An evaluation of the innate and humoral immune response against the opportunistic pathogen Candida albicans.

摘要

In chapter 2 we developed a C. albicans cell surface protein microarray to profile the immunoglobulin G (IgG) response during commensal colonization and candidemia. The antibody response from the sera of patients with candidemia and our negative control groups indicate that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans. We identified antigens that are specific to different phases (i.e. acute, early and mid convalescence) of candidemia. We also identified a set of thirteen cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. In addition, we identified 33 antigenic proteins enriched in convalescent sera of the candidemia patients. These findings have important implications for the mechanisms of C. albicans colonization and the development of candidemia.;The study in chapter 3 examined the mode of action of the salivary antimicrobial peptide histatin-5 (Hst-5). To understand how Hst-5 is internalized, we investigated the cellular localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low physiological concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm. Using cell sorting, we show that cells with vacuolar localization of Hst-5 survived while cells with cytoplasmic Hst-5 did not. Using time-lapse microscopy we observed Hst-5 bind the cell surface and induce a single perturbation on the cell surface, causing an immediate and rapid internalization of Hst-5 from the site. The formation of a spatially restricted site in the membrane causes the initial injury to C. albicans, and offers a mechanism for its internalization into the cytoplasm.;The study of chapter 4 examined using planar bilayer assays whether Hst-5 is able to disrupt bilayer membranes using a strong trans-negative membrane potential. We found that Hst-5 alone is unable to translocate across the lipid bilayer, however using the conductance probe nonactin we observed that Hst-5 alters the biophysical properties of lipid bilayers. The results indicate that Hst-5 binds to negative lipids and alters the membrane surface potential and possibly increases the driving force for Hst-5 translocation across the plasma membrane.