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Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis
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The ESX-5 associated eccB-EccC locus is essential for Mycobacterium tuberculosis viability.

Mariagrazia Di Luca1, Daria Bottai, Giovanna Batoni

  • 1Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Pisa, Italy.

Plos One
|January 4, 2013
PubMed
Summary

The ESX-5 secretion system

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Immunology

Background:

  • The ESX-5 secretion system in Mycobacterium tuberculosis is vital for host-pathogen interactions, influencing PPE protein secretion, cell wall integrity, and virulence.
  • While some ESX-5 components are known, key elements of this secretion machinery require further characterization.

Purpose of the Study:

  • To investigate the roles of EccB(5) (a transmembrane protein) and EccC(5) (a membrane-bound ATPase) as potential core components of the M. tuberculosis ESX-5 secretion system.
  • To determine the essentiality of the eccB(5)-eccC(5) operon for M. tuberculosis viability and ESX-5 substrate secretion.

Main Methods:

  • In vitro expression studies to analyze the eccB(5)-eccC(5) operon.
  • Construction and characterization of M. tuberculosis conditional mutant strains (Mtb(Pptr)eccB(5) and Mtb(Pptr)eccC(5)) using an anhydrotetracycline-repressible promoter.
  • Analysis of bacterial growth in vitro and within human macrophages (THP-1 cell line).
  • Secretome analysis to identify ESX-5 specific substrates.

Main Results:

  • EccB(5) and EccC(5) genes form an operon essential for M. tuberculosis growth, as evidenced by the inability to delete the genomic segment without a functional copy.
  • Repression of the eccB(5)-eccC(5) operon or eccC(5) gene significantly impairs M. tuberculosis growth both in vitro and in macrophages.
  • Both EccB(5) and EccC(5) are indispensable for the secretion of ESX-5 specific substrates, confirming their role in the ESX-5 machinery.

Conclusions:

  • EccB(5) and EccC(5) are essential components of the Mycobacterium tuberculosis ESX-5 secretion system.
  • A functional ESX-5 system is critical for the viability of M. tuberculosis.
  • These findings offer potential avenues for developing novel antimycobacterial therapeutic strategies targeting the ESX-5 system.