Understanding DNA transport by topo IV and gyrase to counter antimicrobial resistance

Antibiotic resistance is a worldwide problem and a major challenge for the treatment of infection. Bacteria are becoming resistant to our most important antibiotics jeopardising even straightforward medical procedures. One serious threat is posed by Streptococcus pneumoniae, a global pathogen that causes life-threatening pneumonia and meningitis in childern and the elderly. Increasingly, S. pneumoniae isolates are found to be resistant to penicillins and to fluoroquinolones, which are key members of our antibiotic arsenal. It is known that fluoroquinolones interfere with DNA breakage by gyrase and topoisomerase (topo) IV, two enzymes that untangle DNA and are required for bacterial DNA replication and growth. Remarkably, these enzymes make a transient break in DNA and cross another DNA through the break. By this means, gyrase can remove twists when DNA is copied in the cell, and topo IV can unlink tangled chromosomes ahead of cell division. By using X-ray crystallography to solve the structure of these enzymes bound to DNA and fluoroquinolones, we now understand in detail how quinolones bind and trap the DNA break and how resistance arises. However, almost nothing is known about the nature of the transported DNA, how it is recognised, captured and then crossed through the DNA break. To fill this gap in knowledge, and guided by our recent structure of a topo IV-transport DNA complex, we shall use a combination of techniques including X-ray crystallography, cryo-electron microscopy, fluorescence and protein biochemistry to establish the mechanism of DNA transport by topo IV and gyrase from S. pneumoniae and from Klebsiella pneumoniae, another pathogen highly resistant to quinolones. Completion of the work will be a major contribution in understanding these fascinating molecular machines and will provide new opportunites for design of new drugs that overcome resistance by targeting DNA transport. New drugs will be essential in addressing the global emergency of antimicrobial resistance.

DNA gyrase and topo IV change DNA topology to regulate DNA supercoiling and chromosome segregation and are the validated targets of clinically important antibacterial fluoroquinolones. The enzymes act by capturing a DNA helix (the so-called T- or transport segment) and crossing it through a transient break in a second DNA segment (the so-called G-segment) via the formation of a 'cleavage complex'. From our solving the first structure of a cleavage complex, that of Streptococcus pneumoniae topo IV with bound quinolones, much is now known about the DNA gate. By contrast, the nature of the T-segment DNA and how it is captured and transported has remained elusive. We have now solved a second key structure - that of a Transport DNA segment captured in the hole formed by dimerisation of the ATPase domains of S. pneumoniae topo IV- the first glimpse of this important intermediate and which reveals intriguing features. The captured DNA is kinked, has a intercalated nucleotide and is bound asymmetrically by positively-charged residues lining the cavity whose mutagenesis severely inhibits or abrogates catalysis by topo IV. In the proposed research, we address four major questions. First, we shall use FRET, genetic and X-ray structural approaches to establish what identifies a transported DNA and whether kinking/ATP intercalation are integral to binding in the topo IV annulus. To illuminate the disposition of transport and gate DNAs and how DNA is navigated through the enzyme, we aim to solve an X-ray or cryo-EM structure of the full complex. Using mutagenesis of the cavity, we will identify residues essential to topo IV (and gyrase) activity suggesting a stratgy to guide drug design targeting transport. Finally, we shall translate these approaches to topo IV/gyrase of the ESKAPE pathogen, Klebsiella pneumoniae. Addressing these gaps in knowledge will advance topoisomerase mechanism and open new opportunities for design of novel agents able to combat quinolone resistance.

Who will benefit from the research?
1. Researchers in academic institutions:
Structural biologists and nano-engineers studying molecular machines
Those in a range of scientific disciplines with a focus on DNA-protein interactions
Academic researchers interested in collaborating with industry to translate basic research into drugs
Integrative biologists

2. Industry:
The pharmaceutical sector developing new antibacterial topo IV/gyrase inhibitors as anti-infectives. Potential also for novel anticancer therapeutics targeting human topo II and building on insights provided by bacterial gyrase and topo IV orthologues
Microbiologists and drug developers interested in combatting antimicrobial drug resistance

3. The general public:
In the longer term, fundamental work will translate into new medicines to counter disease

How will they benefit from the research?
1. Academics:
Training of first-rate postdocs in a cutting edge research laboratory
Access to new knowledge of wide relevance to many scientific disciplines
Multidisciplinary collaboration to enrich basic and translation approaches

2. Industry:
Gyrase and topo IV are established and active targets in the discovery programmes of nearly all pharmaceutical companies interested in developing anti-infective therapeutics. The research will provide novel usable insights on these enzyme mechanisms to inform drug discovery programmes.
Better appreciation of how antibiotics target gyrase and topo IV and how resistance e.g. to quinolones may be overcome through agents with a new mode of action.
Maintaining the competitive strength of teh UK pharmacuetical sector.

3. The general public:
Public engagement through local schools and open public meetings to inform people about antibiotics and resistance.
New drugs for the very young and elderly with pneumococcal disease
Society at large- through effective disease management reducing the societal and economic costs of healthcare and hospitalisation.
Enhanced quality of life.