Kugadas, Abi2011-05-092011-05-092011-01https://hdl.handle.net/11299/104195University of Minnesota Master of Science thesis. January 2011. Major: Veterinary Medicine. Advisor: Srinand Sreevatsan. 1 computer file (PDF); vi, 61 pages.Pathogenic mycobacteria survive in the acidic environment of the phagosome. We hypothesize that a serine protease of Mycobacterium avium subspecies paratuberculosis (MAP), encoded by MAP0403, aids in the resistance to phagosomal acidification and is critical for survival in macrophages. The modulation of expression of MAP0403 within macrophages by MAP K-10 was studied. Bafilomycin treatment was used to block phagosomal acidification. Gene encoding the MAP serine protease was significantly up regulated in the acidified phagosomes. Highest levels of MAP0403 expression correlated with peak phagosome acidification in macrophages. Bioinformatically predicted genes that encode proteins interacting or are co-expressed with MAP0403 during phagosomal acidification were analyzed by microarray. Results show that the genes involved in DNA repair, protein synthesis and those located immediately up stream of MAP serine protease are up regulated in the acidified phagosome. Inasmuch as Mycobacterium smegmatis cannot resist and persist in the acidified phagosome, we cloned the open reading frame of MAP0403 into M. smegmatis mc2 155. Compared with controls, M. smegmatis mc2 155 transformants carrying the MAP serine protease show increased survival during in vitro acid stress and in monocyte derived macrophages. Further, we show that serine protease carrying M. smegmatis transformants are able to maintain intra-bacterial pH when exposed to an acidic media (pH~5), while controls failed to do so. Our studies suggest that MAP serine protease is critical in resisting the phagosomal acidification by MAP.en-USVeterinary MedicineInfectious DiseasesThesis or Dissertation