Assessing the Role of FtsZ Activity in Bacterial Pathogenicity

An essential component of cell division is the formation of a septum, which separates newly formed cells.  This process is common to all organisms and must be achieved with a high degree of regularity.  In bacteria such as Escherichia coli, this process is controlled by a protein called Filamenting temperature-sensitive mutant Z (FtsZ).  This protein is not present in humans, and is therefore an ideal target for antibiotics. At Rockefeller, a compound was designed as an inhibitor of 2-epimerase, a novel component of bacteriophage-induced cell lysis.   This treatment is fatal to gram-positive bacteria.  Surprisingly, it was discovered that the compound also killed bacteria which lack 2-epimerase; thus, there must be a secondary target of this compound. FtsZ shows striking similarities to the 2-epimerase, and is a known essential component of cell division. The focus of my studies has been to investigate the impact of the compound on FtsZ activity.  We hypothesized that the compound inhibits FtsZ to the extent that cell division can no longer occur.  To explore this concept, we created a bacterial plasmid expressing FtsZ with a Green Fluorescence Protein (GFP) tag. To create the hybrid, the FtsZ gene was isolated using a polymerase chain reaction with primers specific to the FtsZ gene that incorporated restriction endonuclease recognition sites for SmaI and SalI.  This fragment was then digested and ligated into a similarly digested GFP expression vector plasmid.  The ligation product was used to transform competent E. coli.  Out of 24 transformants, one colony was identified to have possessed the FtsZ-GFP fusion. We will use this hybrid to assess the impact of the compound on FtsZ depolymerization.  We also assessed the compound's effects on polymerization and localization of FtsZ.  We found that, while there is no distinct and reproducible inhibition of normal localization, the polymerization was highly affected.  After purifying the protein using High Performance Liquid Chromatography, we conducted a GTPase activity assay with the protein being exposed to the compound for different lengths of time. We concluded that this compound inhibits the polymerization of FtsZ, which when reproduced in vivo should cause the death of the pathogen.