Characterization of α-Enolase in Aggregatibacter actinomycetemcomitans

Objectives: We've identified α-enolase in Aggregatibacter actinomycetemcomitans (Aa) by proteomics. It is a 46 kDa protein that is up-regulated under iron-limited conditions. This study characterized this putative virulence factor. Methods: Proteomics was performed according to the protocols described by Keck Laboratories (New Haven, CT). Aa whole cell protein extracts were obtained following anaerobic incubation in iron-rich or iron-limited media. The labeled proteins were separated using pH 3-10 IPG strips and 12.5% SDS polyacrylamide gel electrophoresis. DeCyder software was used to quantify the gel image and to identify a "pick list" of differentially expressed protein spots to be excised and subjected to MS-based protein identification. Fur regulation was examined using one-step RT-qPCR and gel-shift assays with proteins isolated from Aawt; E. coli H1780 with pUCAafur expressing the cloned Fur protein; and Aafur- strains. To determine the location of α-enolase, cells were fractionated into their subcellular compartments according to the solubility of inner and outer membrane proteins, separated by SDS-PAGE and transferred to a membrane hybridized with anti-α-enolase antibody. Immunofluorescence microscopy of whole cells was performed using anti-α-enolase antibodies with anti-Aa antibodies used as a control. Results: RT-qPCR showed that α-enolase expression in Aa is up-regulated under iron-limited conditions and does not respond to iron concentrations in the Aafur- strain. The Fur protein bound to the promoter region of the α-enolase gene in gel shift assays. Intense staining was observed in whole cell extracts but no reaction to anti-α-enolase antibodies was seen in outer membrane fractions. There was no reaction observable by immunofluorescence microscopy using antibody to α-enolase to the Aa outer membrane using whole cells. Conclusions: α-enolase expression in Aa appears to be iron/Fur-regulated as indicated by gel-shift assays and RT-qPCR. Under our test conditions, α-enolase does not appear to be surface exposed in Aa.