Colony and Single Cell Level Analysis of the Heterogeneous Response of Cryptococcus neoformans to Fluconazole

Abstract

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.

Extreme Osmotolerance and Halotolerance in Food-Relevant Yeasts and the Role of Glycerol-Dependent Cell Individuality

“…Previous studies have established that genetically uniform populations of yeast cells can be very diverse in their resistance to other stressors, with only small sub-populations being highly resistant. This has been demonstrated in resistance to heat (Levy et al, 2012), sorbic acid (Steels et al, 2000; Stratford et al, 2013), drugs (Altamirano et al, 2018; Tindall et al, 2018), and toxic metals (Bishop et al, 2007; Holland et al, 2014), for example. Occasional, rare-cell hyper-resistance means that cell populations which can appear relatively sensitive at intermediate stress levels may persist to very high doses, overtaking the survival of less heterogeneous cell populations (Bishop et al, 2007).…”

“…Previous work had shown than the resistance of yeasts to preservatives or other inhibitors is subject to population heterogeneity (Steels et al, 2000; Holland et al, 2014; Stratford et al, 2014; Altamirano et al, 2018): despite genetic uniformity of the cell population, the bulk population was only moderately resistant and a very small sub-population showed high resistance, i.e., heteroresistance. This indicated that population growth (e.g., associated with spoilage) was largely due to the presence of rare, resistant individuals within the population.…”

Scanning electron microscopy and machine learning reveal heterogeneity in capsular morphotypes of the human pathogen Cryptococcus spp.

“…The methodological advances achieved during the last decade allowed the study of phenotype heterogeneity in clonal cells. Particularly, the use of microorganism models has challenged the classical view of the phenotype determinacy based on the genotype and environmental context [1][2][3][4][5] .…”

Fatores microbiológicos da criptococose e sua importância na carga da doença em pacientes atendidos em um hospital de referência do Estado de São Paulo

“…Observamos neste trabalho que a maioria das subpopulações hFCZ apresentaram menor fluorescência do marcador rodamina 6G, sugerindo que há maior funcionamento das bombas de efluxo nestas células, em comparação com as populações de células originais, Seguindo o objetivo de ampliar o conhecimento da natureza do fenômeno de heterorresistência e investigar sua dimensão em isolados brasileiros, foram analisadas também alterações morfológicas nas subpopulações heterorresistentes e comparadas com aquelas observadas na cepa parental. De fato, pode-se confirmar que estas células apresentam morfologias distintas em relação às células parentais, tais como: células maiores e em filamentação, confirmando, assim, achados de outros autores (SIONOV et al, 2010ALTAMIRANO et al, 2018.…”

“…Células heterorresistentes possuem, também, alteração na composição da membrana celular, especificamente, no conteúdo de ergosterol, que é um esterol essencial para manter a permeabilidade e fluidez da membrana (ALTAMIRANO et al, 2018). O FCZ inibe a enzima 14α-demetilase, codificada pelo ERG11.…”