The proteins and antigens of Cryptococcus neoformans: Implications for pathogenesis and vaccine development
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Extracellular proteins and protein antigens of Cryptococcus neoformans were studied to better understand the pathogenesis of infection and the mechanism(s) of host defense. Initial studies characterized the extracellular protein products of C. neoformans, with emphasis on proteolytic activities. These studies showed that C. neoformans produces extracellular proteins that are dependent upon strain and growth conditions. Some of these extracellular proteins may be associated with esterase, esterase lipase, alkaline and acid phosphatase activities. Extracellular proteolytic activity was studied by protein agar clearance, in vitro spectrophotometric assays, and substrate gel electrophoresis which demonstrated activity associated with ∼200, 100, and 50 kDa proteins. Studies using proteolytic variants of one C. neoformans strain showed that proteolytic activity was not required for the establishment or dissemination of infection but that isolates of the same strain could elicit different inflammatory responses.;Western blotting of human sera against cryptococcal proteins showed the presence of anti-cryptococcal protein antibodies regardless of previous exposure. Human sera exhibit heterogeneous antibody profiles to cryptococcal proteins while rodent sera recognize only a few proteins after infection. The intensity and specificity of the rodent responses are dependent upon the species, the mouse strain, the route of infection/exposure, and the viability of the inoculated organisms.;While studying extracellular protein production, certain conditions were noted to result in incorporation of [35S] into proteins without de novo protein synthesis. Studies with unlabeled competitors, with different [35S] products, varying the proteins and incubation time confirmed this observation. Amino acid analysis of acid precipitated proteins showed that sufficient [35S] could be incorporated into proteins to generate an autoradiographic signal as well as be detected by scintillation counting. This data suggests that a mechanism for the transfer of [ 35S] to a protein exists that does not rely on protein synthesis.;In summary, the main results are: (1) C. neoformans produces strain and growth condition dependent extracellular proteins which are associated with enzymatic, specifically proteolytic, activities; (2) Proteolytic activity does not appear to be required for virulence; (3) There is a high prevalence of C. neoformans reactive antibodies in humans with or without HIV infection, and in rodents after cryptococcal infection, and (4) proteins can be labeled with [35S] labeled amino acids in the absence of cells or enzymes.
- Theses and Dissertations