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

Author:

Bates Timothy A1ORCID,Trank-Greene Mila1ORCID,Nguyenla Xammy1ORCID,Anastas Aidan1ORCID,Gurmessa Sintayehu K1ORCID,Merutka Ilaria R1ORCID,Dixon Shandee D1ORCID,Shumate Anthony2ORCID,Groncki Abigail R1,Parson Matthew AH3ORCID,Ingram Jessica R4ORCID,Barklis Eric1ORCID,Burke John E35ORCID,Shinde Ujwal2ORCID,Ploegh Hidde L4ORCID,Tafesse Fikadu G1ORCID

Affiliation:

1. Department of Molecular Microbiology and Immunology, Oregon Health & Sciences University

2. Department of Chemical Physiology and Biochemistry, Oregon Health & Science University

3. Department of Biochemistry and Microbiology, University of Victoria

4. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Harvard Medical School

5. Department of Biochemistry and Molecular Biology, The University of British Columbia

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.

Publisher

eLife Sciences Publications, Ltd

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