ABSTRACT
Antimicrobial resistance (AMR) is a serious global threat demanding innovations for effective control of pathogens. The bacterial SOS response, regulated by the master regulators, LexA and RecA, contributes to AMR through advantageous mutations. Targeting the LexA/RecA system with a novel inhibitor could suppress the SOS response and potentially reduce the occurrence of AMR. RecA presents a challenge as a therapeutic target due to its conserved structure and function across species, including humans. Conversely, LexA which is absent in eukaryotes, can be potentially targeted, due to its involvement in SOS response which is majorly responsible for adaptive mutagenesis and AMR. Our studies combining bioinformatic, biochemical, biophysical, molecular, and cell-based assays present a unique inhibitor of mycobacterial LexA, wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA of Mycobacterium tuberculosis (Mtb), consequently hindering its cleavage, suppressing SOS response thereby reducing mutation frequency and AMR.
Subject(s)
Bacterial Proteins , Drug Resistance, Bacterial , Mycobacterium tuberculosis , SOS Response, Genetics , Serine Endopeptidases , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , SOS Response, Genetics/drug effects , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Serine Endopeptidases/metabolism , Serine Endopeptidases/genetics , Rec A Recombinases/metabolism , Rec A Recombinases/genetics , Rec A Recombinases/chemistry , Humans , Mutagens/pharmacology , Anti-Bacterial Agents/pharmacologyABSTRACT
Transcriptional repressor, LexA, regulates the 'SOS' response, an indispensable bacterial DNA damage repair machinery. Compared with its Escherichia coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA-binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in 'SOS' regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different 'SOS' boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, the present study provides insights into the kinetics of the interaction between Mtb LexA and its target 'SOS' boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of 'SOS' regulation and activation in Mtb.
Subject(s)
Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Interferometry/methods , Mycobacterium tuberculosis/enzymology , Serine Endopeptidases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Kinetics , Phylogeny , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SOS Response, Genetics/physiology , Serine Endopeptidases/chemistry , Serine Endopeptidases/geneticsABSTRACT
Transcriptional repressor, LexA, regulates the "SOS" response, an indispensable bacterial DNA damage repair machinery. Compared to its E.coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in "SOS" regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different "SOS" boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, this study provides insights into the kinetics of the interaction between Mtb LexA and its target "SOS" boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of "SOS" regulation and activation in Mtb.