Actinobacillus seminis GroEL-homologous protein agglutinates sheep erythrocytes

Actinobacillus seminis, a commensal of ovine and caprine reproductive organs, is able to induce epididymitis in the small ruminants that it infects. In this work, we characterised two protein bands of approximately 150 kDa and 65 kDa. These proteins cross-reacted with a polyclonal serum against Gallibacterium anatis hemagglutinin and with a polyclonal serum from sheep with epididymitis, indicating that the proteins are expressed in vivo; the two proteins also interacted with biotin-labeled sheep fibrinogen and fibronectin, suggesting that they may function as adhesins. The participation of these proteins as adhesins was confirmed by a cultured human bladder cell-A. seminis adhesion assay and adherence inhibition by preincubation of A. seminis with polyclonal antiserum to the 150 kDa protein. Both proteins presented sequence identity with an A. seminis GroEL protein by mass spectrometry analysis and agglutinated glutaraldehyde-fixed sheep red blood cells. Immunogold labeling was observed by transmission electron microscopy on bacterial cells that were negatively stained, and a peroxidase reaction was detected in A. seminis biofilms, when an anti-A. seminis 150 kDa protein serum was used, indicating the presence of this protein on the surface of A. seminis and in biofilms. The A. seminis GroEL-homologue is a multifunctional protein that likely acts as a hemagglutinin.