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

Abstract

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 (1-7). 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 broadspectrum 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. Molecular Cloning-B. subtilis was transformed by the method described by Anagnostopoulos and Spizizen (8) as modified by Jenkinson (9) or the method described by Kunst and Rapoport (10), 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 g/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 (11). DNA manipulations and E. coli transformations were carried out as described by Sambrook et al. (12). All cloning was done in E. coli DH5␣ (Invitrogen). EXPERIMENTAL PROCEDURESCell Division Dual Reporter Assay-B. subtilis PL16 was grown in hydrolyzed casein medium to exponential phase and centrifuged, and cell pellets were frozen at Ϫ80°C. Frozen cell aliquots of strain PL16 were resuspended in ...

A 4‐Aminofurazan Derivative—A189—Inhibits Assembly of Bacterial Cell Division Protein FtsZ In Vitro and In Vivo

“…Several small-molecule inhibitors of FtsZ have already been reported (11,21,23,24). However, only one inhibitor with antibacterial activity, i.e.…”

The re-emergence of natural products for drug discovery in the genomics era

“…This approach is exemplified by the discovery of inhibitors of the FabF and FabB enzymes (for example, platensimycin 215 , which has been reviewed by Martens and Demain 216 ) and cell division protein FtsZ inhibitors 217 . Collections of bacterial and yeast strains with knockdown or overexpression of particular genes are available, along with information on identified genes that are essential for, for example, E. coli 218 , S. aureus 219 or Saccharomyces cerevisiae 220,221 .…”

Inhibition of Septation in Bacillus subtilis by a Peptide Antibiotic, Edeine B1

“…Inhibitors of FtsZ have been reported. [26][27][28][29][30][31] Edeine B 1 is a potentially useful tool for studying the mechanism of bacterial cell division.…”