Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2000
Publication Date: N/A
Citation: N/A Interpretive Summary: Mycobacterium paratuberculosis is the causative agent of Johne's disease. Previous studies in our laboratory have shown that a portion of the gene sequence, termed hspX, specifically detects M. paratuberculosis and not those other species of mycobacteria in a DNA-based assay. In this communication, we cloned and expressed hspX by itself and as a fusion with the maltose binding protein (MBP) in Escherichia coli in order to characterize the protein. HspX was detected by sera from 4 of 24 paratuberculous cows (17%) as determined by immunoblot of E. coli lysates expressing HspX. No reactivity was observed in any of the three control cows. The MBP/HspX fusion was purified and used to produce monospecific antibody in rabbits. Immunoblot and immunofluorescence studies with anti-MBP/HspX sera showed that HspX is present within M. paratuberculosis-infected macrophages of bovine and murine origin. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. These experiments shed light on the diagnostic utility of this antigen as well as its potential role in pathogenesis.
Technical Abstract: Little is known about the bacterial factors that enable Mycobacterium avium subspecies paratuberculosis (hereafter referred to as M. paratuberculosis) to survive within the macrophages of infected animals. A portion of the gene encoding HspX has been previously identified as a sequence specific to M. paratuberculosis based on DNA hybridization experiments. Rabbit antisera were raised against a recombinant protein of HspX fused to the E. coli maltose binding protein (MBP/HspX). Immunoblots of lysates of M. paratuberculosis-infected macrophages probed with the rabbit antisera showed that HspX was present within infected macrophages of bovine and murine origin. This observation was confirmed by immunofluorescence microscopy of infected macrophages. Lysates of E. coli expressing HspX without the MBP fusion partner were loaded onto preparative SDS-PAGE gels and used to determine whether infected cattle generated a humoral immune response to the antigen. Sera from 4 of 24 paratuberculous cows (17%) detected HspX. No reactivity was present in sera from control cows. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. Collectively, these data give a preliminary characterization of the first described M. paratuberculosis protein identified within infected macrophages.