Number of transcripts and proteins related to metabolism and energy subcategories regulated in <i>Paracoccidioides</i>, <i>Pb</i>01, under carbon starvation.

<p>The number of transcripts and proteins, in percentage (%), regulated in <i>Paracoccidioides</i>, <i>Pb</i>01, under carbon starvation was calculated based on number of transcripts/proteins in each category shown in Figures S3 and S9, panels A. (A) Metabolism. The subcategories were represented by: amino acid; nitrogen/sulfur; C- compound and carbohydrates; lipid/fatty acid, and isoprenoid; purin nucleotide/nucleoside/nucleobase; secondary and phosphate metabolism. (B) Energy. The subcategories were represented by glycolysis/gluconeogenesis; TCA cycle; electron transport and membrane associated energy; ethanol production; pentose phosphate pathway and glyoxylate cycle. Black and gray bars indicate genes and proteins, respectively.</p

Transcriptional and Proteomic Responses to Carbon Starvation in <i>Paracoccidioides</i>

<div><p>Background</p><p>The genus <i>Paracoccidioides</i> comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis.</p><p>Methodology/Principal Findings</p><p>The fungal pathogen <i>Paracoccidioides</i> spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of <i>Paracoccidioides</i>, <i>Pb</i>01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MS^E) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to <i>Paracoccidioides</i> inside macrophages.</p><p>Conclusions/Significance</p><p>For a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of <i>Paracoccidioides</i> spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding to carbon sources play a major role in adaptation and persistence in the host niche.</p></div

Growth of <i>Paracoccidioides</i> under carbon starvation.

<p>A total of 5.10⁷ cells/50 mL of <i>Paracoccidioides</i> yeast cells were incubated in minimal medium (MVM) with carbon (4% glucose) or not (0% glucose) up to 72 h. At time points 0, 24, 48 and 72 h, cells were collected, killed by heat, and lyophilized to determine the cell dry weight. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments.*, significantly different from the carbon condition, at a p-value of ≤0.05.</p

Ethanol detection in <i>Paracoccidioides</i>, <i>Pb</i>01 yeast cells, under carbon starvation.

<p>The concentration of ethanol (g/L) in <i>Paracoccidioides</i> yeast cells under carbon or carbon-starved conditions was determined. A total of 10⁶ cells were used for each sample, and the ethanol compound was quantified using the enzymatic detection kit (UV-test for ethanol, RBiopharm, Darmstadt, Germany). Data are expressed as the mean ± standard deviation of the biological triplicates of independent experiments. Student's <i>t</i>-test was used.*, significantly different from the carbon condition, at a p-value of ≤0.05.</p

The most abundant down-regulated proteins of <i>Paracoccidioides</i> (<i>Pb</i>01) yeast cells under carbon starvation detected using NanoUPLC-MS^E.

a<p>Identification of differentially regulated proteins from <i>Paracoccidioides</i> genome database (<a href="http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html</a>) using the ProteinLynx Global Server (PLGS) version 3.0 (Waters Corporation. Manchester. UK);</p>b<p>Proteins annotation from <i>Paracoccidioides</i> genome database or by homology in NCBI database (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>);</p>c<p>Protein expression profiles in <b>log2</b>-fold change (<b>5×-threshold</b>) obtained from ProteinLynx Global Server (PLGS) analysis normalized with internal standard.</p>d<p>Biological process of differentially expressed proteins from MIPS (<a href="http://pedant.helmholtz-muenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01" target="_blank">http://pedant.helmholtz-muenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01</a>) and Uniprot database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>).</p><p>*: identified only in the presence of glucose (carbon condition).</p

Overview of metabolic responses of <i>Paracoccidioides</i> to carbon starvation.

