Objectives: The high lethality of cerebral aspergillosis delivers evidence for efficient immune evasion mechanisms of Aspergillus. An important target for evasion mechanisms is the complement system as a key player of innate immunity in the CNS. As our studies indicate that secreted fungal proteases can degrade complement proteins we aim to target these proteolytic enzymes by new therapeutic approaches. Methods: Aspergillus spp was grown in medium or cerebrospinal fluid (CSF). Different supplements were used to study their influence on protease secretion. The degradation of soluble immune proteins was investigated by Western Blot, cellular expression of surface proteins was quantified by FACS. Fungal opsonization and subsequent phagocytosis by primary microglia was examined by immunofluorescence. Results: When cultivated in CSF pathogenic Aspergillus species secreted proteolytic enzymes that are able to degrade a broad spectrum of complement factors of all three activation pathways. Besides soluble complement proteins, also complement receptor CR3 (CD11b/CD18) and MHC molecules on membranes of immune cells were affected. The consequences were striking: any new opsonization of fungal hyphae was markedly reduced by CSF/Aspergillus supernatant, and complement factor C3 already bound to the surface was removed. In consequence microglial cells showed less phagocytic activity compared to conidia opsonized in CSF without fungal growth. In order to prevent the secretion of fungal proteases, different supplements were tested. While sugars, phosphate and iron compounds showed no effect, various nitrogen sources were able to suppress the proteolytic activity. Amino acids as endogenous substances were of particular interest and turned out to be very effective; the number of the nitrogen atoms within the molecule seems to be crucial for the relevant concentration. Glutamine and arginine appear as the most promising candidates for a future therapeutic use as they are present in the brain and act in comparatively low concentrations. Conclusions: The degradation of complement and surface-bound molecules of immune cells by secreted proteases turned out to be an important mechanism for immune evasion in cerebral aspergillosis. Inhibition or suppression of these proteases are promising approaches for supportive therapeutic approaches in the future.
Full conference title:
19th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 19th (2009)