The Role of Ceramide Synthases in the Pathogenicity of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans) is estimated to cause about 220,000 new cases every year in patients with AIDS, despite advances in antifungal treatments. C. neoformans possesses a remarkable ability to disseminate through an immunocompromised host, making treatment difficult. Here, we examine the mechanism of survival of C. neoformans under varying host conditions and find a role for ceramide synthase in C. neoformans virulence. This study also provides a detailed lipidomics resource for the fungal lipid research community in addition to discovering a potential target for antifungal therapy. Cell Rep 2018 Feb 6; 22(6):1392-140

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.post-print10768 K

Differences in Sirtuin Regulation in Response to Calorie Restriction in Cryptococcus neoformans

Cryptococcus neoformans successfully replicates in low glucose in infected patients. In the serotype A strain, H99, growth in this condition prolongs lifespan regulated by SIR2, and can be modulated with SIR2-specific drugs. Previous studies show that lifespan modulation of a cryptococcal population affects its sensitivity to antifungals, and survival in an infection model. Sirtuins and their role in longevity are conserved among fungi; however, the effect of glucose starvation is not confirmed even in Saccharomyces cerevisiae. Lifespan analysis of C. neoformans strains in low glucose showed that 37.5% exhibited pro-longevity, and lifespan of a serotype D strain, RC2, was shortened. Transcriptome comparison of H99 and RC2 under calorie restriction demonstrated differences, confirmed by real-time PCR showing that SIR2, TOR1, SCH9, and PKA1 expression correlated with lifespan response to calorie restriction. As expected, RC2-sir2Δ cells exhibited a shortened lifespan, which was reconstituted. However, shortened lifespan from calorie restriction was independent of SIR2. In contrast to H99 but consistent with altered SIR2 regulation, SIR2-specific drugs did not affect outcome of RC2 infection. These data suggest that SIR2 regulation and response to calorie restriction varies in C. neoformans, which should be considered when Sirtuins are investigated as potential therapy targets for fungal infections

The Role of Ceramide Synthases in the Pathogenicity of Cryptococcus neoformans

Summary: Cryptococcus neoformans (C. neoformans) is estimated to cause about 220,000 new cases every year in patients with AIDS, despite advances in antifungal treatments. C. neoformans possesses a remarkable ability to disseminate through an immunocompromised host, making treatment difficult. Here, we examine the mechanism of survival of C. neoformans under varying host conditions and find a role for ceramide synthase in C. neoformans virulence. This study also provides a detailed lipidomics resource for the fungal lipid research community in addition to discovering a potential target for antifungal therapy

List of genes upregulated in old <i>C</i>. <i>glabrata</i> cells known to be involved in antifungal resistance.

<p>List of genes upregulated in old <i>C</i>. <i>glabrata</i> cells known to be involved in antifungal resistance.</p

Gene ontology enrichment analysis for genes upregulated in old <i>C</i>. <i>glabrata</i> cells.

<p>Gene ontology enrichment analysis for genes upregulated in old <i>C</i>. <i>glabrata</i> cells.</p

Enhanced resilience of older <i>C</i>. <i>glabrata</i> cells.

<p>(A) Older cells contributed to increased virulence in <i>Galleria</i> (n = 20 worms compared by Log-Rank test). (B) CFU counts continuously increased after <i>Galleria mellonella</i> infection with old BG2 cells (10 generations). Young BG2 cells (0–3 generations) were cleared before establishing infection within the first few hours post infection (n = 25 worms per time point). (C) Resistance to neutrophil-mediated killing was observed in older cells (experiments were run in duplicates, with six replicates, and compared by Student’s t-test). (D and E) NET induction was higher by old (14 and 28 generations) compared to young <i>C</i>. <i>glabrata</i> cells, but still significantly lower compared to that seen with hyphal <i>C</i>. <i>albicans</i>. % NETs indicates neutrophil nuclei >1,000 μm² over total neutrophils, and upper panel in D shows sytox staining of nuclei (experiments were run in duplicates, with six replicates, and compared by Student’s t-test). (F) Neutrophil Elastase nuclear localization was higher in old (14 and 28 generations) compared to young <i>C</i>. <i>glabrata</i> cells and unstimulated neutrophils (10 neutrophils were analyzed per condition and compared by one-way ANOVA) (G) H₂O₂ disc diffusion assay shows smaller zone of inhibition for both 14 and 28 generation old cells (experiments were run in triplicates and compared by Student’s t-test). (H) Epithelial cell adhesion assays demonstrate decreased adhesion of older cells (experiments were run in triplicates and compared by Student’s t-test). <i>*P</i> < 0.05, **<i>P <</i> 0.01, ***<i>P <</i> 0.001, ****<i>P <</i> 0.0001.</p