| Candida albicans, a polymorphic yeast, has become one of the most common agents of nosocomial infection in immunocompromised patients. C. albicans is a normal commensal flora of mucosal surfaces, including oropharynx, gastrointestinal tract, and vagina. However, when local or systemic host defence mechanisms are impaired, this microorganisms can cause a variety of mucosal diseases. In susceptible hosts, C. albicans can also penetrate the mucosa epithelium and blood vessel endothelium causing life-threating disseminated candidiasis.During both mucosal colonization and induction of disease, C. albicans will interact with epithelial and endothelial cells. But our understanding of Candida-host interactions at human epithelial and endothelial surfaces is elementary. Investigations into this complex interaction would be helpful in understanding the pathogenesis of C. albicans and finding new target to prevent diseases causing by this microorganisms.The fungus cause disease through direct interaction of its cell wall with host cells. There are various antigens in the cell wall of C. albicans that can be recognized by host immunity. The 80-90% cell wall of C. albicans is carbohydrate. The outer layer of the cell wall predominantly consists mannosepolymers that are covalently associated with proteins to form glycoproteins. Previous studies revealed that the structure of the glycan component of C. albicans cell wall was al,6-linked mannosides backbone and an oligomannose side chain mainly containing β-1,2-linked mannose residues with a small number of phosphate groups. It has been showed that antibodies from mice immunized by fungal cell wall phosphomannan complex could protect mice against the challenge of live C. albicans. However, mannan are poorly immunogenic T-cell independent type 2 antigens that make them inefficient antigens for inducing antibody responses.Aiming at the development of effective antifungal mannan vaccines, we synthesized and evaluated P-mannan di-, tri-, and tetrasaccharide conjugate reagents that coupled with a carrier protein, keyhole limpet hemocyanin (KLH), to form new semi-synthetic glycoconjugate vaccines. One protein (Alslp) derived from Alsl, which was cell surface protein that mediates adherence of C. albicans to endothelial cells, was also selected to conjugate with polysaccharide to provide extra epitope. Mice were successively immunized with these synthetic reagents. Serum were obtained two weeks after the final boost. Immunogenicity and immunoreactivity of the synthetic mannan vaccine were valued.The KLH conjugates of the synthesized β-mannan di-. tri-, and tetrasaccharides could elicit high titers of antigen-specific total IgG antibodies in mice. Mice that received the KLH conjugates without adjuvant could also respond well. Tetrasaccharides were not more immunogenic than the di-and trisaccharides. Antibodies from mice immunized with trisaccharides, not di-or tetrasaccharides could recognize C. albicans (including yeast and hyphae), Candida tropicalis, Candida glabrata, and Candida lusitaniae. However, except β-(Man)3-KLH immunized without adjuvant, all of other mannan polysaccharides could not provoke protective immunities against disseminated infection model of C. albicans in mice. These results revealed that trisaccharide of β-mannan was required for producing antibody that recognized C. albicans. Longer or shorter oligosaccharide sequence of P-mannan could not induce proper antibodies that recognized C. albicans.The invasion of normally nonphagocytic host cells, epithelial cells and endothelial cells, is a key early event during C. albicans infection. However, the pathogen-host cells interactions are yet to be fully visualized dynamically. We applied time-lapse live cell imaging method to capture snapshots of the early stages of the interaction. Two different mechanisms of C. albicans invading nonphagocytic host cells, induced endocytosis and active penetration were also compared in epithelial cells and endothelial cells. Using live cell imaging, we found that pseudopods producing was important to induced endocytosis. While active penetration relied on hyphae growth. Interaction of C. albicans with different cell lines of epithelial and endothelial cells was also compared to investigate the differences of induced endocytosis and active penetration in these cells. We found that induced endocytosis was the main route of C. albicans invasion FaDu epithelial cells. In OKF6/Tert2 epithelial cell, the invasion depended on both routes. In HUVECs endothelial cells, induced endocytosis played a more important role at the early time point of invasion, while at the late time point, active penetration was more important. Unlike HUVECs, C. albicans invasion of TIME endothelial cells almost relied on induced endocytosis. The differences among them could be caused by different cell membrane receptors (EGFR, ErBb2) distribution, different signal pathway (PI3K pathway), and cytoskeleton (actin and microtubule). At last, we also found that various proteins in the surface of C. albicans may play a different role in the induced endocytosis and active penetration. |
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