Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.

We conclude that ache, ankle edema, and skin changes in limbs with reflux confined to the superficial venous system are predominantly associated with reflux in the below-knee veins. Ulceration is found only when the whole of the LSV is involved (8%) or when reflux is extensive in both LSV and SSV (14%).

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a ‘pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.

The pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density-dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid-based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density-dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxI. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxI homologue from both E. carotovora (carI) and E. agglomerans (eagI) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri LuxI. Despite this, carI, eagI and luxI are shown to be biologically equivalent. An insertion mutant of eagI demonstrates that this gene is essential for OHHL production in E. agglomerans.

Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 μM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 μM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or with intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound.

Staphylococcus aureus is a major human pathogen, which produces a variety of virulence determinants. To study environmental regulation of virulencedeterminant production, several transcriptional reporter gene fusions were constructed. Chromosomal fusions were made with the staphylococcal accessory regulator (sarA), a-haemolysin (hla), surface protein A (spa) and toxic-shock syndrome toxin-I (tst) genes. The effect of many different environmental conditions on the expression of the fusions was examined. Expression of hla, t s t and spa was strongly repressed in the presence of sodium chloride (1 M) or sucrose (20 mM), but sarA was relatively unaffected. The global regulator of expression of virulence-determinant genes, agr (accessory gene regulator) was not involved in the salt or sucrose repression. Novobiocin, a DNA gyrase inhibitor, did not significantly increase the expression of tst in wild-type or agr backgrounds and failed to relieve the salt suppression. Expression of tst was strongly stimulated in several low-metal environments, independently of agr, whilst spa levels were significantly reduced by EGTA. The complex, interactive role of environmental factors in the control of expression of the virulence determinants is discussed.Keywords : Staphylococcus aureus, virulence, regulation, toxin, surface protein INTRODUCTIONStaphylococcus aureus is an important human pathogen implicated in a wide range of diseases including septicaemia, meningitis, endocarditis, osteomyelitis, septic arthritis, toxic-shock syndrome (TSS) and food poisoning (Waldvogel, 1995). The pathogenic diversity of the bacterium reflects its ability to successfully colonize, adapt and survive in many different host tissues during infection. The production of a repertoire of virulence determinants such as extracellular proteins [including a-haemolysin (encoded by the hla gene) and toxic-shock-syndrome toxin-1 (tst gene)] and cellsurface-associated proteins [such as protein A (spa gene) and fibronectin-binding protein] contributes to the virulence of the bacterium (Iandolo, 1990). The exo-and surface proteins are expressed coordinately in a growth-phase-dependent manner and are controlled by at least two well-characterized global regulatory loci, sar and agr (Cheung et al., 1992 Morfeldt et al., 1988;Peng et al., 1988). Other putative pleiotropic regulators such as xpr and sae have also been described (Hart et al., 1993 ;Giraudo et al., 1994). The agr locus controls virulence-determinant gene expression by way of a regulatory RNA molecule, RNAIII, as an effector (Janzon & Arvidson, 1990; Novick et al., 1993). This both upregulates production of extracellular toxins and represses expression of surface proteins primarily at the transcriptional level (Kornblum et al., 1990;Vandenesch et al., 1991 ; Novick et al., 1993 ;Morfeldt et al., 1995). The agr locus encodes a two-component sensor-regulator pair, agrC and agrA, respectively Parkinson & Kofoid, 1992). The sensor protein (AgrC) has been proposed to sense the extracellular concentration...

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