NUCLEOTIDE-SEQUENCE ANALYSIS AND SEROREACTIVITIES OF THE 65K HEAT-SHOCK PROTEIN FROM MYCOBACTERIUM-PARATUBERCULOSIS

Mycobacterium paratuberculosis is the causative agent of Johne's disea se, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of u nknown etiology, The mycobacterial 65K heat shock proteins (hsp-65K) a re among the most extensively studied mycobacterial proteins, and thei r 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 our M. paratube rculosis PTB65K genomic library. A high degree of identity was found b etween the open reading frame (ORF) of the PTB65K gene and those of My cobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and My cobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tubercu losis and M. leprae proteins each differed by 36 amino acid residues a nd that the M. avium 18 protein differed by 8 residues. We also invest igated the humoral immune responses of animals with Johne's disease an d patients with Crohn's disease against the recombinant PTB65K antigen . Immunoblot analysis showed that sera from only 3 of 10 clinically il l and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptom s of Johne's disease also reacted with PTB65K; 0 samples from 10 norma l cows reacted. In humans, sera from 7 of 13 patients with Crohn's dis ease; 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with noni nflammatory 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 comp ared with that from noninflammatory bowel disease patients showed a po sitive correlation with mycobacterial diseases.