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

The AGC kinase YpkA regulates sphingolipids biosynthesis and physically interacts with SakA MAP kinase in Aspergillus fumigatus

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the "AGC kinases" Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkA(YPK1) in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the 1 ypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP462383/2014-82009/53546-5; 2015/17541-0; 2017/19694-3This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Grant Numbers: 2009/53546-5, 2015/17541-0, and 2017/19694-3) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant Number 462383/2014-8), Brazil to IM. This work was also supported by NIH grants (AI116420 and AI125770) and by the VA Merit Award grant (I01BX002624) to MDP. The funding body had no role in designing the study or in collecting, analyzing and interpreting the data, or in writing the manuscrip

The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the “AGC kinases” Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkAY PK1 in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the ΔypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways

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

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

Summary of the phenotypic characteristics of 14-generation-old <i>C</i>. <i>glabrata</i> cells compared to young cells (strain BG2).

<p>Summary of the phenotypic characteristics of 14-generation-old <i>C</i>. <i>glabrata</i> cells compared to young cells (strain BG2).</p

Older <i>C</i>. <i>glabrata</i> cells accumulate <i>in vivo</i>.

<p>(A) Calcofluor stain shows BG2 cells with increased budscars in mouse kidneys (inset). Significantly more BG2 cells with high budscar counts (mean budscars represented by line through plot) were found at day 2 and 4 in kidneys of WT compared to neutropenic mice injected intravenously, and also compared to day 0 in either host. Higher budscar counts were also found in <i>C</i>. <i>glabrata</i> from candiduric patient when compared to the same strain grown <i>in vitro</i> (#42). (B) BG2 cells were larger at days 2 and 4 in kidneys of WT mice compared to neutropenic mice and inoculum (day 0). (C) Model of expansion or contraction of a fungal cell population with five age classes: 0–2, 3–5, 6–8, 9–11 and 12–16 replications, whose populations are given by N₀, N₁, N₂, N₃ and N₄, respectively. <i>r</i>_<i>0</i>…<i>r</i>_<i>3</i> are replication rates, and <i>m</i>_<i>0</i>…<i>m</i>_<i>4</i> are mortality rates of each age class. (D) Theoretical model can reproduce experimental results and confirm differential selection hypothesis. Upper panel: Probability distributions of replicative ages found experimentally (shaded areas) overlaid with results from the model parametrized with the most optimal parameters found using optimization (lines). Lower panel: Mortality rate profiles that yielded the best fits of the model to the data for each condition. Precise mortality rates were <i>m</i> = [6.9 3.0 2.8 4.1 4.0] (WT host); <i>m</i> = [2.8 2.3 9.3 2.9 2.8] (neutropenic host); <i>m</i> = [0.0, 10.4, 10.4, 0.0, 0.5] (<i>in vitro</i>).</p