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Published on: September 27, 2018
ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth
Timothy A Bates1, Mila Trank-Greene1, Xammy Nguyenla1
1Department of Molecular Microbiology and Immunology, Oregon Health & Sciences University, Portland, Oregon, United States.
Abstract:
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.
Insights
Mycobacterium tuberculosis survives in macrophages by disrupting phagosomes using the ESAT-6 protein. This study reveals ESAT-6
Area of Science:
- Microbiology
- Cell Biology
- Structural Biology
Background:
- Mycobacterium tuberculosis (Mtb) evades host immunity by surviving within macrophages.
- Mtb compromises phagosomal integrity, crucial for intracellular survival.
- The ESX-1 secretion system, particularly ESAT-6 and CFP-10, is vital for this process.
Approach:
- Biochemical analyses to determine ESAT-6/CFP-10 binding kinetics.
- Protein modeling and molecular dynamics simulations to elucidate ESAT-6 structure and interactions.
- Utilized a novel anti-ESAT-6 nanobody to investigate ESAT-6 function in vivo.
Key Points:
- ESAT-6 forms a stable tetrameric complex under acidic conditions.
- Identified the probable interaction interface of ESAT-6.
- A pH-dependent mechanism for ESAT-6 action is proposed.
- Blocking ESAT-6 function inhibits Mtb replication within macrophages.
Conclusions:
- ESAT-6 plays a critical role in Mtb's intracellular survival strategy.
- ESAT-6 mediates pH-dependent communication between the phagosome and cytoplasm.
- Understanding ESAT-6's mechanism offers potential therapeutic targets against tuberculosis.
