Molecular characterisation of the FtsK DNA motor and its interaction with topo IV in chromosome segregation

Lead Research Organisation: St George's, University of London
Department Name: Basic Medical Sciences

Abstract

FtsK is a multifunctional enzyme with the key role of coupling bacterial chromosome segregation and cell division. It is fixed to the DNA septum, a ring structure separating daughter cells, and functions as an ATP-driven DNA motor to pump chromosomal DNA through the closing septal ring. Moreover, it interacts with topo IV, an enzyme that separates topologically interlocked chromosomes and whose activity is blocked by quinolone drugs. These agents are widely used to treat infections caused by Streptococcus pneumoniae and other Gram-positive pathogens. Quinolones trap a topo IV-DNA complex that is converted into a lethal double stranded DNA break by motor proteins that track on DNA. Despite its fundamental scientific and pharmaceutical importance, little is known about topo IV, its interactions with FtsK and quinolones. By using a soluble truncated FtsK protein that retains the DNA motor activity, we aim to study how pneumococcal FtsK directs and modulates topo IV and its targeting by quinolones. The work will lead to significant advances in our understanding of chromosome segregation and cell division, and how antimicrobial quinolones disrupt these processes. In the longer term, the work should aid the development of more effective antibacterials.

Technical Summary

FtsK is a DNA motor protein that acts at the bacterial septum to coordinate chromosome segregation and cell division. FtsK activates topo IV, the chromosome decatenase, and target of quinolone drugs used against Streptococcus pneumoniae and other Gram-positives. Antipneumococcal quinolones stabilise a DNA cleavage complex of topo IV that is converted into a lethal lesion by DNA replication and other tracking processes on DNA. We aim to understand the mechanisms by which the DNA motor domain of pneumococcal FtsK directs topo IV functions. These studies will provide important new insights on the role of FtsK-topo IV interactions in chromosome segregation and quinolone action relevant to the design of new antibacterials.
 
Description The DNA motor FtsK and the enzyme topoisomerase IV are partners in bacterial chromosome segregation. Moreover, topo IV is a target for clinically important quinolones.

We have investigated the interactions of recombinant FtsK translocase and other molecules with the quinolone-arrested cleavage complex of topoisomerase IV. We have shown:
1. Collision with FtsK tracking on DNA did not convert a quinolone-arrested topo IV cleavage complex into an irreversible form suggesting that other mechanisms account for quinolone lethality in bacteria.
2. Clerocidin and quinazolinediones, small molecule antimicrobial inhibitors of topo IV- do arrest topoisomerase IV (and gyrase) in irreversible cleavage complexes on DNA through novel mechanisms involving covalent modification of the DNA gate and very tight binding, respectively. (Pan et al. Nucl Acids Res 36, 5516, 2008 and Pan et al Antimicrob Agents Chemother, 53, 3822-3831, 2009).
3. We have generated a number of useful reagents including plasmids allowing facile expression and purification of E. coli/S. pneumoniae FtsK and S. pneumoniae ParE metal binding domain of S. pneumoniae topo IV. New methodology developed to investigate covalent trapping and modification of topoisomerase cleavage complexes by alkylating poisons such as the antibiotic clerocidin leading to mechansitic advances.
Exploitation Route The main beneficiary from our work is the pharmaceutical industry which through new insights on topo IV arrest by drugs developed in the work will be aided in new development of novel antibacterial agents, so keenly needed in a climate of increasing drug resistance. In particular our work on clerocidin provides a prototype for new drugs acting through new mechanisms. Finally, our accumulated expertise on bacterial topo IV/gyrase was important in the FDA approval of besifloxacin as a new opthalmic quinolone (see Narrative Impact section)
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description In collaboration with Prof Emmanuelle Cambau, we established the target specificity of besifloxacin, a novel quinolone developed by Bausch and Lomb for the treatment of ocular infections (Cambau E et al J Antimicrob Chemother 63, 443-450, 2009. The work and publication were instrumental in the FDA approval of besifloxacin (Besivance) for treatment of bacterial conjunctivitis in May 2009. The properties of the drug established by these genetic and biochemical means were consistent with the desired dual targeting of gyrase and topo IV and lower resistance development. The Fisher group used recombinant human topoisomerase II (cloned by them and expressed in yeast) to show that besifloxacin was highly selective in inhibiting bacterial topoisomerases whilst having little effect on human topoisomerase. Their work contributed significantly to the FDA approval for the drug (NDA 22-308) granted in May 2009 - extended in Sept 2012 to other indications including virulent sight-threatening pathogens such as Pseudomonas aeruginosa. Besifloxacin (Besivance) has been shown to resolve bacterial conjunctivitis more rapidly and efficiently than other treatments on the market providing significant patient benefit. InSite Vision (which took a single-figure royalty for providing the mucoadhesive formulation) recorded $1.2 million besifloxacin royalty revenues in 2011, and $2.1 million for 20129. 400,000 prescriptions for besifloxacin were filled in the US from November 2010 through March 2012. The work was included as a Case Impact Study in the 2014 REF (L M Fisher)
First Year Of Impact 2009
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic
 
Title Recombinant enzymes 
Description Were able for the first time to produce highly active recombinant gyrase and topoisomerase IV from Streptococcus pneumoniae in quantity and of high quality. 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact Supplied to a small biotech company engaged in the development of novel antimicrobial agents.