Novel Inhibitors of Bacterial Cytokinesis Identified by a Cell-based Antibiotic Screening Assay

The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery. The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery. Cell division has been of considerable interest as an antibacterial target because it comprises a group of well conserved proteins that are all essential for the viability of a wide range of bacteria, and their activities are completely different from those of the proteins involved in the division of mammalian cells. A number of compounds that act on components of the cell division machinery have been described (1Ohashi Y. Chijiiwa Y. Suzuki K. Takahashi K. Nanamiya H. Sato T. Hosoya Y. Ochi K. Kawamura F. J. Bacteriol. 1999; 181: 1348-1351Crossref PubMed Google Scholar, 2Jennings L.D. Foreman K.W. Rush III, T.S. Tsao D.H.H. Mosyak L. Kincaid S.L. Sukhdeo M.N. Sutherland A.G. Ding W. Kenny C.H. Sabus C.L. Liu H. Dushin E.G. 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Med. Chem. Lett. 2004; 14: 3161-3164Crossref PubMed Scopus (63) Google Scholar). So far, most of the effort has been directed at the FtsZ protein because it has several biochemical activities that can be assayed in vitro. Here we describe a novel approach to the discovery of inhibitors of bacterial cell division using a cell-based reporter assay. We have used the assay to identify a novel class of antibacterial compounds with potential broad-spectrum activity. We show that the compounds act on the highly conserved, essential cell division protein FtsZ in vitro and in vivo. These compounds represent a potential class of new antibiotics that act by a different mechanism than any of the antibiotics currently in clinical use. Bacterial Strains—The bacterial strains used in this work are: Bacillus subtilis 168 (trpC2); B. subtilis 1801 (trpC2 chrI::pJSIZΩpble(Pspac-ftsZ ble)); B. subtilis 2020 (trpC2 Ω(amyE::spc Pxyl-gfp-ftsZ)); B. subtilis PL16 (trpC2 Ω(amyE::spoIIQ-gus neo) Ω105J49 (spoIIA-lacZ cat)); Enterococcus faecalis ATCC 29212; Escherichia coli ATCC 25922; Haemophilus influenzae ATCC 49247; Moraxella catarrhalis ATCC 25240; Pseudomonas aeruginosa 101021; Staphylococcus aureus ATCC 601055; Streptococcus pneumoniae ATCC 49619. Molecular Cloning—B. subtilis was transformed by the method described by Anagnostopoulos and Spizizen (8Anagnostopoulos C. Spizizen J. J. Bacteriol. 1961; 81: 741-746Crossref PubMed Google Scholar) as modified by Jenkinson (9Jenkinson H.F. J. Gen. Microbiol. 1983; 129: 1945-1958PubMed Google Scholar) or the method described by Kunst and Rapoport (10Kunst F. Rapoport G. J. Bacteriol. 1995; 177: 2403-2407Crossref PubMed Google Scholar), except that 20 min after the addition of DNA the transformed cultures were supplemented with 0.66% casamino acids solution. Transformants were selected on Oxoid nutrient agar containing chloramphenicol (5 μg/ml). Sporulation was induced by growth in a hydrolyzed casein medium followed by resuspension in a starvation medium. Starvation medium was as described by Karamata and Gross (11Karamata D. Gross J.D. Mol. Gen. Genet. 1970; 108: 277-287Crossref PubMed Scopus (113) Google Scholar). DNA manipulations and E. coli transformations were carried out as described by Sambrook et al. (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). All cloning was done in E. coli DH5α (Invitrogen). Cell Division Dual Reporter Assay—B. subtilis PL16 was grown in hydrolyzed casein medium to exponential phase and centrifuged, and cell pellets were frozen at –80 °C. Frozen cell aliquots of strain PL16 were resuspended in warm starvation medium, incubated with shaking at 37 °C for 50 min (t50), and then added to Greiner 96-well microtiter plates containing compounds and controls (novobiocin, cephalexin, and 2% Me2SO). Plates were incubated at 37 °C shaking for another 100 min (t150). Assay buffer, containing lysozyme, 4-methyl-umbelliferyl β-d-galactoside, resofurin β-d-glucuronide, and Triton X-100, was added, and after a 60-min incubation at room temperature the fluorescence was measured on a BMG Fluostar Galaxy. Fluorescence was expressed in arbitrary fluorescence units (AFU). 