Short communication Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae Karina Antu ´ nez a, *, Matilde Anido a , Jay D. Evans b , Pablo Zunino a a Departamento de Microbiologı´a, Instituto de Investigaciones Biolo ´gicas Clemente Estable, Avda. Italia 3318, C.P.11600 Montevideo, Uruguay b USDA ARS Bee Research Laboratory, BARC-East Building 476, Beltsville, MD, United States 1. Introduction American Foulbrood is a worldwide distributed disease that affects larvae of the honeybee Apis mellifera, causing a significant decrease in honeybee populations and produc- tivity (Hansen and Brødsgaard, 1999). The causative agent is Paenibacillus larvae, a gram-positive and spore-forming bacterium (Genersch et al., 2006). Larvae are infected by swallowing food contaminated with spores. These spores germinate in the larval midgut, vegetative cells proliferate, move to the haemocoel and spread causing septicemia. As larvae die their tissues decay and sporulation of bacterial vegetative cells occurs (Hansen and Brødsgaard, 1999). So far the only described virulence factors of P. larvae are metalloproteases (Dancer and Chantawannakul, 1997; Hrabak and Martinek, 2007). It has been proposed that metalloproteases are involved in the inhibition of immune system through the degradation of antibacterial polypep- tides (Casteels et al., 1989) and, in the late steps of infection, in the degradation of larval tissues (Katznelson and Lochhead, 1947; Glinski and Jarosz, 1998). Bacterial secreted proteins perform several important functions, such as provision of nutrients, cell-to-cell communication, detoxification of the environment, and killing of potential competitors. Moreover, secreted proteins of pathogenic bacteria seem to play critical roles in virulence. The aim of the present work was to perform a first approach to identify P. larvae secreted proteins and to Veterinary Microbiology 141 (2010) 385–389 ARTICLE INFO Article history: Received 31 March 2009 Received in revised form 13 August 2009 Accepted 4 September 2009 Keywords: Paenibacillus larvae American Foulbrood Secretome Pathogenicity Virulence factors ABSTRACT American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms. ß 2009 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +598 2 4871616; fax: +598 2 4875548. E-mail address: karina@iibce.edu.uy (K. Antu ´ nez). Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.006