scholarly journals Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in Mycobacterium tuberculosis

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Yu-Min Chuang ◽  
Nirmalya Bandyopadhyay ◽  
Dalin Rifat ◽  
Harvey Rubin ◽  
Joel S. Bader ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Stringent Response ◽  
Inorganic Polyphosphate ◽  
Content Type ◽  
P Accumulation ◽  
Wall Permeability ◽  
Altered Cell ◽  
Cell Wall Permeability ◽  
Long Chain

ABSTRACTMycobacterium tuberculosiscan persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here thatM. tuberculosispoly(P) accumulation is temporally linked to bacillary growth restriction. We also identifyM. tuberculosisRv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression ofRv1026(ppx2), we found thatM. tuberculosispoly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, theppx2knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of theppx2knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role inM. tuberculosisgrowth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability.IMPORTANCEThe stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediateMycobacterium tuberculosispersistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in theppx2gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.

scholarly journals 3D-QSAR and Cell Wall Permeability of Antitubercular Nitroimidazoles against Mycobacterium tuberculosis

Molecules ◽  
2013 ◽  
Vol 18 (11) ◽  
pp. 13870-13885 ◽  
Author(s):  
Sang-Ho Lee ◽  
Minsung Choi ◽  
Pilho Kim ◽  
Pyung Myung
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
3D Qsar ◽  
Wall Permeability ◽  
Cell Wall Permeability

scholarly journals Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to IsoniazidIn Vivo

2016 ◽  
Vol 60 (11) ◽  
pp. 6460-6470 ◽  
Author(s):  
Yu-Min Chuang ◽  
Noton K. Dutta ◽  
Chien-Fu Hung ◽  
T.-C. Wu ◽  
Harvey Rubin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biofilm Formation ◽  
Tricarboxylic Acid Cycle ◽  
Antibiotic Sensitivity ◽  
Stringent Response ◽  
Antibiotic Tolerance ◽  
Inorganic Polyphosphate ◽  
Response Factors ◽  
Content Type

ABSTRACTMycobacterium tuberculosisremains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence ofM. tuberculosispersisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in theM. tuberculosisstringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis inM. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulatingM. tuberculosismutant strains deficient inppx1orppk2had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. Theppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency ofppk2was associated with increased sensitivity to plumbagin and meropenem and deficiency ofppx1led to enhanced susceptibility to clofazimine. A DNA vaccine expressingppx1andppk2, together with two other members of theM. tuberculosisstringent response,M. tuberculosisrelandsigE, did not show protective activity against aerosol challenge withM. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of theM. tuberculosisstringent response play an important role inM. tuberculosismetabolism, biofilm formation, and antibiotic sensitivityin vivo.

scholarly journals Both Phthiocerol Dimycocerosates and Phenolic Glycolipids Are Required for Virulence of Mycobacterium marinum

2012 ◽  
Vol 80 (4) ◽  
pp. 1381-1389 ◽  
Author(s):  
Jia Yu ◽  
Vanessa Tran ◽  
Ming Li ◽  
Xinghua Huang ◽  
Chen Niu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Marinum ◽  
Content Type ◽  
Transposon Insertion ◽  
Wall Permeability ◽  
Cell Wall Permeability ◽  
Phthiocerol Dimycocerosates ◽  
Zebrafish Infection ◽  
Phenolic Glycolipids

ABSTRACTPhthiocerol dimycocerosates (PDIMs) and structurally related phenolic glycolipids (PGLs) are complex cell wall lipids unique to pathogenic mycobacteria. While these lipids have been extensively studied in recent years, there are conflicting reports on some aspects of their biosynthesis and on the role of PDIMs and especially PGLs in virulence ofMycobacterium tuberculosis. This has been complicated by the natural deficiency of PGLs in many clinical strains ofM. tuberculosisand the frequent loss of PDIMs in laboratoryM. tuberculosisstrains. In this study, we isolated seven mutants ofMycobacterium marinumdeficient in PDIMs and/or PGLs in which multiple genes of the PDIM/PGL biosynthetic locus were disrupted by transposon insertion. Zebrafish infection experiments showed thatM. marinumstrains lacking one or both of these lipids were avirulent, suggesting that both PDIMs and PGLs are required for virulence. We also found that these strains were hypersensitive to antibiotics and exhibited increased cell wall permeability. Our studies provide new insights into the biosynthesis of PDIMs/PGLs and may help us to understand the role of PDIMs and PGLs inM. tuberculosisvirulence.

scholarly journals The Sterol Carrier Hydroxypropyl-β-Cyclodextrin Enhances the Metabolism of Phytosterols by Mycobacterium neoaurum

