Nosema ceranae, the fungal freeloader

Nosema ceranae, a fungus which parasitizes bees, has recently been detected in the major commercial honey bee species, Apis mellifera (European honeybee). N. ceranae invades epithelial cells in the bee midgut, resulting in the debilitating nosemosis disease. Like all microsporidian fungi, N. ceranae lack mitochondria making them dependent on its honey bee host for its energy source. Thus, Nosema infections results in energetic stress and has been implicated in reduced longevity, immune function, and performance of commercial honey bees, causing decreased hive productivity. Due to the integral role that bees have in agriculture and in the environment, the adverse effects of N. ceranae to honey bees inspires much anxiety among the apiculture community.

Fumagilin: Treatment to bees, or not to be?

Team UAlberta was alerted of the N. ceranae threat by our discussions with local beekeepers and researchers, who expressed a desire for effective treatments against Nosema infections. Current methods of treating N. ceranae infections rely heavily on an antifungal agent called Fumagilin-B. However, Fumagilin-B is mutagenic, toxic to mammals, and has been shown to increase N. ceranae spore count at low doses. Moreover, the only North American supplier of Fumagilin-B has gone out of business and the remaining supply of Fumagilin-B is beginning to run low. Our discussions with the beekeeping community in Alberta, Canada revealed tremendous concern about the lack of an alternative safeguard against N. ceranae.

The honey industry's impact on the Canadian economy also drives much of the concern regarding Nosema. Between honey production and the contribution of their pollination to agriculture, honey bees contributed roughly $2 Billion to the Canadian economy in 2013. The economic impact of the bee industry in Canada is far-reaching and a lack of fumagillin alternatives poses a serious threat to industry and individual livelihoods. The developments regarding the supply of Fumagilin-B and the effects of nosemosis on hive productivity motivates Team UAlberta to develop an alternative treatment against Nosema.

Porphyrins

Recent research has found that porphyrins, a class of organic compounds, are capable of deactivating N. ceranae spores. Porphyrin antifungal action is attributed to its disruption of spore cell walls (Figure 1). When bee diets were supplemented with chemically synthesized porphyrin species, spore count in the bee's midgut significantly decreased while no adverse effects on the bees were observed. Particularly, a porphyrin derivative, PP(Asp)2 was successful in reducing the spore load in treated bees. Therefore, using porphyrin-type molecules like PP(Asp)2 may be a feasible method of treating N. ceranae infections.

Plan Bee

Luckily, PP(Asp)2 is structurally similar to protoporphyrin IX, a porphyrin-type molecule produced endogenously in E. coli. Rather than feeding bees chemically synthesized PP(Asp)2, we aim to genetically engineer a strain of E. coli capable of living in the bee midgut to biosynthesize protoporphyrin IX. We will build off of the work of a previous iGEM team (BeeColi; NYMU-Taipei , 2013) that developed an alginate coat to allow E. coli to travel through the harsh environment of the bee stomach to colonize the bee midgut. Once in the midgut, our E. coli will secrete excess porphyrin IX using an endogenous efflux pump (TolC). Given that ingested porphyrin IX has been shown to damage N. ceranae, we hypothesize that porphyrin IX secreted directly into the midgut will also damage N. ceranae, allowing us to "bee" part of the Nosema solution.

Objective One: Engineer constructs exploiting C5 heme biosynthesis pathway to selectively express and secrete porphyrin intermediates

First, a liquid chromatography mass spectroscopy assay will be conducted to analyze the distribution of porphyrin-type species produced in wild-type E. coli, establishing a baseline for manipulation of heme biosynthesis. Next, strains of E. coli will be engineered to produce target porphyrin intermediates in the C5 pathway with high yield, by overexpressing genes encoding heme biosynthesis enzymes (Figure 4). To facilitate the activity of porphyrins in the bee midgut, and to avoid possible complications arising from negative feedback loops, we will engineer the E. coli to constitutively secrete the produced porphyrins. Previous work has shown that the outer membrane channel-tunnel protein, TolC, functions with efflux pumps to export excess porphyrins and maintain homeostasis. Thus, with control over porphyrin intermediate accumulation, secretion of the compounds is achievable through existing cellular machinery. It should be noted that an introduction of a kill switch may be needed to prevent the unwanted proliferation of modified E. coli.

Objective Two: Assay modified strains and associated porphyrin intermediates for their ability to inactivate N. ceranae spores in bees.

This assay will be performed using spores from an in vivo source, A. mellifera worker bees, and an in vitro source, Sf9 cells which are permissive to N. ceranae infection. Spores will be incubated with the engineered E. coli and infectivity will be quantified, using conventional PCR and fluorescent cell staining, which are consistent with established methods. A previous iGEM team (NYMU-Taipei 2013) has shown that modified E. coli are capable of persisting within the gut of bees. A similar protocol will be employed to introduce the E. coli into live bees and the capability of the E. coli to confer extended resistance against N. ceranae will be measured by spore count. The total load of E. coli will be observed to evaluate the response of the microbiome to the modified strain.