FKS1 is required for Cryptococcus neoformans fitness in vivo: application of copper-regulated gene expression to mouse models of cryptococcosis

Abstract

There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used to modulate gene expression in vivo previously. The premise for these experiments is that copper concentrations vary depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied to the gene encoding 1,3-{beta}-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through post-transcriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection. ImportanceConditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied it to the study of FKS1, the gene encoding for the target of the echinocandin class of 1,3-{beta}-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-{beta}-glucan synthase inhibitors toward C. neoformans.