EVIDENCE FOR PRESENCE IN THE CELL-WALL OF CANDIDA-ALBICANS OF A PROTEIN RELATED TO THE HSP70 FAMILY

We have previously reported the isolation of several clones from a cDN A expression library from Candida albicans, one of which was associate d with a constitutively expressed 70-kDa protein, The moiety was prese nt in the beta-mercaptoethanol extracts of cell walls from both blasto conidia and germ tubes, The surface expression of this moiety was reve aled by an indirect immunofluorescence assay using affinity-purified a ntibody to the fusion protein produced by the clone, The 0.68-kb cDNA insert was sequenced, A database search revealed extensive homology wi th the 70-kDa family of stress or heat shock proteins (hsps), The 77% homology with another C. albicans HSP70 sequence suggested that this f ragment represented a second member of the HSP70 family in this organi sm, Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae H SP70 gene family, The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recogniz ing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to disting uish extracellular from cytosolic proteins, Biotinylated hsp70 was det ected by Western blotting (immunoblotting) among the biotinylated comp onents affinity purified by chromatography on streptavidin, thereby es tablishing its presence in the cell wall, Immunoelectron microscopy sh owed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through ch annels, Northern (RNA) blot analysis showed that the gene was expresse d in yeast cells growing in yeast extract-peptone medium at both 25 an d 37 degrees C and in Lee medium at 25 degrees C and during formation of germ tubes in Lee medium 37 degrees C. No obvious increase in the e xpression level was detected after the temperature shift. Members of t he hsp70 family have been reported to be immunoreactive. The fusion pr otein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and transl ocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.