3The abbreviations used are: AFUarbitrary fluorescence unitsMICminimum inhibitory concentrationMOPS4-morpholinepropanesulfonic acidGFPgreen fluorescent proteinIPTGisopropyl β-d-thiogalactopyranoside. Percentage ratio was calculated using the following formula,%Ratio=AFU(LacZ/GusMe2SO)-AFU(LacZ/Guscompound)AFU(LacZ/GusMe2SO)-AFU(LacZ/Gusantibiotic)×100 arbitrary fluorescence units minimum inhibitory concentration 4-morpholinepropanesulfonic acid green fluorescent protein isopropyl β-d-thiogalactopyranoside. Cell Division Phenotype Assays—Overnight cultures were grown in starvation medium supplemented with 1% hydrolyzed casein and then diluted in starvation medium supplemented with 3% hydrolyzed casein (B. subtilis) or in Mueller-Hinton medium (E. faecalis, S. aureus) and grown at 37 °C. The culture was diluted to A600 of ∼0.06, and 10-μl aliquots were added to transparent 96-well microtiter plates (BD Biosciences Falcon) containing dilutions of compounds in 100-μl volumes of medium. Concentrations of Me2SO, cephalexin, and 3-methoxybenzamide were included as controls. To examine the effects of ftsZ overexpression, B. subtilis 1801 was grown in the same medium, supplemented with 0.05–2 mm isopropyl β-d-thiogalactopyranoside (IPTG). After incubation for approximately 5 h (4–5 generations) at 37 °C, 20-μl culture samples were transferred to poly-l-lysine-coated slides for microscopy. Cell morphology was assessed by phase-contrast light and fluorescence microscopy on a Zeiss Axiovert 200 M inverted microscope equipped with a Sony Coolsnap HQ cooled charge-coupled device camera (Roper Scientific) and using Metamorph v6.1 software. Cell length and diameter measurements were determined using Metamorph software. Minimum Inhibitory Concentration (MIC) Testing—MICs for compounds against each strain were determined by a broth microdilution method according to the National Committee for Clinical Laboratory Standards (now the Clinical Laboratory Standards Institute) guidelines, for which the MIC was defined as the lowest concentration inhibiting visible growth. Absorbance (A600) readings were also used to calculate a percentage value for each compound using average A600 values for growth and no growth controls. Inhibition was characterized as a reduction in absorbance of ≥85% relative to the controls for all bacterial species, with the exception of S. pneumoniae (≥65%). GFP-FtsZ Localization Experiments—A colony of B. subtilis strain 2020 was resuspended in Oxoid antibiotic medium no. 3 (Pennassay broth) supplemented with 0.05% xylose and grown at 37 °C to A600 ∼0.6. Samples of the culture were mixed with an equal volume of medium containing twice the final desired concentration of compound. Ten-microliter volumes were harvested at appropriate time intervals, combined with an equal volume of prewarmed Pennassay broth containing 1% w/v agarose, and 10-μl samples of these were pipetted onto microscope slides, covered, and visualized as soon as possible. Fluorescein isothiocyanate or enhanced GFP settings were used to capture fluorescent images, with a 1500-ms exposure time and binning set to 1. Resistance Frequencies—To determine the resistance frequency B. subtilis 168 cells were spread on Mueller-Hinton agar (108-109 colony-forming units/plate) containing compound at 1×, 1.5×, 2×, and 4× the MIC. To determine the number of viable cells in the inoculum, dilutions of the culture were plated on compound-free Mueller-Hinton agar. Compound plates were incubated for up to 1 week to allow resistant mutants to grow. By dividing the number of resistant colonies on these compound plates by the number of colony-forming units originally plated the spontaneous resistance frequency was calculated. Characterization of PC58538-resistant Mutants—The ftsZ gene was PCR-amplified from chromosomal DNA of B. subtilis 168 and resistant mutants using oligos ftsZ.fw1 (5′-AAACTCGAGCCGAACATAATAAACAGAGC-3′) and ftsZ.rv1 (5′-CTAGAATTCTGTTTTGTTACTAAGCGAAGG-3′). Two separately amplified PCR products were sequenced using the PerkinElmer Life Sciences ABI PRISM BigDye terminator cycle sequencing kit. ftsZ.fw1 and ftsZ.rv1 were used as primers, and gels were run on an ABI PRISM 377 DNA sequencer (Sir William Dunn School of Pathology Sequencing Service, Oxford, UK). PCR products of wild-type and mutant ftsZ alleles were digested and ligated into pJPR1 and pSG1301. Ligated plasmids were transformed into B. subtilis 1801 or B. subtilis 168 with selection for chloramphenicol resistance in the presence of 0.2% xylose as necessary. FtsZ Sedimentation Assay—B. subtilis FtsZ protein and sedimentation assays were prepared generally as described elsewhere (13Haeusser D.P. Schwartz R.L. Smith A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (103) Google Scholar). Samples of protein were prespun at 25 p.s.i. (127,000 × g) for 20 min in an Airfuge (Beckman) to remove pre-existing polymers and aggregates. FtsZ was diluted to 5 μm final concentration in polymerization buffer containing Me2SO or compound at the indicated concentration. GTP was added to a final concentration of 1 mm followed by 0.1 mg/ml DEAE-dextran hydrochloride. Total reaction volumes were 100 μl. Reactions were incubated at 37 °C for 10 min, and 90-μl samples were centrifuged at 22 p.s.i. (119 000 × g) for 10 min to collect polymers. Supernatants were carefully removed and combined with sample loading buffer. Pellets were resuspended in 100-μl volumes