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

Abstract

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.

Exploring non-toxic co-evolutionary docking

“…The stochastic nature of raw children generations, the differently limited protein cavity shapes / volumes, the hydrophobicity variations among children and/or other unknown target steric limitations, could explain those differences. Overall, these results highlight the existence of a vast chemical space to be further explored 21,22 .…”

The Search for Antibacterial Inhibitors Targeting Cell Division Protein FtsZ at Its Nucleotide and Allosteric Binding Sites

“…New cellular screens may be devised, for example, yeast cells expressing bacterial FtsZ-GFP fusions have been recently employed to monitor the effects of inhibitors on FtsZ assembly while assessing their toxicity of eukaryotic cells in a single-step microscopy screen. This approach [ 75 ] has revealed how PC190723 can bind into the interdomain cleft of FtsZ from Gram-negative Helicobacter pylori, as opposed to the case of EcFtsZ.…”

“…PC190723 and its analogs typically have narrow-spectrum antibacterial activity on several Gram-positive organisms, related to differences in the amino acid side chains lining the FtsZ interdomain cleft in other bacterial species [ 37 ]. Although this cleft is thought to open as well in T-state EcFtsZ filaments [ 26 ], two charged residue pairs have been proposed to form salt bridges between helix H7 and the C-terminal beta sheet, preventing cleft access to PC190723 in resistant species [ 75 ]. An allosteric modulator equivalent to PC190723 is thus missing for E. coli FtsZ, in addition to the general problem in crossing the outer membrane of Gram-negative bacteria [ 76 ], although the analog TXA6101 is active on Klebsiella pneumoniae [ 77 ].…”

Mechanistic insights into GTP-dependence and kinetic polarity of FtsZ filament assembly

“…The cultures were grown to an OD 600 between 0.3 - 0.6- and 1.5-mL culture was centrifuged at 2800 x g for 3 minutes. Microscopy was performed similar to what has been shown in (63). The cell pellets were gently resuspended in 150 µL of fresh LB medium of which 1 – 2 µL were spotted on 1.7 % LB-agarose pads and imaged.…”