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Published online 28 November 2008 Nucleic Acids Research, 2009, Vol. 37, No. 3 731–737 doi:10.1093/nar/gkn936 SURVEY AND SUMMARY Bacterial topoisomerase I as a target for discovery of antibacterial compounds Yuk-Ching Tse-Dinh* Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA Received October 1, 2008; Revised and Accepted November 5, 2008 ABSTRACT barriers, DNA topoisomerases play vital roles in DNA replication, transcription, repair and recombination (1). Bacterial topoisomerase I is a potential target for Topoisomerases are divided into different subfamilies discovery of new antibacterial compounds. Mutant based on their mechanisms and sequence similarities topoisomerases identified by SOS induction screen- (2–4). Type I topoisomerases cleave and rejoin one ing demonstrated that accumulation of the DNA strand of DNA while type II topoisomerases cleave and cleavage complex formed by type IA topoisome- rejoin a double strand of DNA during catalysis. Human rases is bactericidal. Characterization of these topo IB, IIA and bacterial topo IIA enzymes are well mutants of Yersinia pestis and Escherichia coli utilized clinical targets for anticancer and antibacterial topoisomerase I showed that DNA religation can chemotherapy (5–9). These topoisomerase targeting com- be inhibited while maintaining DNA cleavage activity pounds initiate the cell killing process by either stabilizing by decreasing the binding affinity of Mg(II) ions. This or increasing the accumulation of the covalent complex can be accomplished either by mutation of the formed between the enzyme and cleaved DNA and are called ‘topoisomerase poisons’ (9–11). Compounds that TOPRIM motif involved directly in Mg(II) binding or interact with type IA topoisomerases with high specificity by altering the charge distribution of the active site to increase the level of the covalent complex remain to be region. Besides being used to elucidate the key ele- identified. The emergence of bacterial pathogens resistant ments for the control of the cleavage-religation to multiple antibacterial drugs in both the hospital and equilibrium, the SOS-inducing mutants of Y. pestis community setting is a serious global public health pro- and E. coli topoisomerase I have also been utilized blem, presenting an urgent need for discovery of new as models to study the cellular response following classes of antibacterial compounds. Based on the simila- the accumulation of bacterial topoisomerase I rities in the topoisomerase mechanisms, it should be pro- cleavage complex. Bacterial topoisomerase I is ductive to identify small molecules that can act as poisons required for preventing hypernegative supercoiling of bacterial type IA topoisomerases. of DNA during transcription. It plays an important role in transcription of stress genes during bacterial TYPE IA TOPOISOMERASES BACTERIA stress response. Topoisomerase I targeting poisons may be particularly effective when the bacterial There is at least one type IA topoisomerase found in each pathogen is responding to host defense, or in the bacterial genome (12). Topo I is present in all bacteria and presence of other antibiotics that induce the bacte- is the major activity responsible for removal of excess neg- ative supercoiling (13). In Escherichia coli, the promoters rial stress response. of topo I and gyrase genes are under homeostatic control by DNA supercoiling to maintain the global level of supercoiling (14,15). Topo III is found in only some INTRODUCTION of the bacterial genomes. In vitro it is much more efficient Topoisomerases catalyze the interconversion of DNA in catalyzing DNA decatenation reaction than relaxation topological isomers via coupling of DNA phosphodiester (13). Escherichia coli topo III has been proposed to play a bond cleavage and religation with the passage of DNA role in resolving RecQ associated recombination inter- through the break. By maintaining global DNA supercoil- mediates (16). Transposon insertion or deletion mutants ing at optimal level and removing local topological in the topA gene coding for topo I could be isolated from *To whom correspondence should be addressed. Tel: +1 914 594 4061; Fax: +1 914 594 4058; Email: [email protected] ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 732 Nucleic Acids Research, 2009, Vol. 37, No. 3 E. coli, Salmonella typhimurium and Shigella flexneri IA topoisomerase poison, the ability of the pathogen to (17–20). Attempts to isolate transposon insertion mutants survive in the host environment may be compromised. in the topA gene were unsuccessful in Mycobacterium Bacterial genes related to pathogenesis and virulence tuberculosis and Helicobacter pylori (21,22) suggesting have been shown to be sensitive to topA mutation. These that topo I might be essential in these bacteria. This include the fooB gene for fimbriae F1651 in pathogenic should be further investigated by additional genetic