of sample loading buffer. Samples were boiled for 5 min and resolved by 12% PAGE. Gels were stained with Coomassie Brilliant Blue, dried, and scanned. FtsZ GTPase Assay—Conversion of GTP to GDP by FtsZ was measured by monitoring the release of phosphate using malachite green dye in an end point enzyme assay. Reactions contained 5 μm FtsZ and compound at the concentrations indicated in reaction buffer (50 mm MOPS, 5 mm 200 mm Reactions were by the addition of mm GTP and incubated at 37 °C for 10 min, and samples were added to 100-μl volume of malachite green After min the A600 was measured using a E. coli FtsZ protein was from A Assay the of Sporulation on Cell cell-based assay the use of or more that are to specific of cell division. E. coli J. Bacteriol. 2004; PubMed Scopus Google Scholar) and B. A. and J. at to have any that gene in to of division. we and in J. Microbiol. PubMed Scopus Google Scholar) have that of B. of the that gene in the is on of the the and it from the The of the division is on all of the proteins that are to be for division J. Microbiol. Mol. Biol. 2003; PubMed Scopus Google Scholar). The of the mechanism that in to division is but we that this be in a assay for inhibitors of division. The of the assay is in 1. The assay on a strain of B. subtilis reporter The of by a of the to a reporter The as a for inhibitors and is a of the of the to The but is to of is soon after is enhanced by of division J. J. J. Gen. Microbiol. Google Scholar). the presence of a specific inhibitor of cell the of the reporter gene be that of the reporter be inhibitors the of of the ratio of the reporter provide a of specific inhibitors of division. We a microtiter assay for of cell division. The assay was for and using a of and Me2SO in The assay was by the and values for this were in the range of to for the of the assay as J. Biomol. 1999; PubMed Scopus Google Scholar). The of compounds was assessed by the ratio of controls we used cephalexin, which is a antibiotic that in E. at has a for the protein and at concentrations division C. L. J. Bacteriol. 2003; PubMed Scopus Google Scholar), and which to be an inhibitor of it is at concentrations (1Ohashi Y. Chijiiwa Y. Suzuki K. Takahashi K. Nanamiya H. Sato T. Hosoya Y. Ochi K. Kawamura F. J. Bacteriol. 1999; 181: 1348-1351Crossref PubMed Google Scholar). and 3-methoxybenzamide values for the ratio a range of concentrations antibiotics also a specific as by the assay to be specific for cell in the but have a more on cell We that this is because the starvation of cell is and in the is the of A wide range of antibiotics and growth inhibitors that and of which are in reporter and a on the reporter except for effects at a concentration were to be and be by assays that we to at cell division by of a of Cell microtiter assay was used to a of compounds at a concentration or μg/ml). hit compounds were in and then at that and against the assay were to a for of cell division in B. on phase-contrast microscopy. A hit which cell was as a of this the original hit to compound The reporter ratio was by this with the level the with and that compound also division in cells of wild-type B. cells a that been with compound the of was The average length of cells was μm at different time After h generations) in the presence of the average cell length with a in cell division. FtsZ the for seven proteins are to be for cell division in B. subtilis J. Microbiol. Mol. Biol. 2003; PubMed Scopus Google Scholar). or more of these be the target for We used several to identify the specific The division proteins are to into a at the of division. have a for this in which FtsZ as all proteins are on it for with GFP to division proteins and that all of these proteins were from by with the compound be with the compound at the of the to of the FtsZ A and a for cells an the of compound the of was with of fluorescence at the of each of the cells After 10 min of with the were completely FtsZ were the target for it is that of the protein the inhibitory effects of the compound. To this we used a strain of B. subtilis in which the ftsZ gene has been the of the the presence of of strain 1801 well with that FtsZ is for division this level of addition of in an cell length the same of compound but of FtsZ mm the cell length was to another approach to FtsZ as the target for we to isolate resistant also with resistance. of B. subtilis 168 were plated in the presence of concentrations of and incubated to allow the growth of resistant resistant mutants were by on the same concentration of compound. The MIC of compound the used was mutants were on agar containing and 4× MIC and a spontaneous resistance frequency of × colonies were after culture on agar containing that were to on and in the of were further DNA was from the and the ftsZ gene of