This project was initiated to study the molecular genetics of cell cycle associated genes in C. neoformans. In 2006-2007, we deleted the TUP1 gene, a fungal global repressor known to regulate the rate of cell proliferation, mating and morphogenesis, in H99, a serotype A strain of C. neoformans. The H99 strain is of the VN1 molecular type which significantly differs from serotype D strains in capsular antigenicity and the function of various genes. Unlike in serotype D strains, deletion of the tup1 gene in H99 did not cause a quorum sensning phenotype but the growth on complex media was clearly retarded compared to the wild type strains. Tup1 ko isolates of H99 showed a drastic increase in capsule formation. Complementation of the tup1 ko strain with the wild type TUP1 gene decreased the capsule size significantly. These findings indicate that TUP1 represses capsule formation in vitro. The capsule size in vivo, however, was not affected by the TUP1 deletion. Mice infected with the tup1 ko strain survived much longer than either the wild type or the tup1 ko strain complemented with the TUP1 gene. In the same period, we carried out microarray experiments to compare the differences in the expression of known or novel capsule related genes between the wild type and the tup1 ko strain.Interestingly,the microarray data indicated that expression of multiple genes related to iron homeostasis were affected by the deletion of the tup1 gene. This could explain the cause of the observed hypercapsular phenotype of the tup1 ko strain in serotype A background. A direct relationship between iron homeostasis and the degree of capsule formation is known to exist in Cryptococcus neoformans. Since tup1 deletion in H99 caused no quorum sensing phenotype, we attempted to determine whether such a phenotype is widespread in strains of serotype A of different molecular types. The reference strains of molecular type VNI, VNII and VNBt were chosen to study in 2007-2008. Contrary to what has been known in strains of serotype D, expression of the TUP1 gene in strains of serotype A, irespective of molecular type, is unrelated to production of the quorum sensing peptides. These findings support the observed genetic diversity between strains of serotype D and A of C. neoformans even though they produce identical disease in humans. During the period of 2008-2009, we found other pleiotropic phenotypes in serotype A tup1 null strains that had not been observed in serotype D strains. These include increased susceptibility to azole drugs, the most widely used antimycotics to treat cryptococcosis, sensitivity to SDS and a reduction in the formation of melanin, one of the major virulence factors in C. neoformans. These results suggested the pathobiological importance of TUP1 in C.neoformans.C. neoformans is notorious for not behaving like other yeasts in many aspect. One of such pattern is found in cell cycle and a method for synchronization has not been achieved. In many studies , cell cycle synchronization is required to resolve the questions on biological phenomenon that is associated with cell cycle. During 2009-2010, we were able to use a sucrose gradient to isolate cells enriched in G1 phase (>95% purity), but these cells did not exit the G1 phase in synchronized fashion. We used chemicals that have previously been used to achieve synchronization in other organisms, such as hydoxyurea, nocodazole, benomyl, azetidine 2-carboxylic acid and methylmethane sulfonate, but without success. Mutants of cell cycle genes known to cause cell cycle arrest in other systems were also generated in our laboratory. We have constructed a temperature sensitive allele of CDC28 and an ATP-analog sensitive allele of CDC15. However, both mutants failed to cause specific arrest in the cell cycle at restricted conditions. We are currently focusing on pheromone-mediated cell cycle arrest and are testing the G1 exit pattern in differennt strains in order to achieve synchronization of the cell cycle. We expanded our research further during 2010-2011 to uncover the signaling pathways that are pertinent to pathogenicity and drug resistance in C. neroformans. The first study focused on the signaling pathways involved in azole resistance by screening a deletion library of the serotype A genome sequenced strain, H99. Several genes involved in the conserved PDK1 and PKC signaling pathways were found to contribute to the basal tolerance of FLC. Moreover, genes in these two pathways were also found to play different roles in response to salt and rapamycin treatment as well as in controlling homeostasis of complex sphingolipids. We also found evidence that suggests involvement of the TOR pathway in the basal tolerance of FLC. Deletions of PDK1, SIN1, or YPK1 but not MPK1 were deleterious to cell viability in the presence of compounds that inhibit sphingolipid biosynthesis. These mutants accumulated higher amounts of FLC compared with wild-type suggesting a malfunction of the influx/efflux systems in the mutants. We speculate that the FLC hypersensitivity phenotype of pdk1, sin1, and ypk1 is associated with impaired membrane composition due to modified sphingolipid content. Interestingly, the reduced virulence of these three strains in mice suggests that cryptococcal PDK1, PKC, and likely the TOR pathways not only play important roles in managing stress exerted by FLC but also by the host's environment.During 2011-2012, we discovered that Sac6, an actin-binding protein, plays an important role in C. neoformans cell growth at low oxygen conditions.

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Chang, Yun C; Khanal Lamichhane, Ami; Garraffo, H Martin et al. (2014) Molecular mechanisms of hypoxic responses via unique roles of Ras1, Cdc24 and Ptp3 in a human fungal pathogen Cryptococcus neoformans. PLoS Genet 10:e1004292
Chang, Yun C; Lamichhane, Ami Khanal; Kwon-Chung, Kyung J (2012) Role of actin-bundling protein Sac6 in growth of Cryptococcus neoformans at low oxygen concentration. Eukaryot Cell 11:943-51
Lee, Hyeseung; Khanal Lamichhane, Ami; Garraffo, H Martin et al. (2012) Involvement of PDK1, PKC and TOR signalling pathways in basal fluconazole tolerance in Cryptococcus neoformans. Mol Microbiol 84:130-46