Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection Karina Antúnez a, * , Matilde Anido a , Geraldine Schlapp a , Jay D. Evans b , Pablo Zunino a a Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, CP 11600 Montevideo, Uruguay b USDA ARS Bee Research Laboratory, BARC-East Building 476, Beltsville, MD, United States article info Article history: Received 27 March 2009 Accepted 12 July 2009 Available online 26 July 2009 Keywords: Paenibacillus larvae American Foulbrood Proteases Pathogenicity abstract Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different pro- teases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I – BOX A presented PP2, most isolates belonging to ERIC I – BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic dis- tribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, iso- lates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Paenibacillus larvae is the causative agent of American Foul- brood (AFB), the most severe bacterial disease that affects larvae of the honeybee Apis mellifera (Genersch et al., 2006; Hansen and Brodsgaard, 1999). AFB presents a worldwide distribution, causing a significant decrease in honeybee populations and production (honey, pollen, propolis, royal jelly and beeswax) (Hansen and Brodsgaard, 1999). Besides their importance for the beekeeping industry, honeybees play an essential role in the ecology of differ- ent environments throughout pollination, being essential for the production of agricultural systems and conservation of natural ecosystems. Apis mellifera larvae become infected by swallowing food con- taminated with spores. These spores germinate in the larval mid- gut, vegetative cells proliferate, move to the haemocoel and spread causing septicemia. As larvae die, their tissues decay and the consistency of the infected larval body changes to a brownish and viscous mass that then is dehydrated forming a scale. Sporula- tion of bacterial vegetative cells occurs during the whole infection process (Yue et al., 2008; Hansen and Brodsgaard, 1999). AFB infected ropes and scales are highly proteolytic, a feature that is often used for a quick diagnosis of the disease. Inoculation of infected material in milk produces protein coagulation in a few hours. These proteases are produced by P. larvae during the growth and sporulation processes (Hrabak and Martinek, 2007; De Graaf et al., 2006; Holst and Sturtevant, 1940) and are probably involved in the degradation of antibacterial peptides and in the degradation of larval tissues (Glinski and Jarosz, 1998; Casteels et al., 1989; Katznelson and Lochhead, 1947). Dancer and Chantawannakul (1997) reported the production by P. larvae of three different extracellular zinc-dependent metallo- proteases, that belong to an atypical type of multimeric proteases, which vary according to the geographic origin of different isolates (Dancer and Chantawannakul, 1997). The aim of the present work was to characterize the proteases secreted by P. larvae isolates from different geographic regions and to evaluate their potential role in pathogenicity. 2. Methods 2.1. Bacterial strains and culture media Fifty P. larvae isolates were randomly selected from the collec- tion of the Department of Microbiology, Instituto de Investigaci- ones Biológicas Clemente Estable. These isolates were obtained 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2009.07.010 * Corresponding author. Fax: +598 2 4875548. E-mail address: karina@iibce.edu.uy (K. Antúnez). Journal of Invertebrate Pathology 102 (2009) 129–132 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa