Characterization of virulence factors targeting Apis mellifera: Varroa Toxic Protein and LarvinA

Abstract

Apis mellifera populations are declining due to a variety of factors, including the parasitic mite, Varroa destructor, and the bacterial disease, American foulbrood (AFB). This thesis describes the biochemical characterization of two important virulence factors: LarvinA from AFB, and Varroa Toxic Protein (VTP) from the mite. LarvinA was kinetically characterized and confirmed to be a functional C3-like mono-ADP-ribosyltransferase toxin. N-terminal deletions and single-residue variants led to the identification of novel interactions between the α1-helix and the active-site of LarvinA, including the role of net charge in cell entry. Next, VTP was purified using glutathione-based affinity and size-exclusion chromatography. Analysis of the primary sequence and circular dichroism spectra revealed that VTP is primarily an α-helical protein; however, it is thermally labile based on the temperature analysis of the protein. These findings can be combined with future studies to elucidate novel therapeutics.