stud- E. coli 4787 (44) and the thermally regulated invasive ies. In E. coli, topo III is also not essential but absence of genes of S. flexneri (20). The invA gene of S. typhimurium both type IA topoisomerases resulted in chromosomal was poorly expressed in a topA mutant, and this correlated segregation defect (23). It was proposed that a type IA with the ability of S. typhimurium to penetrate tissue cul- topoisomerase activity is required in general for resolving ture cells (45). Signature-tagged transposon mutagenesis recombination intermediates involving single strand DNA has identified topA to be one of the genes affecting survival passage (1). A broad spectrum type IA topoisomerase of Yersinia enterocolitica in animal host (46). In addition, poison that can act on both topo I and topo III would topA gene was among those found to be highly expressed have a target always present in any bacteria. by avian pathogenic E. coli (APEC) in infected tissues Even though E. coli topA mutants are viable, growth at (47). low temperature requires topo I function (24,25). During The lethal mechanism of many bactericidal antibiotics transcription, the movement of the RNA polymerase based on different mechanisms of action, including quino- complex generates positive supercoils in the DNA tem- lones and ampicillin, have been shown to involve at least plate ahead of it and negative supercoils behind it (26). in part the formation of reactive oxygen species (48–50). Topo I activity is needed for removal of the negative Since bacteria in general would respond to such antibac- supercoils to prevent hypernegative supercoiling and R- terial antibiotics with transcription of stress regulons, topo loop formation (27,28). The importance of topo I function I is expected to interact extensively with negatively super- in transcription is also illustrated by the direct protein– coiled DNA at the induced transcription loci, and provide protein interaction between E. coli topo I and RNA poly- additional opportunity for the topo I cleavage complex to merase (29,30). be trapped by an inhibitor. Topo I inhibitors that act as poisons may therefore be particularly effective in combi- nation therapy with other antibiotics. FUNCTION OF BACTERIAL TOPOISOMERASE I IN STRESS RESPONSE AND PATHOGENESIS The role of topo I in relaxation of transcription-induced CELL KILLING BY BACTERIAL TOPOISOMERASE negative supercoiling is probably especially important I MUTANTS THAT ACCUMULATE THE COVALENT during stress response when a large number of stress CLEAVAGE COMPLEX genes have to be induced rapidly for survival (31). Since molecules that can act effectively as bacterial topo I Transcription of E. coli topA gene is under control of mul- poisons have not been previously identified, it is important tiple promoters recognized by s32, s38 in addition to s70 to demonstrate that trapping of topo I cleavage complexes (32,33). Besides these alternative s factors, topA transcrip- on single-stranded DNA will indeed lead to bacterial cell tion is also regulated by binding of Fis to the promoter death, just as tapping of type IIA topoisomerases on region (34). Topo I function and regulation have been double-stranded DNA would. This would validate the tar- shown to be important for E. coli response to high tem- geting of bacterial topo I in the search for novel antibac- perature and oxidative stress (35–38). RNase H overpro- terial compounds. It is known from previous work that duction can partially restore the s32–dependent stress the SOS response of E. coli is induced by the trapping of genes transcription defect in the absence of topA, indicat- gyrase cleavage complex by quinolones (51). Topo I muta- ing that R-loop formation from hypernegative supercoil- tions that mimic the action of a topoisomerase poison and ing at heat shock genes loci is responsible for the effect of result in increased accumulation of the cleavage complex the topA mutation. The response to high temperature and may be expected to also induce the SOS response. The oxidative stress is an important element of bacterial patho- isolation of such topo I mutants was achieved by screening gen adaptation against host defense. In H. pylori, the topA for SOS-inducing recombinant mutant Yersinia pestis gene has been shown to be up-regulated by prolonged acid topo I expressed in E. coli under the control of the exposure (39). Loss of topo I function in E. coli affects tightly-regulated BAD promoter (52). A pool of mutagen- transcription of the acid resistance genes gadA and gadBC ized plasmid pYTOP expressing random mutants of involving a mechanism independent of R-loop suppres- Y. pestis topo I was first isolated in the presence of 2% sion (40). It was shown recently that a pathway of s38 glucose to suppress the expression of any potentially lethal stress response involves unwrapping of the poised inactive mutant that accumulate the cleavage complex.

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