No abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis primarily in immunocompromised individuals. In order to survive and proliferate during infection, C. neoformans must adapt to a variety of stresses it encounters within the host. Patient outcome depends on the interaction between the pathogen and the host. Understanding the mechanisms that C. neoformans uses to facilitate adaptation to the host and promote pathogenesis is necessary to better predict disease severity and establish proper treatment. Several virulence phenotypes have been characterized in C. neoformans, but the field still lacks a complete understanding of how genotype and phenotype contribute to clinical outcome. Furthermore, while it is known that C. neoformans genotype impacts patient outcome, the mechanisms remain unknown. This lack of understanding may be due to the genetic heterogeneity of C. neoformans and the extensive phenotypic variation observed between and within isolates during infection. In this review, we summarize the current understanding of how the various genotypes and phenotypes observed in C. neoformans correlate with human disease progression in the context of patient outcome and recurrence. We also postulate the mechanisms underlying the genetic and phenotypic changes that occur in vivo to promote rapid adaptation in the host.

Azoles are antifungals that are widely utilized due to relatively low toxicity and cost of treatment. One of their drawbacks, however, is that azoles are primarily cytostatic, leaving fungal cells capable of developing drug resistance. The human pathogen Cryptococcus neoformans acquires resistance to the azole drug fluconazole (FLC) through the development of aneuploidy, leading to elevated expression of key resistance genes, a mechanism that is also common for Candida albicans (K. J. Kwon-Chung and Y. C. Chang, PLoS Pathog 8:e1003022, 2012, https://doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser, J Microbiol 54:192–201, 2016, https://doi.org/10.1007/s12275-016-5628-4). However, the exact ways in which FLC contributes to increased resistance in either of these important fungal pathogens remain unclear. Here we found that FLC treatment leads to an increase in DNA content in C. neoformans through multiple mechanisms, potentially increasing the size of a pool of cells from which aneuploids with increased resistance are selected. This study demonstrated the importance of FLC’s inhibitory effects on growth and cytokinesis in the generation of cell populations with decreased sensitivity to the drug.

Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease.

Cryptococcus neoformans is a human fungal pathogen that can cause fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug administered to treat cryptococcosis. When exposed to the inhibitory concentration of FLC, C. neoformans exhibits heteroresistance where a small subpopulation of cells develops into FLC-resistant colonies. FLC-resistant cells are aneuploids with regard to specific beneficial chromosomal regions. Factors underlying the potential for only certain C. neoformans cells in a genetically isogenic population to become FLC-resistant are unknown. In this study, we systematically examine the heterogeneous response of C. neoformans to FLC at a colony and individual cell level. We find that the heterogeneity in response to FLC is reflected by variable diminishment of the ergosterol at the plasma membrane. A population of C. neoformans spread on a semi-solid medium displays two types of outcomes following FLC exposure. The first outcome is colonies consisting of non-resistant cells (survivors). The size of colonies consisting of survivors ranges from a few cells to visible colonies, which reflects intrinsic phenotypic heterogeneity of the C. neoformans population. The second outcome is FLC-resistant cells forming colonies of sizes significantly larger as compared to colonies made of survivors. We propose a model that describes how a distribution of these types of cellular responses within a population changes depending on FLC concentration and factors that influence the rate of cellular growth including temperature, media type, growth phase, and the age of cells. Our findings highlight a complex nature of the response to a fungistatic drug and provide insights that may help to optimize FLC therapy.