Cytological Profile of Antibacterial FtsZ Inhibitors and Synthetic Peptide MciZ.

Lidia Araújo-Bazán,Laura B Ruiz-Avila,David Andreu,José M Andreu,Sonia Huecas

doi:10.3389/fmicb.2016.01558

Abstract

Cell division protein FtsZ is the organizer of the cytokinetic ring in almost all bacteria and a target for the discovery of new antibacterial agents that are needed to counter widespread antibiotic resistance. Bacterial cytological profiling, using quantitative microscopy, is a powerful approach for identifying the mechanism of action of antibacterial molecules affecting different cellular pathways. We have determined the cytological profile on Bacillus subtilis cells of a selection of small molecule inhibitors targeting FtsZ on different binding sites. FtsZ inhibitors lead to long undivided cells, impair the normal assembly of FtsZ into the midcell Z-rings, induce aberrant ring distributions, punctate FtsZ foci, membrane spots and also modify nucleoid length. Quantitative analysis of cell and nucleoid length combined, or the Z-ring distribution, allows categorizing FtsZ inhibitors and to distinguish them from antibiotics with other mechanisms of action, which should be useful for identifying new antibacterial FtsZ inhibitors. Biochemical assays of FtsZ polymerization and GTPase activity combined explain the cellular effects of the FtsZ polymer stabilizing agent PC190723 and its fragments. MciZ is a 40-aminoacid endogenous inhibitor of cell division normally expressed during sporulation in B. subtilis. Using FtsZ cytological profiling we have determined that exogenous synthetic MciZ is an effective inhibitor of B. subtilis cell division, Z-ring formation and localization. This finding supports our cell-based approach to screen for FtsZ inhibitors and opens new possibilities for peptide inhibitors of bacterial cell division.

Highlights

Cell division protein FtsZ, a tubulin-like GTPase conserved in most bacteria, is a target for new antibiotics
Bacillus subtilis 168 cells were grown in cation adjusted MuellerHinton broth (CAMHB; Becton, Dickinson and Company) at 37◦C to an absorbance 0.1–0.2 at 600 nm and the culture was divided into new flasks containing the compound at the desired concentration
In order to quantitatively characterize this effect we measured the length of wild type B. subtilis 168 cells incubated with our FtsZ inhibitor panel, as well as with several known antibiotics of different mechanisms of action

Summary

Introduction

Cell division protein FtsZ, a tubulin-like GTPase conserved in most bacteria, is a target for new antibiotics. At the earliest step of cell division, FtsZ undergoes assembly at mid-cell forming a dynamic membrane-attached ring structure (Bi and Lutkenhaus, 1991). Other bacterial division proteins are recruited to this Z-ring to form the divisome, a complex that constricts between the future daughter cells (Adams and Errington, 2009; Lutkenhaus et al, 2012; Egan and Vollmer, 2013; Meier and Goley, 2014; Haeusser and Margolin, 2016). FtsZ assembles into polar tubulin-like protofilaments in which the GTP-binding site of one monomer is at the association interface with the monomer completing the GTPase site (Oliva et al, 2004; Matsui et al, 2012). The Z-ring is stabilized by a protein network connecting the cell membrane to the chromosome in Escherichia coli cells (Buss et al, 2015), where the constriction force has been suggested to come mainly from the septal cell wall synthesis (Coltharp et al, 2016)

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Conclusion