Characterization of Putative FUR Proteins in Prevotella intermedia

Prevotella intermedia has been implicated in many forms of human periodontal disease, but little is known about its virulence features. Many microbes (e.g. Escherichia coli) have developed specific and efficient systems to assimilate iron, an essential nutrient, from host tissues. These pathways may promote expression of virulence factors as well as proteins and non-protein siderophores involved in iron uptake and regulation. Iron-regulated gene expression is regulated at the transcription level, primarily through negative regulation by the Ferric Uptake Regulator (FUR) protein. Homologues of E. coli FUR protein are found in numerous bacteria. We hypothesize that P. intermedia has virulent iron-acquisition and utilization pathways that are regulated by a FUR-like protein. Objective: Identify, clone, and characterize a P. intermedia FUR homolog and determine its ability to complement E. coli FUR activity. Methods: We identified possible FUR-homologues by comparing E. coli FUR protein sequence to P. intermedia genomic DNA sequences using BLAST. We cloned and expressed these proteins in E. coli. Using E. coli 1780 cells (FUR null) we examined the ability of P. intermedia FUR homologues to complement missing FUR activity. Results:P. intermedia contains three genes that encode FUR-like proteins, each expressed from a single open reading frame, and containing conserved DNA- and metal-binding regions. In 1780 cells, the three FUR-like proteins partially complement E. coli FUR function to varying degrees, although none as efficiently as the re-introduced E. coli FUR. Conclusions: Unlike E. coli, P. intermedia contains three distinct FUR-like genes. Partial complementation of E. coli FUR function by each of these suggests complex iron-regulated systems in P. intermedia. While genetic manipulation of P. intermedia is not currently possible, we are continuing to characterize its FUR-like proteins to discover and possibly target virulent iron-regulated pathways in this oral pathogen.