Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Several assays have been designed to screen for small molecules targeting FtsZ but rely upon a multitude of steps to validate the target and to ensure minimum toxicity to the eukaryotic cells. Here, we have utilized the fission yeast heterologous expression system to develop a single step cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ and are non-toxic to eukaryotic cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-sheet S10 and S7 mediate resistance to PC190723 in FtsZ. The single step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay functions as a powerful tool to rapidly identify new molecules that specifically target the bacterial cell division protein FtsZ, or other polymeric bacterial cytoskeletal proteins, understand how they affect polymerization dynamics and study resistance determinants in targets.

FtsZ, a tubulin homolog, forms the Z-ring at the division site in bacteria. FtsZ filaments guide peptidoglycan synthesis machinery to drive cell division, while their role in cell wall-less bacteria is unclear. We report the structure and biochemical properties of FtsZ from the cell wall-less bacterium Spiroplasma melliferum (SmFtsZ). Compared to Escherichia coli FtsZ (EcFtsZ), SmFtsZ possessed lower GTPase activity and higher critical concentration (CC) of polymerization. In FtsZs, a conformational switch from R- to T-state favours polymerization. In the crystal structures of SmFtsZ captured as domain-swapped dimers, conformation of the GTP-bound N-terminal domain (NTD) was in the T-state, while the relative orientation of the NTD and the C-terminal domain (CTD) matched the R-state. The occurrence of GTP-bound NTD in the T-state and CTD in the R-state captures a conformational state that facilitates preferential binding of the NTD of the monomeric FtsZ to the CTD-exposed end of the FtsZ filament. CTD of the nucleotide-bound monomer cannot bind to the NTD-exposed end of the filament unless relative rotation of the domains leads to cleft opening. Mutation of SmFtsZ-Phe224 in the cleft to methionine, the corresponding residue in EcFtsZ (Met226), resulted in higher GTPase activity and lower CC. Mutation of EcFtsZM226F resulted in cell division defects. These results suggest that the relative rotation of the two domains leading to cleft opening is a rate-limiting step of polymerization. Hence, addition of monomers to the CTD end of the filament is hypothesized to be faster than to the NTD end, thus explaining the kinetic polarity.

Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-strands S9 and S10 mediate resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

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