Biology of Int1p in Canadida albicans Fungemia

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Candida albicans fungemia leads to excess medical costs of 800 million dollars per year. Both neutropenic and non- neutropenic patients are affected; more than 85 percent harbor a central venous catheter, through which blood products, antibiotics, hyperalimentation fluids, and heparin are infused. Although more than 20 percent of infected patients die, predisposing factors other than neutropenia are poorly understood. The C. albicans gene INT1 links adhesion, morphogenesis and virulence in vitro and in vivo, but the mechanism of its virulence has not been elucidated. Preliminary data demonstrate Int1p-dependent superantigen-like effects. Both C. albicans and S. cerevisiae expressing Int1p activate CD4 T lymphocytes and expand the Vbeta2 subset; TNFalpha and IL-6 are released in response to C. albicans. Superantigen-like activity was originally localized to the first 434 amino acids of the Int1p amino terminus. This proposal focuses on Pep263, a polypeptide that is exposed and cleaved from the amino terminus of Int1p in the presence of heparin. 100 picomolar concentrations of purified, soluble Pep263 activate human and murine T lymphocytes and ex and T lymphocyte populations bearing Vbeta2, Vbeta17, and Vbeta22 subsets more rapidly and more potently than 105 C. albicans cells. In Specific Aim One we shall map Int1p domains essential for the expression and cleavage of Pep263 using INT1 mutants and human lymphocytes in vitro. The response of additional Vbeta subsets, the relative contributions of CD4/CD8 T lymphocytes, and their cytokine profiles will be determined. A newly discovered identity of Pep263 with the MHC Class II binding site of Mycoplasma arthritidis MAM, a well-known superantigen, will be exploited to identify relevant MHC Class II alleles on human and murine antigen-presenting cells. The effects of Pep263 and mutants lacking the MHC Class II binding site will be studied in mice transgenic for human HLA-DR and -DQ alleles. The crystal structure of Pep263 will be determined. In Specific Aim Two we shall test whether Kex2p or Int1p itself is the proprotein convertase that cleaves Pep263. Anti-proteases, heparin analogs, and antibodies to Pep263 will be studied as potential inhibitors. Direct and indirect mechanisms by which heparin accelerates cleavage of Pep263 will be analyzed using INT1-GFP constructs and INT1 mutants missing a putative heparin-binding site. These studies address the cellular, structural, and biochemical mechanisms underlying the heparin-accelerated generation of a C. albicans superantigen.