Dynamics of interdomain rotation facilitates FtsZ filament assembly.

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, bound to GDP and GMPPNP respectively, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase with a higher critical concentration (CC) compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified that Phe224, located at the interdomain cleft of SmFtsZ, is crucial for R- to T-state transition. SmFtsZF224M exhibited higher GTPase activity and lower CC, whereas the corresponding EcFtsZM225F resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis suggests that the rotation is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study highlights the importance of interdomain interactions and conformational changes in regulating FtsZ assembly dynamics.

Disruption of salt bridge interactions in the inter-domain cleft of the tubulin-like protein FtsZ of Escherichia coli makes cells sensitive to the cell division inhibitor PC190723.

FtsZ forms a ring-like assembly at the site of division in bacteria. It is the first protein involved in the formation of the divisome complex to split the cell into two halves, indicating its importance in bacterial cell division. FtsZ is an attractive target for developing new anti-microbial drugs to overcome the challenges of antibiotic resistance. The most potent inhibitor against FtsZ is PC190723, which is effective against all strains and species of Staphylococcus, including the methicillin- and multi-drug-resistant Staphylococcus aureus and strains of Bacillus. However, FtsZs from bacteria such as E. coli, Streptococcus, and Enterococcus were shown to be resistant to this inhibitor. In this study, we provide further evidence that the three pairwise bridging interactions, between residues S227 and G191, R307 and E198 and D299 and R202, between S7, S9, S10 ß-strands and the H7 helix occlude the inhibitor from binding to E. coli FtsZ. We generated single, double and triple mutations to disrupt those bridges and tested the effectiveness of PC190723 directly on Z-ring assembly in vivo. Our results show that the disruption of S227-G191 and R307-E198 bridges render EcFtsZ highly sensitive to PC190723 for Z-ring assembly. Ectopic expression of the double mutants, FtsZ S227I R307V results in hypersensitivity of the susceptible E. coli imp4213 strain to PC190723. Our studies could further predict the effectiveness of PC190723 or its derivatives towards FtsZs of other bacterial genera.

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

A mechanism of salt bridge-mediated resistance to FtsZ inhibitor PC190723 revealed by a cell-based screen.

Bacterial cell division proteins, especially the tubulin homologue 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. On the basis of sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt bridge interactions between the central H7 helix and ß-strands S9 and S10 mediates 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.