2020 ◽  
Vol 86 (15) ◽  
Author(s):  
Liqiu Su ◽  
Shuangping Xu ◽  
Yanbing Shen ◽  
Menglei Xia ◽  
Xiaoxian Ren ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Growth ◽  
Low Cost ◽  
Inhibitory Effect ◽  
Sterol Metabolism ◽  
Content Type ◽  
Mycobacterium Neoaurum ◽  
Complex Functions ◽  
Wall Permeability ◽  
Cell Wall Permeability

ABSTRACT Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium. Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-β-CD (HP-β-CD) on phytosterol metabolism in Mycobacterium neoaurum TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-β-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-β-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-β-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism. IMPORTANCE Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by Mycobacterium. However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1 mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-β-CD (HP-β-CD) on the expression of enzymes related to steroid catabolic pathways in Mycobacterium neoaurum were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production.

scholarly journals Alginate-Capped Silver Nanoparticles as a Potent Anti-mycobacterial Agent Against Mycobacterium tuberculosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Cheng-Cheung Chen ◽  
Yih-Yuan Chen ◽  
Chang-Ching Yeh ◽  
Chia-Wei Hsu ◽  
Shang-Jie Yu ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Therapeutic Option ◽  
Infectious Agent ◽  
Drug Susceptibility ◽  
Drug Resistant ◽  
Wall Permeability ◽  
Cell Wall Permeability

Tuberculosis (TB) is a leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb). Although progress has been made in TB control, still about 10 million people worldwide develop TB annually and 1.5 million die of the disease. The rapid emergence of aggressive, drug-resistant strains and latent infections have caused TB to remain a global health challenge. TB treatments are lengthy and their side effects lead to poor patient compliance, which in turn has contributed to the drug resistance and exacerbated the TB epidemic. The relatively low output of newly approved antibiotics has spurred research interest toward alternative antibacterial molecules such as silver nanoparticles (AgNPs). In the present study, we use the natural biopolymer alginate to serve as a stabilizer and/or reductant to green synthesize AgNPs, which improves their biocompatibility and avoids the use of toxic chemicals. The average size of the alginate-capped AgNPs (ALG-AgNPs) was characterized as nanoscale, and the particles were round in shape. Drug susceptibility tests showed that these ALG-AgNPs are effective against both drug-resistant Mtb strains and dormant Mtb. A bacterial cell-wall permeability assay showed that the anti-mycobacterial action of ALG-AgNPs is mediated through an increase in cell-wall permeability. Notably, the anti-mycobacterial potential of ALG-AgNPs was effective in both zebrafish and mouse TB animal models in vivo. These results suggest that ALG-AgNPs could provide a new therapeutic option to overcome the difficulties of current TB treatments.

Cell Wall Permeability of Pinto Bean Cotyledon Cells Regulates In Vitro Fecal Fermentation and Gut Microbiota

2021 ◽  
Author(s):  
Yanrong Huang ◽  
Sushil Dhital ◽  
Feitong Liu ◽  
Xiong Fu ◽  
Qiang Huang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Structural Changes ◽  
Microbiota Composition ◽  
Colonic Fermentation ◽  
Pinto Bean ◽  
Wall Permeability ◽  
Cell Wall Permeability ◽  
Cotyledon Cells ◽  
Whole Foods

Processing induced structural changes of whole foods on regulation of colonic fermentation rate and microbiota composition are least understood and often overlooked. In the present study, intact cotyledon cells from...

scholarly journals A Mutation of RNA Polymerase β′ Subunit (RpoC) Converts Heterogeneously Vancomycin-Intermediate Staphylococcus aureus (hVISA) into “Slow VISA”

2015 ◽  
Vol 59 (7) ◽  
pp. 4215-4225 ◽  
Author(s):  
Miki Matsuo ◽  
Tomomi Hishinuma ◽  
Yuki Katayama ◽  
Keiichi Hiramatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Rna Polymerase ◽  
Stringent Response ◽  
Whole Genome ◽  
Multicopy Plasmid ◽  
Β Subunit ◽  
Whole Genome Analysis ◽  
Content Type ◽  
Peptidoglycan Biosynthesis

ABSTRACTVarious mutations in therpoBgene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediateStaphylococcus aureus(VISA) and heterogeneously VISA (hVISA) strains. We reported thatrpoBmutations are also linked to the expression of the recently found “slow VISA” (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024–5035, 2014,http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of therpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, inrpoC. Introduction of the wild-type (WT)rpoCgene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that therpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: therpoCgene itself as regulatory mutations, peptidoglycan biosynthesis genes, andrelQ, which is involved in the stringent response. It appears that therpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

scholarly journals Synergistic Lethality of a Binary Inhibitor ofMycobacterium tuberculosisKasA

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
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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