In 2010, we initiated the construction of an insertional mutant library by the Agrobacterium mediated transformation of Cryptococcus gattii and isolated 30,000 mutant clones by the summer of 2011. This library will serve as a tool for identification of the genes that are involved in the manifestation of the pathobiological differences between the two species. During 2011-2012, we compared the pattern of nitrogen assimilation between C. gattii and C. neoformans and found striking differences in the utilization of D-amino acids such as D-proline and D-alanine. Furthermore, we discovered that the primary target organ for the two species in mice is different: the primary target organ for C. neoformans is the brain while for C. gattii, it is the lung. Since the host response toward the two species is clearly different, we initiated a study to compare the host-paracite relationship between the two species in 2013. Using the plasma samples from immunocompetent patients with cryptococcal menigitis in China and Australia, we found the presence of anti-GM-CSF autoantibodies in plasma to be a risk factor for C. gattii infection and not necessarily for C. neoformans infection. During 2015, we found an isolate of C. neoformans from an otherwise healthy GM-CSF autoantibody positive patient to be molecular type VNI which is the most common type of C. neoformans. This indicates that GM-CSF autoantibody is a more risk for C. gattii infection, it also can be a risk factor for C. neoformans. During 2015-2016, we compared the effect of exogenous Type 1 IFN in mice infected with the two species. For the exogenous type 1 IFN, we used Poly-IC which activates Type 1 IFN and found that poly-IC mediates protection of mice from both species. However, the immunological bases for poly-IC mediated protection was different between the two species. During C. neoformans infection, poly-IC treatment altered polarization of CD4 T helper cells from Th2 toward more protective Th1 and Th17 resulting in a corresponding change of polarization in recruited lung macrophages from M2 toward M1 polarization. Overall these immunological changes resulted in containment of the fungus within granuloma-like lung structures, thus preventing cryptococcal dissemination to central nervous system. However, these CD4 T helper cells were entirely dispensable for protection from C. gattii. During 2016-2017, we discovered that poly-IC mediated protection of mice from C. gattii required beta-2 macroglobulin (B2m)as B2m deficient mice displayed severly diminished protection. However, the immunological factors known to be affected by B2m (CD8 T-cells, NK cells and IgG antibody) were also shown to be dispensable for poly-IC protection. We found that B2m deficient mice had elevated levels of serum iron. We found that addition of exogenous unbound iron totally reversed poly-IC induced protection.
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