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Updated: Aug 19, 2026
Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells
Published on: March 3, 2015
Genome-wide interaction screen for Mycobacterium tuberculosis ClpCP protease reveals toxin-antitoxin systems as a
Michal Ziemski1, Julia Leodolter1, Gabrielle Taylor1
1Institute of Molecular Biology & Biophysics, ETH Zurich, Switzerland.
Abstract:
In Mycobacterium tuberculosis (Mtb), the Clp protease degradation pathway, mediated by the modular ClpCP and ClpXP protease complexes, is essential for growth and presents an attractive drug target. Employing a bacterial adenylate cyclase two-hybrid (BACTH) screening approach that we adapted to screen the proteome of an Mtb ORF library, we identify protein interaction partners of the ClpC1 chaperone on a genome-wide level. Our results demonstrate that bipartite type II toxin-antitoxin (TA) systems represent a major substrate class. Out of the 67 type II TA systems known in Mtb, 25 appear as ClpC1 interaction partners in the BACTH screen, including members of the VapBC, MazEF, and ParDE families, as well as a RelBE member that was identified biochemically. We show that antitoxins of the Vap and Rel families are degraded by ClpCP in vitro. We also demonstrate that ClpCP is responsible for mediating the N-end rule pathway, since the adaptor protein ClpS supports ClpC-dependent degradation of an N-end rule model substrate in vitro.
Insights
Mycobacterium tuberculosis ClpC1 chaperone interacts with type II toxin-antitoxin systems. The ClpCP protease degrades antitoxins and mediates the N-end rule pathway, offering potential drug targets.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The Clp protease system in Mycobacterium tuberculosis (Mtb) is crucial for bacterial growth and represents a potential drug target.
- Understanding the substrates and regulation of Clp proteases is key to developing new anti-TB therapies.
Purpose of the Study:
- To identify genome-wide protein interaction partners of the Mtb ClpC1 chaperone.
- To investigate the role of ClpCP in the degradation of type II toxin-antitoxin systems and the N-end rule pathway.
Main Methods:
- Adaptation and application of the bacterial adenylate cyclase two-hybrid (BACTH) screening assay.
- Genome-wide screening of an Mtb open reading frame (ORF) library.
- In vitro biochemical assays to confirm protein degradation.
Main Results:
- The BACTH screen identified 25 type II toxin-antitoxin (TA) systems as ClpC1 interaction partners.
- Interacting TA systems include VapBC, MazEF, ParDE, and RelBE families.
- In vitro studies confirmed ClpCP degrades Vap and Rel family antitoxins.
- ClpCP, with adaptor ClpS, mediates the N-end rule pathway in vitro.
Conclusions:
- Bipartite type II TA systems are a major substrate class for the Mtb ClpC1 chaperone.
- The ClpCP protease plays a significant role in degrading TA antitoxins and executing the N-end rule pathway.
- These findings highlight the Clp protease system as a promising target for novel tuberculosis drugs.
