Western-blot detection of RickA within spotted fever group rickettsiae using a specific monoclonal antibody

Abstract

Spotted fever group rickettsiae are obligate intracellular pathogens able to manipulate the actin cytoskeleton, thus enabling cell-to-cell spreading during infection. While the RickA protein, which has similarity to the WASP family of Arp2/3-complex activators, was described as being responsible for actin-based motility, recent studies demonstrated that another factor, still unidentified, is also involved in this phenomenon. Here, using recombinant protein of Rickettsia conorii as an antigen, we produced a monoclonal antibody (mAb) directed against RickA. Its specificity was checked using two-dimensional polyacrylamide gel electrophoresis coupled with MS analysis. In Western-blot assays, our antibody recognized the RickA protein from all spotted fever group rickettsiae tested. This mAb would be useful to monitor the expression of RickA in spotted fever group rickettsiae grown under various culture conditions, associated or not with the motility phenotype, and thus to gain better knowledge about the molecular mechanisms involved in their cell-to-cell spreading.