Testosterone and estradiol regulate the expression of proteases and a hemoglobinase in Actinobacillus seminis

Actinobacillus seminis is a causative agent of epididymitis, infertility, and sterility in sexually mature ruminants. Previous studies suggest that sex hormones regulate the expression of A. seminis virulence factors, promote its growth, and support adhesin expression and biofilm formation; however, the effects of these hormones on protease expression are unknown. The effects of testosterone (1-5 ng/ml) and estradiol (5-25 pg/ml) were evaluated on the A. seminis protease expression. Zymograms revealed that both hormones enhanced the secretion of a 50 kDa metalloprotease and a 65 kDa serine protease. The 65 kDa serine protease showed optimal activity at a pH of 6-8, was stable at temperatures up to 70°C, and hydrolyzed bovine hemoglobin and casein; interestingly, this hemoglobin protease was expressed after treatment with sex steroid hormones but not in the presence of catecholamines. This serine-protease presents identity with two A. seminis serine proteases of 50 kDa. The metalloprotease has previously been shown to hydrolyze bovine IgG and fibrinogen and presented identity with a carboxy-terminal protease. Both proteases showed immune cross-reactivity with hyperimmune sera against metalloproteases from A. seminis and Actinobacillus pleuropneumoniae, and with serum against a Mannheimia haemolytica serine protease. Our results suggest that hormones affect the expression of different A. seminis virulence factors, such as proteases, and may play a key role in bacterial pathogenesis.