Abstract:
Mycobacterium avium is a ubiquitous environmental organism found in both water and soil. It can cause disease in patients with previous pulmonary conditions, as well as immunosupressed patients, with the most prevalent being AIDS patients. Studies have indicated that passage through amoeba, a common environmental protozoa, increases virulence of M. avium. Using an M. avium GFP-promoter library in M. smegmatis, and real-time PCR, this study seeks to examine genes expressed during amoeba infection. First, M. avium and M. smegmatis survivability in Acanthamoeba castellani were determined. It was found that both species were able to infect and multiply within amoeba for at least 4 days. After screening the GFP-promoter library using A. castellani, it was determined that 20 genes were expressed at various timepoints. These included genes involved in metabolic pathways, protein transcription and translation, and macromolecule degradation. Eight of these genes were also found to be upregulated in macrophage infection. Finally, real-time PCR was used to confirm expression of five chosen genes. Genes were found to be upregulated by at least two-fold. Through these studies we have determined that the M. avium GFP-promoter library is an effective tool for studying gene upregulation. We have also determined at least some of the genes used by M. avium during amoeba infection, as well as the ones that are shared in macrophage infection.
