Chaperone-mediated control of toxin-antitoxin systems in Mycobacterium tuberculosis. – mycoTAC

Task 1
-Protein purification
-Cloning and mutagenesis
-Affinity purification methods
-proteomic analyses

Task 2 :
-Protein purification
-Cloning and mutagenesis
-Genetic engineering

Task 3 :
-Protein purification
-Cloning and mutagenesis
-Affinity purification methods
-In vitro degradation assay

Task 4 :
-Protein purification
-Affinity purification methods
-SEC-MALS/X-ray crystallography

-Exploring the chaperone function in protein export: We have purified the chaperone and different Sec proteins and identified a direct interaction between the chaperone and one of the Sec proteins, using an in vitro pull-down assay. We have performed affinity purification followed by quantitative proteomic analyses of his-tagged chaperone pre-incubated with wild type or ?TAC cell lysates and identified several proteins, potentially new substrates of the chaperone.
-Toxin target(s) and role in virulence: We have developed a Tet-based promoter replacement cassette allowing the one-step engineering of mutant with a conditional expression of selected chromosomal genes. This cassette was used to construct isogenic sets of strains with TAC operon under the control of anhydrotetracycline. In addition, we have deleted TAC in the presence of the chaperone under the control of anhydrotetracycline at a different locus. We have established a procedure to purify the toxin.
- Regulation by proteases: We have shown that proteolysis is key to the activation of the system. A conditional protease mutant was constructed and we have identified a specific motif that is recognized by the proteases in vivo.
-TAC complex formation and chaperone addiction:We have identified and characterized the chaperone dependent domain of the antitoxin in vivo and in vitro. In addition, through a directed evolution experiment of solitary chaperone, we have now identified a previously uncharacterized but conserved region of the chaperone, which modulates its ability to control toxin-antitoxin systems. We have explored the structure and dynamics of TAC members—successfully purified chaperone and chaperone-antitoxin complexes. Important crystallization campaigns using commercial and home-made screening kits have been conducted on the chaperone alone and on both constructs of the complex, and also in the presence of trace amounts of protease.

-Structural determinant of Sec involved in the interaction with the chaperone will be determined. Interplay between Sec and the toxin antitoxin chaperone components will be assess in vitro, using purified proteins already available in the Lab. The role played by these new partners in vivo will be investigated as well, using either substrates identified above or known exported proteins.
-The Tet-inducible strains will be used to investigate whether expression of the components during infection of macrophages and during the persistence phase in mouse model. Preliminary experiments in vivo have been initiated to set up the conditions. We will use an in vitro approach to characterize the toxin toxic mechanism.
-Interplay between combination of TAC components and proteases and detailed analysis of the degron.
-Further seek for structure of TAC members using other functional TAC systems from different bacteria as well as complexes between the chaperone and purified peptides.

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Newly synthesized proteins emerging from the ribosome are assisted by essential molecular chaperones, which facilitate their folding and/or partition them as cytosolic, inner-membrane, or exported proteins. In most Gram-negative bacteria, newborn precursors of exported proteins are targeted to the Sec translocon at the inner membrane with the assistance of the chaperone SecB, which binds non-native substrates and maintains them in a translocation-competent state.
The major human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, is an unusual Gram-positive bacterium with a well-defined outer membrane (the mycomembrane) and mostly unidentified outer membrane proteins. Nothing is known yet about the molecular chaperones involved in assisting the export of such proteins. Remarkably, we have recently revealed the presence of an atypical SecB-like chaperone Rv1957 in every strain within the M. tuberculosis complex, which seems to combine two important cellular functions. Indeed, in spite of very little sequence similarity with the canonical SecB chaperone from Escherichia coli, Rv1957 is able (i) to efficiently replace SecB during protein export in E. coli and (ii) to specifically control a functional toxin-antitoxin system relevant for M. tuberculosis adaptive response. This novel system, named TAC for toxin-antitoxin-chaperone, associates for the first time a molecular chaperone with stress-responsive toxin-antitoxin element. How the mycobacterial SecB-like chaperone responds to stress and controls the toxin activation cascade, and to what extent such activation is important for M. tuberculosis persistence and virulence are so far unresolved questions. An attractive hypothesis is that the chaperone represents an intimate link between export stress and activation of a toxin-antitoxin system.
The aim of the mycoTAC project is to unravel the molecular mechanism of TAC activation and its potential role in M. tuberculosis physiology and virulence. We will investigate whether the mycobacterial chaperone from TAC is indeed involved in protein export and whether such a property is linked to the activation of the associated stress-responsive toxin-antitoxin system (Task 1). Furthermore, we will identify the cellular process(es) targeted by the toxin from TAC (potentially metal homeostasis), and examine its role in persistence and virulence (Task 2). In addition, we will search for the stress protease(s) and the associated degradation signals involved in TAC activation (Task 3). Finally, we will dissect the molecular mechanism of TAC complex formation and attempt to identify the structure of TAC members (Task 4). The proposed combination of biochemical, biophysical, structural biology and in vivo approaches presented in this project will hopefully shed light on the mechanism and the function of this atypical stress-responsive element.