Pathogenic differences between Cryptococcus neoformans and C. gattii

National Institute Of Allergy And Infectious Diseases

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Abstract

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-parasite 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 severely 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. During 2017-2018, our study confirmed the important role of iron levels in the lung for the protection of mice from C. gattii by exogenous activation of type I IFN. 2019, we have concluded that the reason for AIDS patients primarily suffer from cryptococcosis due to C. neoformans is the depletion of CD4+ T-cells while rarely getting infected by C. gattii which is unaffected by low CD4+ T-cells. During 2019-2020, we have identified a hither to unknown ecological source for C. gattii but not for C. neoformans. In addition, we sought to examine the mechanism mediating Type 1 IFN (t1IFN) protection of mice from C. gattii. We found that induction of t1IFN by poly-IC induced the expression of serum proteins into the airspace fluids. Poly-IC induced the leak of intravenously injected FITC-dextran into lung air space fluids suggesting that serum protein reach the lung airspace via t1IFN -induced vascular permeability. During the FY22 we studied the mechanism of t1IFN-induced vascular permeability. Interestingly, pICLC induced a pro-inflammatory signature with significant expression of IL-1 and IL-6 which depended on MDA5 and t1IFN. Vascular permeability depended on MDA5, t1IFN, IL-1, and IL-6. T1IFN also induced MDA5 and other MDA5 signaling components suggesting that positive feedback contributes to t1IFN dependent expression of the pro-inflammatory signature. Vascular permeability, induced by pICLC or another compound, inhibited Cg by limiting iron. These data suggest that pICLC induces t1IFN which potentiates pICLC-MDA5 signaling increasing IL-1 and IL-6 resulting in leakage of antimicrobial serum factors into lung airspace. Thus, induced vascular permeability may act as an innate defense mechanism against opportunistic fungal infection, such as cryptococcosis, and may be exploited as a host-directed therapeutic target. In 2022 we also investigated the frequencies of pulmonary alveola proteinosis in cryptococcosis patients with positive GM-CSF autoantibody. In 2023 to 2024, we have identified avirulent C. gattii isolates from environment which convert to virulent forms by animal passage. In 2025, we are in the process of identifying genetic differences between avirulent environmental isolates and virulent isolates resulted from animal exposure.