Published December 1, 2023 | Version v1
Dataset Open

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages

Authors/Creators

  • 1. ROR icon Oregon Health & Science University
  • 2. ROR icon University of Victoria
  • 3. ROR icon Boston Children's Hospital
  • 4. The University of British Columbia
  • 5. Oregon Health and Science University

Description

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6’s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

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BLI_dimer_long_pH.zip

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Additional details

Related works

Is published in
Journal article: 10.7554/eLife.91930 (DOI)