Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.
DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
U H
UH
Manjunatha
S
S
Mahadevan
S S
SS
Visweswariah
V
V
Nagaraja
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000276:immunology*; D004278:DNA, Superhelical / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D009161:Mycobacterium / Q000201:enzymology*; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D004278:DNA, Superhelical; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II