Synthetic developmental regulator MciZ targets FtsZ across Bacillus species and inhibits bacterial division

Abstract

Summary Cell division in most bacteria is directed by FtsZ, a conserved tubulin‐like GTPase that assembles forming the cytokinetic Z‐ring and constitutes a target for the discovery of new antibiotics. The developmental regulator MciZ, a 40‐amino acid peptide endogenously produced during Bacillus subtilis sporulation, halts cytokinesis in the mother cell by inhibiting FtsZ. The crystal structure of a FtsZ:MciZ complex revealed that bound MciZ extends the C‐terminal β‐sheet of FtsZ blocking its assembly interface. Here we demonstrate that exogenously added MciZ specifically inhibits B. subtilis cell division, sporulation and germination, and provide insight into MciZ molecular recognition by FtsZ from different bacteria. MciZ and FtsZ form a complex with sub‐micromolar affinity, analyzed by analytical ultracentrifugation, laser biolayer interferometry and isothermal titration calorimetry. Synthetic MciZ analogs, carrying single amino acid substitutions impairing MciZ β‐strand formation or hydrogen bonding to FtsZ, show a gradual reduction in affinity that resembles their impaired activity in bacteria. Gene sequences encoding MciZ spread across genus Bacillus and synthetic MciZ slows down cell division in Bacillus species, including pathogenic Bacillus cereus and Bacillus anthracis. Moreover, B. subtilis MciZ is recognized by the homologous FtsZ from Staphylococcus aureus and inhibits division when it is expressed into S. aureus cells.

Nucleotide‐induced folding of cell division protein FtsZ from Staphylococcus aureus

“…It provides a solid starting point to robotic high‐throughput screening for high‐affinity inhibitors targeting the GTP‐binding site of Gram‐positive S. aureus FtsZ. Fluorescence and ITC binding measurements should be combined with phenotypically characterizing inhibitor effects on bacterial division . On the other hand, very high‐affinity ligands, binding to the GTP or other site, may induce apo‐SaFtsZ T folding in the absence of osmolytes, so that they could be picked up by an appropriate fluorescence thermal shift screen designed to detect apo‐SaFtsZ T stabilization.…”

Targeting the Achilles Heel of FtsZ: The Interdomain Cleft

“…However, MciZ does not bind to the IDC either, and instead interacts with H10 and beta strand 9 of FtsZ. This results in occlusion of subunit-subunit contacts that causes capping of the growing FtsZ protofilament end ( Araujo-Bazan et al, 2019 ). Other peptide inhibitors of FtsZ, including Kil from bacteriophage lambda and GP0.4 from bacteriophage T7, disrupt assembly of Ec FtsZ protofilaments, but their binding sites on FtsZ are not yet known ( Kiro et al, 2013 ; Haeusser et al, 2014 ; Hernandez-Rocamora et al, 2015 ).…”

Intermolecular Communication in Bacillus subtilis: RNA-RNA, RNA-Protein and Small Protein-Protein Interactions

“…Moreover, it can also cause filamentation in vitro. A recent study showed that MciZ can affect B. subtilis sporulation: Excessive amounts of MciZ produced intracellularly or added exogenously can not only decrease spore formation efficiency but also inhibit spore germination (Araújo-Bazán et al, 2019).…”