Comparative Genomics and Description of Putative Virulence Factors of Melissococcus plutonius, the Causative Agent of European Foulbrood Disease in Honey Bees

In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees. Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors (endo-alpha-N-acetylgalactosaminidase, enhancin and epsilon toxin) was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.