Identification of Diagnostic Proteins in <i>Mycobacterium avium</i> subspecies <i>paratuberculosis</i> by a Whole Genome Analysis Approach

Abstract

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne's disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these animals. As with many other animal diseases, efforts need to be concentrated on the development of simple, rapid, noninvasive tests that can be performed by veterinarians or animal producers without expensive laboratory equipment. With the genome sequence of M. paratuberculosis now complete, we have taken a different strategy to identify novel proteins that are present uniquely in M. paratuberculosis and are antigenic in the context of infected cattle. Through a whole genome comparison of M. paratuberculosis with other sequenced mycobacterial genomes, we identified a collection of more than 90 genes that are present uniquely in M. paratuberculosis. This list has been further trimmed to 39 after amplification using polymerase chain reaction of unique genes using the genomic deoxyribonucleic acid template from several mycobacterial species and isolates. A selection of the remaining genes has been cloned and expressed in Escherichia coli and purified by affinity chromatography. Successfully purified proteins were analyzed using sera from rabbits immunized with M. paratuberculosis. Furthermore, to identify antigens in the context of disease, sera from cattle with Johne's disease as well as healthy control cattle are used in immunoassays. Using this methodology, we identified the first protein antigens specific to M. paratuberculosis.

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle

“…L5P is a lipopeptide, its structure presumably involves specific CD1 antigen presentation to T cells (Young et al, 2009) but its hydrophobicity would imply a non-specific cell membrane affinity which could hamper the expected presentation, thus partly explaining the weak IFN-γ response. Furthermore, the small antigenic peptide (5 amino acids) represented only a small part of the Map specific antigen (Bannantine and Paustian, 2006). A combination of lipid transport molecules (van den Elzen et al, 2005) and a selected recombinant antigen pool (Mikkelsen et al, 2011a) could enhance the IFN-γ response at the same time conserving high specificity.…”

Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling

“…Cloning, protein production, and purification were described in detail previously (4). Briefly, a maltose binding protein (MBP) fusion of M. tuberculosis Rv0348 was constructed in Escherichia coli using the pMAL-c2 vector (New England Biolabs, Beverly, MA).…”

Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays

“…The cloning, protein production, and purification is described in detail previously (5). Briefly, maltose binding protein (MBP) fusions of M. avium subsp.…”