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System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
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Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

Mamta Singh1, Prabhakar Tiwari1, Garima Arora1

  • 1Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Haryana, India.

Scientific Reports
|June 10, 2016
PubMed

Insights

The Mycobacterium tuberculosis PPK-2 enzyme is crucial for disease progression and drug resistance. Inhibiting PPK-2 may offer a new strategy against tuberculosis infections.

Area of Science:

  • Microbiology
  • Biochemistry
  • Pathogenesis

Background:

  • Inorganic polyphosphate (PolyP) is vital for microbial stress adaptation, virulence, and drug tolerance.
  • Mycobacterium tuberculosis possesses two polyphosphate kinases (PPK-1, PPK-2) and polyphosphatases (ppx-1, ppx-2) to regulate intracellular PolyP levels.
  • Polyphosphate kinases enable microorganisms to use PolyP as a phosphate donor for ATP synthesis.

Purpose of the Study:

  • To investigate the role of the PPK-2 enzyme in Mycobacterium tuberculosis pathogenesis and drug tolerance.
  • To develop and standardize a high-throughput screening assay for identifying inhibitors of M. tuberculosis PPK-2.

Main Methods:

  • Construction of a ppk-2 mutant strain of M. tuberculosis.
  • Infection of guinea pigs with wild type and ppk-2 mutant strains to assess virulence.
  • Evaluation of ppk-2 mutant strain's tolerance to isoniazid and survival in THP-1 macrophages.
  • Development of a luciferase-based assay for M. tuberculosis PPK-2 inhibition screening.

Main Results:

  • The ppk-2 mutant strain exhibited significantly reduced bacterial loads and tissue pathology in infected guinea pigs compared to the wild type.
  • The ppk-2 mutant strain showed increased tolerance to isoniazid and impaired survival in THP-1 macrophages.
  • A standardized luciferase-based assay system was established to identify non-cytotoxic inhibitors of M. tuberculosis PPK-2.

Conclusions:

  • The PPK-2 enzyme is a significant contributor to M. tuberculosis virulence and drug tolerance.
  • Targeting M. tuberculosis PPK-2 presents a potential therapeutic strategy for tuberculosis.
  • This study demonstrates the feasibility of high-throughput screening for small molecule PPK-2 inhibitors.

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