The Mycobacterium avium subsp. Paratuberculosis Fur Element’s Interactions with an in vivo Environment

Abstract

Johne’s disease is a chronic digestive disease in dairy cattle that is caused by the bacteria, Mycobacterium avium subsp. paratuberculosis (MAP). Currently, there is no effective vaccine for Johne’s disease, and the detection testing for the disease is a long process. In order to shorten the testing and create a vaccine, we need to better understand how MAP interacts with its environment. Like most living organisms, MAP needs iron to survive, and it uses mycobactin for iron uptake. The ferric uptake regulator, Fur element, a gene on a horizontal gene transfer island unique to MAP, is likely utilized in-vitro to regulate an alternate iron acquisition pathway. To further study MAP’s Fur element, we undertook studies to first demonstrate that the Fur box was functional in MAP. MAP3773C, or the Fur of MAP, was cloned into M. smegmatis on a MSMEG optimized vector. The colonies were grown, and protein production was induced in vitro. A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to determine protein expression and a Western Blot was used for confirmation. If MAP3773C was produced by the clones, an electrophoretic mobility shift assay (EMSA) was used to study the interaction of Fur to its binding sites or motifs. The starting method of protein expression was not successful, so different methods were studied with still no fur protein expression. Further work needs to be done with the protein expression. Optimization of the EMSA test was undertaken to get DNA-Fur protein binding to occur. The modified EMSA test shows DNA-protein binding occurs. In future studies, to further study the EMSA results, DNase footprinting and chromatin immunoprecipitation (ChiP-seq) will be used to identify the regulon of Fur.