Abstract:
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.