A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of <i>Cryptococcus neoformans</i> in Mammals

<div><p>The pathogenic yeast <i>Cryptococcus neoformans</i> causes cryptococcosis, a life-threatening fungal disease. <i>C</i>. <i>neoformans</i> has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of <i>C</i>. <i>neoformans</i> strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as <i>HVA1</i> (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of <i>HVA1</i> was negatively correlated with virulence and was reduced <i>in vitro</i> and <i>in vivo</i> in both mouse- and <i>Galleria</i>-passaged strains of <i>C</i>. <i>neoformans</i>. Phenotypic analysis in <i>hva1</i>Δ and <i>hva1</i>Δ+<i>HVA1</i> strains revealed no significant differences in established virulence factors. Mice infected intravenously with the <i>hva1</i>Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the <i>hva1</i>Δ+<i>HVA1</i> strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the <i>hva1Δ</i> strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.</p></div

Crystal structure of Hva1.

<p>(A) The two monomers of the asymmetric unit are distinguished in cyan and magenta. Regions corresponding to β-strands and α-helix are shown as arrows and a coil, respectively. The β-strands that form the twisted sheet are labelled as β1–5 in the magenta model of this figure with β5 not represented in the arrow format to improve clarity. Overall, the architecture of Hva1 is very ordered and the two monomers are associated with each other via an extensive interface. (B) Connecting topology of the various secondary structural motifs of Hva1. The two monomers of the asymmetric unit are distinguished in cyan and magenta. Arrows represent β-strands which are labelled as indicated in (A). The regions that form the monomer-monomer interface are highlighted by dotted circles.</p

Volcano plot comparing the <i>hva1Δ</i> and H99W strains.

<p>Metabolites that are elevated in the <i>hva1Δ</i> strain versus H99W are red, while those that are decreased in the <i>hva1Δ</i> strain versus H99W are blue. To be colored the fold change must be greater than 1.5 and p-value<0.05. These data are suggestive of a block in the in the production of 2-ketoglutarate in the <i>hva1Δ</i> strain compared to H99W.</p

Lung histology at day 7.

<p>Mice were infected with the pre-passage H99W (A), the <i>hva1Δ</i> and <i>hva1Δ+HVA1</i> strains (B and C, respectively). The <i>hva1Δ</i> strain showed lower fungal burden and dense areas compared to H99W and the <i>hva1Δ+HVA1</i> strain, which showed organized inflammation around <i>C</i>. <i>neoformans</i>.</p

The <i>hva1Δ</i> strain has a hypervirulent phenotype.

<p>Mouse fungal burden in the spleen (A), brain (B) and lungs (C) of mice infected intravenously with the pre-passage H99W, the <i>hva1Δ</i> and <i>hva1Δ+HVA1</i> strains. Six mice were infected for each group. Error bars depict standard deviation. D) Survival data for mice infected intravenously with the pre-passage H99W, the <i>hva1Δ</i> and <i>hva1Δ+HVA1</i> strains. Ten mice were infected for each group (only 9 mice reported for H99W).</p

Microarray analysis comparing the <i>hva1Δ</i> and <i>hva1</i>Δ+<i>HVA1</i>strains. All genes shown were up-regulated in the <i>hva1</i>Δ+<i>HVA1</i> strain compared to the <i>hva1Δ</i> strain.

<p>Microarray analysis comparing the <i>hva1Δ</i> and <i>hva1</i>Δ+<i>HVA1</i>strains. All genes shown were up-regulated in the <i>hva1</i>Δ+<i>HVA1</i> strain compared to the <i>hva1Δ</i> strain.</p

Gene expression of <i>HVA1</i> from microarray and qRT-PCR analysis of two mouse-passaged and one <i>Galleria</i>-passaged <i>C</i>. <i>neoformans</i> strains.

<p>Gene expression of <i>HVA1</i> from microarray and qRT-PCR analysis of two mouse-passaged and one <i>Galleria</i>-passaged <i>C</i>. <i>neoformans</i> strains.</p

<i>In vivo</i> gene expression levels of <i>HVA1</i> in the liver are correlated with virulence.

<p><i>In vivo</i> gene expression levels of <i>HVA1</i> were measured using qRT-PCR for six different mouse-passaged strains and plotted against the average time to death in 10 mice caused by each strain.</p

Metabolic Remodeling in Moderate Synchronous versus Dyssynchronous Pacing-Induced Heart Failure: Integrated Metabolomics and Proteomics Study

<div><p>Heart failure (HF) is accompanied by complex alterations in myocardial energy metabolism. Up to 40% of HF patients have dyssynchronous ventricular contraction, which is an independent indicator of mortality. We hypothesized that electromechanical dyssynchrony significantly affects metabolic remodeling in the course of HF. We used a canine model of tachypacing-induced HF. Animals were paced at 200 bpm for 6 weeks either in the right atrium (synchronous HF, SHF) or in the right ventricle (dyssynchronous HF, DHF). We collected biopsies from left ventricular apex and performed comprehensive metabolic pathway analysis using multi-platform metabolomics (GC/MS; MS/MS; HPLC) and LC-MS/MS label-free proteomics. We found important differences in metabolic remodeling between SHF and DHF. As compared to Control, ATP, phosphocreatine (PCr), creatine, and PCr/ATP (prognostic indicator of mortality in HF patients) were all significantly reduced in DHF, but not SHF. In addition, the myocardial levels of carnitine (mitochondrial fatty acid carrier) and fatty acids (12:0, 14:0) were significantly reduced in DHF, but not SHF. Carnitine parmitoyltransferase I, a key regulatory enzyme of fatty acid ß-oxidation, was significantly upregulated in SHF but was not different in DHF, as compared to Control. Both SHF and DHF exhibited a reduction, but to a different degree, in creatine and the intermediates of glycolysis and the TCA cycle. In contrast to this, the enzymes of creatine kinase shuttle were upregulated, and the enzymes of glycolysis and the TCA cycle were predominantly upregulated or unchanged in both SHF and DHF. These data suggest a systemic mismatch between substrate supply and demand in pacing-induced HF. The energy deficit observed in DHF, but not in SHF, may be associated with a critical decrease in fatty acid delivery to the ß-oxidation pipeline, primarily due to a reduction in myocardial carnitine content.</p></div

Heat map of myocardial metabolome.

<p>The data obtained by multi-platform metabolomics (GC/MS, MS/MS, HPLC) and presented as fold change in SHF and DHF as compared to Control. Green indicates a significant decrease, and read indicates a significant increase in the level of metabolite as compared to Control. BCAA: branched-chain amino acid, PPP: pentose phosphate pathway, GSH: glutathione, GC/MS: gas-chromatography/mass-spectrometry, MS/MS: tandem mass-spectrometry, HPLC: high performance liquid chromatography.</p