each mutant was PCR-amplified and All of the mutants have a point in the ftsZ with FtsZ the target for the different were which in the following acid and the of the different mutants to with with the wild-type strain All of the mutants a of the compound on cell of cell length was almost these for mutant the length was to The amplified ftsZ alleles from wild-type B. subtilis 168 and each of the mutants were and transformed into B. and these were in the cell division assay. The same effects were that the mutant alleles were to resistance to the acid resistance all on of the FtsZ protein, on with the of the FtsZ protein J. PubMed Scopus Google Scholar) These are in the of the on the final that the compound on FtsZ we to in vitro with FtsZ a assay was used to polymerization of B. subtilis FtsZ Sedimentation of the protein in the of GTP but in its presence the presence of GTP and was We also the effects of on the GTPase of in GTPase was in a by with an value of of an an of analogues of has been of the more potent of these is was used in GTPase assays against B. subtilis and E. coli FtsZ in this compound was against of and it values of 10 and against the B. subtilis and E. coli indicated that the mechanism of of B. subtilis FtsZ by was and of range of analogues has been for and of against different antibacterial with against of the and these all at for of the more potent analogues are in generally the compounds were more against bacteria, all of the compounds in were against at M. compound with a MIC against this an or in in on B. subtilis and M. and it was also against S. aureus and E. The of against B. subtilis its against FtsZ protein of a selection of cell division inhibitor compounds against and subtilis faecalis ATCC coli ATCC influenzae ATCC catarrhalis ATCC aeruginosa aureus ATCC pneumoniae ATCC in a new We J. Mol. Microbiol. 2003; 50: PubMed Scopus Google Scholar) that of FtsZ in S. aureus has a different than in in that the cells and but and cells because a division have a cell is different from cells in which are because of of FtsZ C. J. J. Bacteriol. PubMed Scopus (108) Google Scholar). The more potent of to for the division machinery in and as by the of compound and of B. subtilis at which also in B. subtilis and FtsZ GTPase in vitro cells of E. faecalis at as in The in cell diameter in the presence of compound was a novel of a in E. faecalis with of cell division. The of resistance to antibiotics is well to this be to identify novel classes of compounds that act on new Cell division has been as a important target by and to inhibitors of cell division have on the FtsZ protein and have been on the use of in vitro assays against the GTPase or specific We to a cell-based assay with the that compounds be in vivo. We that by the assay specific and we compounds with activity. We the that in cells of B. of the is a to which of the is We that the assay up inhibitors of cell division. also to inhibitors of cell a range of which be in of as an important target for is to for division in cells have a growth because of is then the assay as which cell by a different mechanism J. Microbiol. PubMed Scopus Google Scholar). this be as a potential for the the use of microscopy to compound of compounds that in assays are as specific inhibitors of cell division. a with the a specific inhibitor of cell division was this compound antibacterial and was to have been up by assays for the compound was to be a specific inhibitor of cell division. is well that of cell division in bacteria B. subtilis and E. coli to the of which generally as to of that the target for the inhibitor and its analogues is the compound of the FtsZ well as at division and and because FtsZ is at the of the of J. Microbiol. Mol. Biol. 2003; PubMed Scopus Google Scholar), it be the protein that is by the compound. of FtsZ the inhibitory effects of point resistance to all in the ftsZ gene and acid in the The of these a for on the protein and work by the for compound. work for by the GTPase of the protein to for The spontaneous frequency and level of resistance by the point are of this compound from a development we that and a more potent can FtsZ in vitro in sedimentation and GTPase assays the original hit has antibacterial a number of analogues were that and a of activity. for is to more than against and and bacteria were of S. aureus was with these of another species, E. faecalis, with and analogues an with an on cell division compounds were as against clinical with resistance to S. aureus and as were against strains Cell division of the most attractive for novel classes of development of the compound described in this to the of compounds with were by We of for and as well as Levin and of for B. subtilis FtsZ We also of the William Dunn School of of Oxford, for with in vitro FtsZ