CHARACTERIZATION OF THE NUCLEOTIDE-SEQUENCE OF THE GROE OPERON ENCODING HEAT-SHOCK PROTEINS CHAPERONE-60 AND CHAPERONE-10 OF FRANCISELLA-TULARENSIS AND DETERMINATION OF THE T-CELL RESPONSE TO THE PROTEINS IN INDIVIDUALS VACCINATED WITH FRANCISELLA-TULARENSIS

The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characteri zed, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed, The cpn1 0 and cpn60 genes were amplified by PCR with degenerate oligonucleotid es derived from the N-terminal sequence of the two proteins, The seque nce analysis revealed the expected two open reading frames, encoding p roteins with estimated M(r)s of 10,300 and 57,400, The deduced amino a cid sequences closely resembled cpn10 and Cpn60 proteins of ether prok aryotes. The genes constituted a bicistronic operon, the cpn10 gene pr eceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma(70)-dependent bu t not of sigma(32)- dependent promoters of Escherichia coli were found , The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sig ma(32)-dependent promoters. Primer extension analysis disclosed one an d the same transcription start, irrespective of the presence or absenc e of heat or oxidative stress, After separation of lysates of the F. t ularensis LVS organism by two-dimensional gel electrophoresis, DnaK, C pn60, and Cpn10 were extracted and used as antigens in T-cell tests, W hen compared to those from nonvaccinated individuals, T cells from ind ividuals previously vaccinated with live F. tularensis LVS showed an i ncreased proliferative response to DnaK and Cpn60 hut not to Cpn10, Th e present data will facilitate further studies of the involvement of t he heat shock proteins in protective immunity to tularemia.