Genetic and biochemical characterization of the putative xanthine and uric acid transporters from the bacterial causative agent Paenibacillus larvae of the American Foulbrood disease (AFB) in honey bee
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Dr. George Mourad
Department of Biology
Indiana University – Purdue University Fort Wayne
Paenibacillus larvae is the known causative agent of the American Foulbrood disease in Apis mellifera, the honey bee.Worker bees bring in spores of P. larvae and act as vehicles for the transportation of the spores to the hive’s royal jelly.The honeybee larvae feed on the jelly, resulting in the ingestion of P. larvae spores.P. larvae spores recognize environmental cues such as uric acid and L-tyrosine in the midgut which stimulate germination into the vegetative state resulting in the American Foulbrood disease.Current treatments include antibiotics such as oxytetracycline but are not efficient means of treatment as these toxic substances concentrate in the honey.The objective of this research was to identify the substrate specificities and binding properties of the P. larvae putative xanthine transporter and uric acid transporter with the aim of developing targets for inhibiting the process of spore germination in the midgut of the honeybee larvae.The genes encoding the P. larvae putative xanthine and uric acid transporters were PCR-cloned and spliced into a yeast expression vector then transformed into yeast cells lacking their native transporter.The substrate specificity of P. larvae putative transporters expressed in transgenic yeast was assayed by a series of radiolabeled nucleobase uptake experiments.An array of non-radiolabeled substrates were tested for binding to the putative P. larvae transporters by heterologous competition for uptake with a radiolabeled substrate.The results revealed that the putative P. larvae xanthine transporter moves the purines adenine and guanine, and the pyrimidine uracil.The putative P. larvae uric acid transporter solely transports uracil.Heterologous competition experiments revealed that nucleobases adenine, guanine, cytosine, xanthine, hypoxanthine, and uric acid competitively inhibit the transport of radiolabeled adenine by the P. larvae putative xanthine transporter while uracil, uric acid, and xanthine competitively inhibit radiolabeled uracil transport by the P. larvae putative uric acid transporter.
Biology | Life Sciences
Stoffer, Amanda; Mourad, George; and Test, Opus, "Genetic and biochemical characterization of the putative xanthine and uric acid transporters from the bacterial causative agent Paenibacillus larvae of the American Foulbrood disease (AFB) in honey bee" (2015). 2015 IPFW Student Research and Creative Endeavor Symposium. 64.