<p>The figure summarizes the data from transcriptome and proteomic analyses and suggests the mechanism and the first flow of carbon used by this fungus to overcome the carbon starvation stress. HXK: hexokinase; PGM: phosphoglucomutase; PFK: 6-phosphofructokinase; FBPase: fructose-1,6-biphosphatase; ALD: fructose-bisphosphate aldolase; TPI: triosephosphate isomerase; PGK: phosphoglycerate kinase; ENO: enolase; PEPCK: phosphoenolpyruvate carboxykinase; PYC: pyruvate carboxylase; PDC: pyruvate decarboxylase; ADH: alcohol dehydrogenase; ALDH: aldehyde dehydrogenase; ACD: acyl-CoA dehydrogenase; ECH: enoyl-CoA hydratase; THIO: 3-ketoacyl-CoA thiolase; PDH: pyruvate dehydrogenase; ICL: isocitrate lyase; MLS: malate synthase; IDH: isocitrate dehydrogenase; OGDC: 2-oxoglutarate dehydrogenase E1; SUCLA: succinyl-CoA ligase; FUM: fumarate hydratase (fumarase) and MDH: malate dehydrogenase. Enzymes were colored according to their differences in expression and labeled to indicate whether the data were obtained from transcriptome or proteomics. Italic, bold, and underlined labels indicate that the data were obtained from transcriptome, proteome, or both, respectively. Green or red indicate up- or down-regulated proteins, respectively. The numbers 1, 2, 3, and 4 indicate up-regulated amino acids involved in pyruvate, oxaloacetate, succinate, and acetyl-CoA production, respectively. 1) pyruvate production: <i>tryptophan</i> and <b>cysteine</b>. 2) oxaloacetate production: <u>phenylalanine</u>, <b>glutamate</b> and <u>tyrosine</u>. 3) succinate production: <u>threonine</u>. 4) acetyl-CoA production: <u>threonine</u>, <i>tryptophan</i>, <u>tyrosine</u> and <b>leucine</b>. Italic, bold, and underlined labels indicate that the amino acids accumulations were obtained from transcriptome, proteome, or both analyzes, respectively. OXA: oxaloacetate; <i>e⁻</i>: released electrons from enzymatic reaction.</p

Expression of <i>Paracoccidioides fbp</i>, <i>icl</i> and <i>thio</i> genes and susceptibility of yeast cells to macrophages killing during infection.

<p>(<b>A</b>) <i>Pb</i>01 yeast cells were grown without (yeast cells) and with macrophages (yeast cells-macrophages) for 24 h in RPMI medium, and the relative expression of genes <i>fbp</i> (fructose-1,6-biphosphatase), <i>icl</i> (isocitrate lyase), and <i>thio</i> (3-ketoacyl-CoA thiolase) was determined. The data were normalized using the constitutive gene encoding the 60S ribosomal L34 gene as the endogenous control and are presented as relative expression in comparison to the experimental control cells value set at 1. (<b>B</b>) <i>Pb</i>01 yeast cells were previously grown in MMcM medium with carbon (4% of glucose) or absence of glucose (carbon starvation) up to 48 h and then were incubated with macrophages at a 1∶2.5 macrophages: yeast ratio, for both conditions. As demonstrated, the number of viable cells was determined by quantifying the number of colony forming units/mL (CFUs/mL) during infection from culture supernatant (non-internalized cells removed by aspiration prior to macrophages lysis) and after internalization. Data are expressed as the mean ± standard deviation of the biological triplicates of independent experiments. Student's <i>t</i>-test was used. *, significantly different from the control, at a p-value of ≤0.05.</p

Isocitrate lyase activity assay.

<p>The activity was determined by measuring the formation of glyoxylate as its phenylhydrazone derivative in each condition. A total of 50 µg of each total protein extracts of <i>Paracoccidioides</i> under carbon and carbon-starvation (0% glucose) conditions for 48 h in MMcM medium was used. The specific activities were determined as the amount of enzyme required to form 1 µmol of glyoxylate-phenylhydrazone per minute per mg of total protein and are represented as U.mg⁻¹. Errors bars represent standard deviation from three biological replicates while * represents p≤0.05.</p

The most abundant up-regulated proteins of <i>Paracoccidioides</i> (<i>Pb</i>01) yeast cells under carbon starvation detected using NanoUPLC-MS^E.

a<p>Identification of differentially regulated proteins from <i>Paracoccidioides</i> genome database (<a href="http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html</a>) using the ProteinLynx Global Server (PLGS) version 3.0 (Waters Corporation. Manchester. UK);</p>b<p>Proteins annotation from <i>Paracoccidioides</i> genome database or by homology from NCBI database (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>);</p>c<p>Protein expression profiles in <b>log2</b>-fold change (<b>5×-threshold</b>) obtained from ProteinLynx Global Server (PLGS) analysis normalized with internal standard.</p>d<p>Biological process of differentially expressed proteins from MIPS (<a href="http://pedant.helmholtz-muenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01" target="_blank">http://pedant.helmholtz-muenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01</a>) and Uniprot database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>).</p>#<p>: identified only in carbon starvation condition.</p

Genomic Analyses and Transcriptional Profiles of the Glycoside Hydrolase Family 18 Genes of the Entomopathogenic Fungus <i>Metarhizium anisopliae</i>

<div><p>Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen <i>Metarhizium anisopliae,</i> chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the <i>M. anisopliae</i> genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named <i>chit1</i>, <i>chi2</i> and <i>chi3</i>. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-<i>N</i>-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different <i>M. anisopliae</i> developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of <i>M. anisopliae</i> chitinases and ENGases and a more detailed study concerning the enzymes’ roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.</p></div