UBC Theses and Dissertations
A prophylactic probiotic to fight paenibacillus larvae infection in honey bees Romero Guzmán, Atenas Sofia
Honey bees are the most critical crop pollinators worldwide and for the last decades mostly a single antibiotic has been used to protect them from American foulbrood (AFB), a lethal larval disease caused by the bacterium Paenibacillus larvae. Oxytetracycline (OTC) is the only approved prophylactic antibiotic for P. larvae infection in Canada, which has triggered resistant strains. While only the first larvae stages are vulnerable to AFB, consequences are lethal. Early microbiology, genomics and proteomics studies have identified molecular differences between the AFB resistant honey bee stages and the vulnerable larvae. Profiles of the immune factors, such as antimicrobial peptides (AMPs), and a characteristic commensal gut bacteria (CGB) community are key differences. Through this work we want to develop a new prophylactic for AFB, based on the molecular differences between the adult honey bee and the larvae. Specifically, a commensal bacterium strain that expresses and secretes naturally occurring AMPs targeting P. larvae vegetative cells. To identify AMPs candidates to be used as potential prophylactics, we pursued to uncover host-microbe specific responses of the adult honey bee. Through susceptibility assays we tested the inhibitory activity of characteristic honey bee and fruit fly naturally occurring AMPs against P. larvae, CGB (Bartonella apis, S. alvi and Bifidobacterium asteroides), and non-commensal bacteria (Bacillus subtills and Escherichia coli). To look for potential dysbiosis caused on the gut microbiota caused by the candidate AMPs we conducted an in vivo feeding experiment and proceeded with quantitative PCR (qPCR) and 16S rRNA deep amplicon sequencing, determining changes in size and composition. Jelleine and melittin (AMPs) were chosen as final candidates to attempt to be expressed and secreted by S. alvi. Then, we predicted the Sec pathway-dependent extracellular proteins and with a mass spectrometry based-proteomics experiment explored the signal peptides secretion dynamics. Finally, we suggested the signal peptides to be used for the expression and secretion of jelleine and melittin in S. alvi., offering a pioneering approach in the understanding and application of honey bee gut commensals.
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