Identification of honeybee peptide active against Paenibacillus larvae larvae through bacterial growth-inhibition assay on polyacrylamide gel

Abstract

-The inhibition bands showing activity against Gram-positive bacteria were detected by analyses of acidic extracts of honeybee heads, thoraxes, and royal jellies (RJs) using a bacterial growth-inhibition assay on polyacrylamide gel. The presence of antibacterial peptide royalisin and another unknown peptide was found in two detected RJ inhibition bands by N-terminal sequencing. The data suggested that royalisin was the peptide responsible for the activity against Paenibacillus larvae larvae and other tested Gram-positive bacteria. The analyses of RJs collected from individual colonies at two apiaries, one of which showed incidence of American foulbrood, revealed differences in the content of the antibacterial peptide. The results suggest that the differences might be associated with genetic variability between colonies.Apis mellifera / royal jelly / Paenibacillus larvae larvae / American foulbrood / antibacterial peptide

Pharmacophagy and pharmacophory: mechanisms of self-medication and disease prevention in the honeybee colony (Apis mellifera)

“…Two isolated proteins (major royal jelly protein 2 and defensin-1) and the fatty acid 10-hydroxy-2-decenoic acid are discussed as candidates of the antimicrobial activity of royal jelly (Bachanová et al 2002;Bíliková et al 2001Bíliková et al , 2009Chu et al 1992;Hornitzky 1998). The antimicrobial activity of royal jelly is not just dose dependent but also varies significantly among honeybee colonies (Rose and Briggs 1969).…”

“…Honeybees may be able to adjust the quality of royal jelly in response to pathogen infections. Workers of P. larvae -infected colonies produce royal jelly with much higher amounts of antibacterial peptides (Bachanová et al 2002). Most likely, the combination of high-quality proteinous food, enhancing bee health, with increasing antimicrobial activity offers a simple and effective method for reducing or eliminating intra-colony P. larvae titres.…”

Expression of larval jelly antimicrobial peptide defensin1 in Apis mellifera colonies

“…It corresponds to 2.57 and 2.31-fold differences in relative amount of defensin1 normalized to TP and WSP, respectively (Table 1, samples 1-10). Among the tested samples were also RJs whose antimicrobial activity against P. larvae was previously determined by a bacterial inhibition assay on an acidic polyacrylamide gel (Bachanová et al 2002). We found that RJs that contained the highest amounts of defensin1 (samples 4 and 8) yielded also the strongest inhibition bands in the inhibition assay and vice versa those with lowest defensin1 amounts (samples 6 and 10) yielded the weakest inhibition bands.…”

“…Besides defense mechanisms mentioned above, the diet of honeybee larvae-the larval jelly (LJ) has a potential to participate in protection against P. larvae infection (Rose & Briggs 1969;Hornitzky 1998;Bí-liková et al 2001;Bachanová et al 2002). Several proteinaceous and non-proteinaceous antimicrobial substances have been found in royal jelly (RJ), the diet of queen larvae.…”

“…They almost immediately kill bacterial cells by permeabilization of their cytoplasmic membrane (Cociancich et al 1993;Otvos 2000;Wong et al 2007). Defensin1 is expressed in different honeybee tissues as pre-pro-peptide from which active peptide containing 51 amino acids arises by processing (Casteels-Josson et al 1994): (i) fat body of bacterially infected adult bees inducibly expresses hemolymph defensin (Casteels-Josson et al 1994;Casteels 1998), (ii) cephalic hypopharyngeal and mandibular glands of nurse honeybees putatively constitutively express LJ defensin1 (Bachanová et al 2002;Klaudiny et al 2005) and (iii) defensin1 gene is expressed also in thoracic salivary glands of nurses (Klaudiny et al 2005) and in bee larvae (Evans 2004). Two facts support the notion that LJ defensin1 could contribute to the protection of brood against AFB and influence the resistance of colonies against it: (i) the peptide inhibits the in vitro growth of P. larvae (Bíliková et al 2001;Bachanová et al 2002) and (ii) RJs seem to differ in amounts of the peptide as deduced from analyses of RJ samples originating from several colonies (Bachanová et al 2002).…”