ◽

Cited By ~ 1

Author(s):

Mohamed Shaheen ◽

Nicholas J. Ashbolt

Keyword(s):

Legionella Pneumophila ◽

Fluorescent Microscopy ◽

Feeding Preference ◽

Water Systems ◽

Food Sources ◽

Resistant Bacteria ◽

Initial Growth ◽

Intracellular Growth ◽

Free Living ◽

E Coli

Intracellular growth of pathogenic Legionella in free-living amoebae (FLA) results in the critical concentrations that are problematic in engineered water systems (EWS). However, being amoeba-resistant bacteria (ARB), how Legionella spp. becomes internalized within FLA is still poorly understood. Using fluorescent microscopy, we investigated in real-time the preferential feeding behavior of three water-related FLA species, Willaertia magna, Acanthamoeba polyphaga, and Vermamoeba vermiformis regarding Legionella pneumophila and two Escherichia coli strains. Although all the studied FLA species supported intracellular growth of L. pneumophila, they avoided this bacterium to a certain degree in the presence of E. coli and mostly fed on it when the preferred bacterial food-sources were limited. Moreover, once L. pneumophila were intracellular, it inhibited digestion of co-occurring E. coli within the same trophozoites. Altogether, based on FLA–bacteria interactions and the shifts in microbial population dynamics, we propose that FLA’s feeding preference leads to an initial growth of FLA and depletion of prey bacteria, thus increases the relative abundance of Legionella and creates a “forced-feeding” condition facilitating the internalization of Legionella into FLA to initiate the cycles of intracellular multiplication. These findings imply that monitoring of FLA levels in EWS could be useful in predicting possible imminent high occurrence of Legionella.

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Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05742-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1735-1743 ◽

Cited By ~ 44

Author(s):

Thulasi Warrier ◽

Marielle Tropis ◽

Jim Werngren ◽

Anne Diehl ◽

Martin Gengenbacher ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cell Envelope ◽

Acid Synthesis ◽

Multidrug Resistant ◽

Mycolic Acid ◽

Broth Culture ◽

Nuclear Magnetic Resonance Analysis ◽

Transferase Activity ◽

Content Type ◽

Substrate Analogs

ABSTRACTThe antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital forMycobacterium tuberculosisdue to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential forM. tuberculosisvirulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition ofM. tuberculosisdivision in broth culture. I3-AG85 also inhibitedM. tuberculosissurvival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistantM. tuberculosisstrains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of theM. tuberculosisenvelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of theM. tuberculosisenvelope. I3-AG85 inhibited the division of and reduced TDM synthesis in anM. tuberculosisstrain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.

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Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features

Scientific Reports ◽

10.1038/s41598-021-82905-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pakorn Aiewsakun ◽

Pinidphon Prombutara ◽

Tegar Adriansyah Putra Siregar ◽

Thanida Laopanupong ◽

Phongthon Kanjanasirirat ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Resistant Strain ◽

Public Health Problem ◽

Multidrug Resistant ◽

Secretion System ◽

New Drugs ◽

Global Public Health ◽

Intracellular Growth ◽

Cell Infection ◽

Multidrug Resistant Strain

AbstractTuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated “MKR superspreader”, and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR’s intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

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