Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis

Mycobacterium paratuberculosisis the causative agent of Johne’s disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn’s disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from ourM. paratuberculosisPTB65K genomic library. A high degree of identity was found between the openreadingframe(ORF)ofthePTB65KgeneandthoseofMycobacteriumtuberculosis(89.6%),Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that theM. tuberculosisandM. lepraeproteins each differed by 36 amino acid residues and that theM. avium18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne’s disease and patients with Crohn’s disease against the recombinant PTB65Kantigen.Immunoblotanalysisshowedthatserafromonly3of10clinicallyilland5of25subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne’s disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn’s disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with noninflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne’s disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn’s disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases. Mycobacteriumparatuberculosisis the etiologic agent of paratuberculosis or Johne’s disease, an incurable chronic intestinal disease in ruminants (6). Paratuberculosis occurs worldwide and is economically important in the cattle industry. Eradication programs for this disease have been limited by the lack of accurate, reliable, and rapid diagnostic tests to identify and eliminate infected animals (6, 8). Although primarily considered the cause of a ruminant disease, M. paratuberculosis has also been isolated and/or identified from primates suffering from chronic ileitis (33) and patients with Crohn’s disease, an inflammatory bowel disease of unknown etiology but with histopathologic features similar to those of Johne’s disease in animals (4, 11, 23, 29, 31, 36, 44, 53). An association of some immunodominant antigens of M. paratuberculosisand autoimmunity has been suggested for the pathogenesis of both Johne’s disease and Crohn’s disease (29, 30,42).Hence,recentlytherehasbeenanincreasedinterestin the potential role of mycobacteria, especially M. paratuberculosis, in the pathogenesis of Crohn’s disease. The 65K protein is a member of GroEL family of heat shock proteins. They are among the most extensively studied immunogenic mycobacterial proteins. These highly conserved mycobacterial hsp-65K proteins have been shown to be major stimulants of cellular