Identification of Potentially Novel Iron-Regulated Proteins in Actinobacillus actinomycetemcomitans

Objectives: Similar to other bacterial pathogens, environmental iron is thought to regulate the expression of the fur gene and the Ferric Uptake Regulator (Fur) protein in Actinobacillus actinomycetemcomitans (Aa). In the present study, we performed a proteome analysis using differential fluorescence gel electrophoresis to identify additional iron-regulated proteins in this pathogen. Methods: The experiments were performed according to the protocols described by Keck Laboratories (New Haven, CT). Aa whole cell protein extracts were obtained following culture under aerobic or anaerobic conditions on iron-replete or iron-depleted media. The labelled proteins were separated in two dimensions using pH 3-10 IPG strips and 12.5% SDS polyacrylamide gel electrophoresis. Differentially expressed proteins were then subjected to automated in-gel tryptic digestion and MALDI-MS spectra-based identification. Results: Comparison of the two dimensional patterns revealed major differences between cells cultured under iron-replete or iron-depleted conditions. In Aa cells cultured under aerobic conditions in iron-depleted media, the expression of at least 26 proteins was upregulated and the expression of at least 29 proteins was downregulated compared to culture under aerobic conditions in iron-replete media. In Aa cells cultured under anaerobic conditions in iron-depleted media, at least 29 proteins were upregulated and 9 proteins were downregulated compared to culture under anaerobic conditions in iron-replete media. Nine of the upregulated proteins present under both aerobic and anaerobic conditions were compared to the corresponding genes in the published complete Aa genome. Two potential Fur-regulated proteins were identified - enolase and the Tu elongation factor, both of which are iron regulated in other microorganisms - as well as a putative “Fur box” in the promoter region of each gene. Conclusion: Proteome analysis can identify potentially iron-regulated genes in Aa and may be useful in defining genes in the fur regulon.