scholarly journals Withdrawal: Inorganic polyphosphate accumulation suppresses the dormancy response and virulence in Mycobacterium tuberculosis.

2020 ◽  
Vol 295 (43) ◽  
pp. 14791-14791
Author(s):  
Prabhakar Tiwari ◽  
Tannu Priya Gosain ◽  
Mamta Singh ◽  
Gaurav D. Sankhe ◽  
Garima Arora ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inorganic Polyphosphate ◽  
Polyphosphate Accumulation

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

Monitoring of the inorganic polyphosphate accumulation and acid and alkaline phosphatase activity in Cunninghamella elegans strains using factorial design

2012 ◽  
Author(s):  
Luciana de Oliveira Franco ◽  
Thayza Christina Montenegro Stamford ◽  
Marcos Antônio Barbosa de Lima ◽  
Rita de Cassia Carvalho Maia ◽  
Tânia Lúcia Montenegro Stamford ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Factorial Design ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Cunninghamella Elegans ◽  
Inorganic Polyphosphate ◽  
Acid And Alkaline Phosphatase ◽  
Polyphosphate Accumulation

scholarly journals Inorganic Polyphosphate Accumulation inEscherichia coliIs Regulated by DksA but Not by (p)ppGpp

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Michael J. Gray
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Elongation Factor ◽  
Stringent Response ◽  
Inorganic Polyphosphate ◽  
Content Type ◽  
Polyphosphate Accumulation ◽  
Pathogenic Microbes ◽  
Polyp Accumulation

ABSTRACTProduction of inorganic polyphosphate (polyP) by bacteria is triggered by a variety of different stress conditions. polyP is required for stress survival and virulence in diverse pathogenic microbes. Previous studies have hypothesized a model for regulation of polyP synthesis in which production of the stringent-response second messenger (p)ppGpp directly stimulates polyP accumulation. In this work, I have now shown that this model is incorrect, and (p)ppGpp is not required for polyP synthesis inEscherichia coli. However, stringent mutations of RNA polymerase that frequently arise spontaneously in strains defective in (p)ppGpp synthesis and null mutations of the stringent-response-associated transcription factor DksA both strongly inhibit polyP accumulation. The loss of polyP synthesis in a mutant lacking DksA was reversed by deletion of the transcription elongation factor GreA, suggesting that competition between these proteins for binding to the secondary channel of RNA polymerase plays an important role in controlling polyP activation. These results provide new insights into the poorly understood regulation of polyP synthesis in bacteria and indicate that the relationship between polyP and the stringent response is more complex than previously suspected.IMPORTANCEProduction of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress inEscherichia coliand demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

scholarly journals Targeting the Mycobacterium tuberculosis Stringent Response as a Strategy for Shortening Tuberculosis Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Carina Danchik ◽  
Siqing Wang ◽  
Petros C. Karakousis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Species ◽  
Stringent Response ◽  
Therapeutic Benefit ◽  
Current Understanding ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Inorganic Polyphosphate ◽  
Regulatory Molecule ◽  
Response To Stress

The stringent response is well conserved across bacterial species and is a key pathway involved both in bacterial survival and virulence and in the induction of antibiotic tolerance in Mycobacteria. It is mediated by the alarmone (p)ppGpp and the regulatory molecule inorganic polyphosphate in response to stress conditions such as nutrient starvation. Efforts to pharmacologically target various components of the stringent response have shown promise in modulating mycobacterial virulence and antibiotic tolerance. In this review, we summarize the current understanding of the stringent response and its role in virulence and tolerance in Mycobacteria, including evidence that targeting this pathway could have therapeutic benefit.

Inorganic Polyphosphate/ATP-NAD Kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv

2000 ◽  
Vol 276 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Shigeyuki Kawai ◽  
Shigetarou Mori ◽  
Takako Mukai ◽  
Sachiko Suzuki ◽  
Takeshi Yamada ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tuberculosis H37rv ◽  
Nad Kinase ◽  
Inorganic Polyphosphate ◽  
Mycobacterium Tuberculosis H37rv

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 The Polyphosphate Kinase Gene ppk2 Is Required for Mycobacterium tuberculosis Inorganic Polyphosphate Regulation and Virulence

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Yu-Min Chuang ◽  
Deborah A. Belchis ◽  
Petros C. Karakousis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interleukin 2 ◽  
Fold Increase ◽  
Polyphosphate Kinase ◽  
Macrophage Infection ◽  
Inorganic Polyphosphate ◽  
Kinase Gene ◽  
Content Type ◽  
Cytokines And Chemokines ◽  
P Content

ABSTRACT The Mycobacterium tuberculosis gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, MIC of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for M. tuberculosis virulence was assessed in the murine model. The ppk2::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT. Naive ppk2::Tn mutant-infected macrophages showed increased expression of interleukin 2 (IL-2), IL-9, IL-10, IL-12p70, and gamma interferon (IFN-γ) relative to WT-infected macrophages. The ppk2::Tn mutant exhibited significantly lower lung CFU during acute murine infection compared to the control groups. ppk2 is required for control of intrabacillary poly P levels and optimal M. tuberculosis growth and survival in macrophages and mouse lungs. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a highly successful human pathogen because it has developed mechanisms to multiply and survive in the lungs by circumventing the immune system. Identification of virulence factors responsible for M. tuberculosis growth and persistence in host tissues may assist in the development of novel strategies to treat TB. In this study, we found that the mycobacterial enzyme polyphosphate kinase 2 (PPK2) is required for controlling intracellular levels of important regulatory molecules and for maintaining susceptibility to the first-line anti-TB drug isoniazid. In addition, PPK2 was found to be required for M. tuberculosis growth in the lungs of mice, at least in part by suppressing the expression of certain key cytokines and chemokines by inactivated lung macrophages.

Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates

2006 ◽  
Vol 0 (0) ◽  
pp. 060421055900007 ◽  
Author(s):  
T.F. Moriarty ◽  
A. Mullan ◽  
J.W. McGrath ◽  
J.P. Quinn ◽  
J.S. Elborn ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Burkholderia Cepacia Complex ◽  
Inorganic Polyphosphate ◽  
Polyphosphate Accumulation

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.

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