Mycobacterial DNA gyrase : biochemical characterisation and development of a novel gyrase inhibitor

Abstract

Synopsis DNA gyrase is a unique topoisomerase, as it is the only enzyme capable of introducing negative supercoils into DNA. The enzyme has essential roles in cellular DNA transactions such as DNA replication, transcription, and genetic recombination. Consequently, it has received considerable attention as a molecular target against various bacterial infections. In spite of its importance and ubiquitous presence in all bacteria, the enzyme has been studied extensively only from Escherichia coli. The genus Mycobacterium is of immense medical importance, as many pathogenic species belong to this group. Studying DNA gyrase from mycobacteria-a distant eubacterial group-is also important to understand differences in enzyme activity across diverse organisms and to provide insights into its function in mycobacteria. The work embodied in this thesis encompasses the purification and functional characterisation of mycobacterial DNA gyrase. In addition, attempts have been made to understand the mechanism of DNA transport by the enzyme and to develop a new class of inhibitors. The different properties of the mycobacterial gyrase have been compared with the prototype E. coli enzyme. ________________________________________ Chapter 1 - Background and Scope The concept of DNA topology, the classification of DNA topoisomerases, and the physiological roles of various topoisomerases are introduced. The mechanism of DNA transport by type II topoisomerases is discussed in detail along with structural aspects. A detailed account of E. coli DNA gyrase-its structure, function, and mechanism-is presented. A brief account of various type II topoisomerase inhibitors, with emphasis on DNA gyrase, is summarised. Finally, the importance of mycobacterial infections, their epidemiology, diagnosis, and chemotherapy are discussed. The chapter concludes with the scope and objectives of the present study. ________________________________________ Chapter 2 - Evidence for Two Subclasses of Bacterial Gyrases Structural heterogeneity in DNA gyrases from Gram positive and Gram negative bacteria is presented. Polyclonal antibodies produced against Mycobacterium tuberculosis GyrA recognised GyrA from different slow and fast growing mycobacterial species and also from several Gram positive bacteria. However, these antibodies did not cross react with E. coli GyrA or the enzyme from other Gram negative bacteria. The results, together with multiple sequence alignment, pairwise comparisons, and biochemical properties, support the existence of two subclasses of gyrases in the bacterial kingdom. ________________________________________ Chapter 3 - Monoclonal Antibody to M. smegmatis GyrB and Subunit Association A high affinity monoclonal antibody to the GyrB subunit of Mycobacterium smegmatis was developed, which did not cross react with either E. coli GyrB or GyrB subunits from other mycobacterial species. The antibody recognised an epitope in the N terminal, novobiocin binding domain of GyrB. Immunoprecipitation of gyrase from M. smegmatis cell lysate revealed an association of GyrA and GyrB subunits in the cell, mediated by ionic interactions. ________________________________________ Chapter 4 - One Step Immunoaffinity Purification and Biochemical Characterisation Based on observations from Chapter 3, a single step immunoaffinity purification procedure for M. smegmatis DNA gyrase was developed. The mycobacterial enzyme is a ~340 kDa heterotetramer comprising two GyrA and two GyrB subunits, exhibiting subtle differences and similarities to the well characterised E. coli gyrase. Key findings: • Decatenation: In contrast to E. coli gyrase, the M. smegmatis enzyme exhibits strong decatenase activity at physiological Mg² concentrations. • ATPase: The holoenzyme shows very low intrinsic ATPase activity, stimulated ~20 fold in the presence of DNA. DNA stimulated ATPase kinetics yielded apparent K = 0.59 mM and kcat = 0.32 s ¹. • DNA binding: The DNA dissociation constant (K ) is ~10 nM, ~20 fold lower than that of E. coli DNA gyrase. • Drug susceptibility: The enzyme displayed distinct susceptibility profiles to fluoroquinolones (moxifloxacin and ciprofloxacin). Despite these differences, mycobacterial DNA gyrase appears to be functionally and mechanistically conserved. ________________________________________ Chapter 5 - Anti GyrA Monoclonal Antibodies: Neutralisation and Epitope Mapping The generation, characterisation, and molecular interactions of anti GyrA monoclonal antibodies (mAbs) are described. Three mAbs-MsGyrA:C3, MsGyrA:H11, MsGyrA:E9-showed a high degree of cross reactivity with both fast and slow growing mycobacteria. In contrast, none recognised E. coli GyrA, corroborating Chapter 2. All three mAbs were IgG1 isotype, falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, as well as their Fab fragments, inhibited DNA supercoiling catalysed by mycobacterial DNA gyrase. The neutralising epitope appears to involve the region towards the N terminus (residues 351–415) of GyrA. ________________________________________ Chapter 6 - Mechanism of Inhibition by mAb MsGyrA:C3 Mechanistic studies show: • mAb binding does not disrupt GyrA–GyrB subunit interaction or gyrase–DNA binding. • The gyrase–DNA–mAb ternary complex retains DNA stimulated ATP hydrolysis and is competent for quinolone induced DNA cleavage and religation. • DNA gyrase purified from a quinolone resistant M. smegmatis mutant and a quinolone resistant clinical M. tuberculosis isolate showed no cross resistance to antibody mediated supercoiling inhibition. Thus, the mAb’s inhibitory mode is distinct from other known gyrase inhibitors and specific to mycobacterial gyrase. Proposed model: mAb binding near the primary dimer interface likely blocks the exit gate by restricting conformational movements of the gyrase–DNA complex. The mAb induced conformational change favours intermolecular strand passage, leading to catenane formation. ________________________________________ Chapter 7 - Engineering a Single Chain Fv (scFv) Inhibitor To identify the minimal inhibitory region, a single chain Fv (scFv) was engineered by cloning the variable regions of the heavy and light chains as a single gene, followed by expression, purification, and characterisation. Findings: • C3:scFv interacts with M. smegmatis and M. tuberculosis GyrA with affinity comparable to its parental IgG and Fab. • The ~30 kDa C3:scFv inhibits mycobacterial DNA gyrase supercoiling activity. These results provide a basis for designing peptide or protein based inhibitors against DNA gyrase. ________________________________________ Conclusions This thesis reveals multiple features of mycobacterial DNA gyrase that are distinct from E. coli, likely reflecting functional optimisation to mycobacterial physiology. The characterised inhibitory mAb represents a promising lead for novel anti mycobacterial agents, and the work advances our understanding of the DNA transport mechanism of type II topoisomerases. The immunological probes generated here will be valuable tools for structure–function and immunocytological studies of mycobacterial DNA gyrase, with potential applications in the diagnosis of mycobacterial infections.