Topoisomerase I is essential in Cryptococcus neoformans: Role in pathobiology and as an antifungal target

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice an d drosophila. We have cloned the TOPI gene encoding topoisomerase I from th e opportunistic fungal pathogen Cryptococcus-neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the m ammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants an alyzed. When a second functional copy of the TOPI gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologo us recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be u seful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOPI gen e was fused to the GAL7 gene promoter. The resulting GAL7::TOPI fusion gene was modestly regulated by carbon source in a serotype A strain of C. neofo rmans. Modest overexpression of topoisomerase I conferred sensitivity to he at shock, gamma-rays, and camptothecin. In contrast, alterations in topoiso merase I levels had no effect on the toxicity of a novel class of antifunga l agents, the dicationic aromatic compounds (DACs), indicating that topoiso merase I is not the target of DACs. In an animal model of cryptococcal meni ngitis, topoisomerase I regulation was not critically important to establis hed infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the hum an pathogen C. neoformans and represents a novel target for antifungal agen ts.