Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3 a Δ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.

The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans. Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G 1 cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens.

ABSTRACTThe human fungal pathogenCryptococcus neoformansproduces an enlarged “titan” cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells.

iCryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/l, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC 50 of 4 g/ml and an MIC 90 of 8 g/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC 50 to 8 g/ml and an MIC 90 value of 32 g/ml, with 31% of isolates with a fluconazole MIC of >16 g/ml. We observed an amphotericin B MIC 50 of 0.5 g/ml and an MIC 90 of 1 g/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC 50 of 4 g/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P ‫؍‬ 0.52).

To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4⌬ gpr5⌬ mutant, which has minimal titan cell production in vivo. The gpr4⌬ gpr5⌬ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4⌬ gpr5⌬ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.