Paenibacillus larvae protease as a virulence factor in honeybee larvae infection

PELLEGRINI, M. C.; CUGNATA, N. M.; GUASPARI, E.; ALONSO SALCES, ROSA MARIA; PONCE, A. G.; DE CASTRO, R.; FUSELLI, S. R.

Murcia

Conferencia; 6th European Conference of Apidology EURBEE; 2014

Universidad de Murcia y el Campus de Excelencia Internacional 'Mare Nostrum 37/38'

Paenibacillus larvae (PL) is a causative agent of American Foulbrood, a severe disease affecting larvae of Apis mellifera. During the growth and sporulation processes PL produces proteases that could be involved in the degradation of larval tissues. Little is known about growth kinetics of Argentinean PL strains in broth, as well as virulence factors secreted by PL during larval infection. In this work, growth curves are carried out to determine CFU/mL, the total secreted proteins and the secreted proteases, in order to analyse whether the secretation/production of proteases is regulated by a quorum sensing mechanism. In a second step, essential oils are tested as regulators of that secretation/production of proteases, and/or inhibitors of the quorum sensing. Six PL strains were inoculated in J broth and incubated at 37ºC with shaking (170 rpm) for 72h. Aliquots were taken at different times to determine CFU/mL, quantify total secreted proteins by Bradford, test proteolytic activity on skim milk agar, and evaluate the presence of metalloprotease by gelatin zymography +/- EDTA (metalloprotease inhibitor). The bacterial culture went through exponential growth from hour 16 to 40, followed by a 2-4 h stationary phase and a quick death phase. The highest accumulation of total secreted proteins was registered between 67-72 h (0.0013 µg protein/CFU). Proteolytic activity was manifested, and the presence of metalloproteases in a 60 KDa band corroborated by zymography. These results suggest that Argentinian PL strains secrete metalloproteases that may act as virulence factors during A. mellifera larvae infection.