Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain

Keiko Nakamura,Mariko Harada,Kayo Okumura,Masatoshi Okura,Mariko Okamoto,Daisuke Takamatsu

doi:10.1038/s41598-021-88302-8

Abstract

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein and endo-α-N-acetylgalactosaminidase), we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.

Highlights

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees
As all DAT561 mutants constructed and tested in this study lost pMP19, these results suggested again that major virulence factors of DAT561 are not located on pMP19, the same may not necessarily apply to all other virulent strains of CC12
The genes encoding Efp, chitin-binding domaincontaining protein (Cbp) and endo-α-GalNAc-ase were described as putative virulence factors in the previous comparative genomic study of M. plutonius strains[12], their roles in the pathogenesis of EFB were unknown due to a lack of data from isogenic mutants of the genes

Summary

Introduction

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. The midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of bee brood, suggesting that the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain. Irrespective of the presence of pMP19, the CC12 strain DAT561 was virulent and killed more than 80% of the honey bee brood in our in vitro infection model[16], strongly suggesting the presence of unidentified pathomechanisms functioning in the virulence of CC12 strains. In the absence of PlCBP49 expression, PM degradation was markedly reduced and P. larvae virulence was nearly abolished; the PM-degrading protein PlCBP49 is considered a key virulence factor in P. larvae[21]

Methods

Results

Conclusion