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Abstract Retinopathy of prematurity (ROP) is a vasoproliferative retinal disease affecting premature infants. In addition to prematurity itself and oxygen treatment, genetic factors have been suggested to predispose to ROP. We aimed to identify potentially pathogenic genes and biological pathways associated with ROP by analyzing variants from whole exome sequencing (WES) data of premature infants. As part of a multicenter ROP cohort study, 100 non-Hispanic Caucasian preterm infants enriched in phenotypic extremes were subjected to WES. Gene-based testing was done on coding nonsynonymous variants. Genes showing enrichment of qualifying variants in severe ROP compared to mild or no ROP from gene-based tests with adjustment for gestational age and birth weight were selected for gene set enrichment analysis (GSEA). Mean BW of included infants with pre-plus, type-1 or type 2 ROP including aggressive posterior ROP (n = 58) and mild or no ROP (n = 42) were 744 g and 995 g, respectively. No single genes reached genome-wide significance that could account for a severe phenotype. GSEA identified two significantly associated pathways (smooth endoplasmic reticulum and vitamin C metabolism) after correction for multiple tests. WES of premature infants revealed potential pathways that may be important in the pathogenesis of ROP and in further genetic studies.

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.

ABSTRACT Cryptococcus neoformans is a pathogenic yeast that causes lethal cryptococcal meningitis in immunocompromised patients. One of the challenges in treating cryptococcosis is the development of resistance to azole antifungals. Previous studies linked azole resistance to elevated numbers of copies of critical resistance genes in aneuploid cells. However, how aneuploidy is formed in the presence of azole drugs remains unclear. This study showed that treatment with inhibitory concentrations of an azole drug, fluconazole (FLC), resulted in a significant population of cells with increased DNA content, through the following defects: inhibition of budding, premature mitosis, and inhibition of cytokinesis followed by replication in the mother cell. Inhibition of and/or a delay in cytokinesis led to the formation of cells with two or more daughter cells attached (multimeric cells). To investigate which part of cytokinesis fails in the presence of FLC, the dynamics of the actomyosin ring (AMR), septins, and Cts1, a protein involved in cell separation, were analyzed with time-lapse microscopy. Following the constriction of the AMR, septins assembled and the septum was formed between the mother and daughter cells. However, final degradation of the septum was affected. Enlarged cells with aberrant morphology, including multimeric cells, exhibited an increased potential to proliferate in the presence of FLC. These findings suggest that pleiotropic effects of FLC on growth and mitotic division lead to an increase in DNA content, resulting in cells less sensitive to the drug. Cells with increased DNA content continue to proliferate and therefore increase the chance of forming resistant populations. IMPORTANCE 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.