Functional Analysis of Fic Domain Bearing Proteins in Klebsiella pneumoniae

Fic domain bearing proteins are characterized with the presence of a domain carrying the conserved amino acid sequence (HPFX(D/E)GNGR). These proteins are present in all walks of life but are more concentrated within prokaryotes and more so within bacteria. In recent years these proteins have been increasingly characterized and their role in bacterial virulence is being elucidated. Fic proteins are usually secreted by bacteria and once delivered into the host cell they usually target cytoskeleton regulating G proteins that affect the target host in different ways depending on the target G protein. In Klebsiella pneumoniae, we have identified 5 homologous genes that code for five different Fic domain bearing proteins in this bacteria. Our first effort was to determine if these proteins were secreted, to verify if they adhere to the toxin-secretion system paradigm. The work then focused on determining the virulence effect of these proteins in an in vivo assay, an in vitro assay and an enzymatic assay for the Fic-RL protein. Fic- RL is one of the five proteins in K. pneumoniae have been shown to be conserved in all 80 sequenced strains of this bacteria. In our secretion assay this protein have been shown to be secreted by the bacteria, moreover, by utilizing different mutants that lacked different parts of secretion systems, we have been able to determine that the T4SS present on the Integrative and Conjugative Element 1 (icekp1) to be responsible for the secretion of this protein and not the other 4 homologs. Virulence assays showed that when this protein was expressed with eukaryotic cells by means of transfection, confocal and fluorescent microscopy revealed that cytotoxic effect are evident as cell rounding and actin cytoskeletal collapse in transfected cells, which does not occur when a mutated version of the protein is instead expressed. Survival killing assays utilizing the larvae of the Galleria mellonella wax moth, revealed significant attenuation of strains lacking the gene coding for Fic domain bearing proteins, which is partially complemented by re-introducing the genes in plasmid constructs. The enzymatic function for the Fic-RL protein was shown in a Guanine Exchange Factor assay, designed to reveal if a protein is able to utilize fluorophore bound substrates in its reaction of provided G protein targets (Cdc42, Rac1, RhoA), showed that Fic-RL is able to cause an increase in relative fluorescence measured that was similar to a known Guanine Exchange Factor (hDbs) but only when used on Cdc42 and not the other two G proteins.