Characterization of the <i>Paracoccidioides</i> Hypoxia Response Reveals New Insights into Pathogenesis Mechanisms of This Important Human Pathogenic Fungus

<div><p>Background</p><p>Hypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome <i>in vivo</i> microenvironmental stress conditions including low oxygen tension; however nothing is known how <i>Paracoccidioides</i> species respond to hypoxia. The genus <i>Paracoccidioides</i> comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America.</p><p>Methodology/Principal Findings</p><p>In this work, a detailed hypoxia characterization was performed in <i>Paracoccidioides</i>. Using NanoUPLC-MS^E proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that <i>Paracoccidioides</i> species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the <i>srbA</i> null mutant in <i>Aspergillus fumigatus</i>. <i>Paracoccidioides srbA</i> (<i>PbsrbA</i>), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses.</p><p>Conclusions/Significance</p><p>In this study, the hypoxia was characterized in <i>Paracoccidioides</i>. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.</p></div

Overview of metabolic responses of the <i>Paracoccidioides</i> upon hypoxia.

<p>The figure summarizes the data from proteomic analysis and shows the main changes in <i>Paracoccidioides Pb</i>01 metabolism during hypoxia, for 12 and 24 h. Up-regulated proteins detected at both time points are indicated by black, and down-regulated proteins, by grey colours. Proteins are indicated by letters: <b>ACD</b> [long-chain specific acyl-CoA dehydrogenase]; <b>ALDH</b> [aldehyde dehydrogenase]; <b>GLYP</b> [glycogen phosphorylase]; <b>PYC</b> [pyruvate carboxylase]; <b>PDHX</b> [pyruvate dehydrogenase protein X component]; <b>ACL1</b> [ATP-citrate synthase subunit 1]; <b>ACLY</b> [ATP-citrate lyase]; <b>ACO</b> [aconitate hydratase]; <b>IDH</b> [isocitrate dehydrogenase]; <b>MTND</b> [NADH-ubiquinone oxidoreductase]; <b>COX</b> [cytochrome-c oxidase chain VI]; <b>ATPG</b> [ATP synthase gamma chain]; <b>GPY</b> [glucose-6-phosphate isomerase]; <b>PGAM</b> [2,3-bisphosphoglycerate independent phosphoglycerate mutase]; <b>HBD</b> [3-hydroxybutyryl-CoA dehydrogenase]; <b>FAS1</b> [fatty acid synthase subunit beta dehydratase]; <b>FAS2</b> [fatty acid synthase subunit alpha reductase]; <b>GDH</b> [NADP specific glutamate dehydrogenase]; <b>GABD</b> [succinate-semialdehyde dehydrogenase]; <b>ATP1</b> [ATPase alpha subunit]; <b>ATPB</b> [ATP synthase subunit beta]; <b>F1F0ATP</b> [mitochondrial F1F0 ATP synthase subunit]; <b>ACS</b> [acetyl-coenzyme A synthetase]; <b>FUMA</b> [fumarate hydratase].</p

<i>PbsrbA</i> is required for hyphal growth under hypoxic conditions.

<p>The <i>A</i>. <i>fumigatus</i> reconstituted strains (<i>PbsrbA</i>), wild type (CEA10) and <i>ΔsrbA</i> were plated on GMM plates and incubated at 37°C under normoxia and hypoxia. The diameter of the colony was measured over 96 h every 24 h and are expressed in inches (cm). Under normoxia, no significant difference in growth speed and colony size could be observed (P ≤ 0.01) except less conidiation in the reconstituted strains (upper panel). In contrast, under hypoxic conditions only the mutant strain (<i>ΔsrbA</i>) did not demonstrate any detectable growth. The reconstituted strains 1, 2 and 3 (Rec1, 2 and 3) and the wild type (CEA10) showed comparable growth; P ≤ 0.01 (lower panel).</p

Predicted protein domains for SREBPs component orthologs.

<p>The amino acid sequences were obtained from GenBank (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>) to <i>Paracoccidioides Pb</i>01 (XP_002794199); <i>Pb</i>03 (KGY15961); <i>Pb</i>18 (XP_010758341); <i>Aspergillus fumigatus</i> (XP_749262); <i>Schizosaccharomyces pombe</i> (NP_595694); <i>Cryptococcus neoformans</i> (XP_567526) and <i>Homo sapiens</i> (P36956). The SREBPs bHLH (<i>basic helix-loop- helix leucine zipper DNA-binding domain</i>) protein domains and the length of each protein (indicated on the right of each protein in amino acids [aa]) were predicted by SMART tool (<a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>) and the transmembrane segments (blue rectangles) were predicted using the Phobius (<a href="http://phobius.sbc.su.se/" target="_blank">http://phobius.sbc.su.se/</a>) and SACS MEMSAT2 Prediction softwares (<a href="http://www.sacs.ucsf.edu/cgi-bin/memsat.py" target="_blank">http://www.sacs.ucsf.edu/cgi-bin/memsat.py</a>). *: conserved tyrosine residue specific to the SREBP family of bHLH transcription factors.</p

Quantitative RT-PCR revealing <i>PbsrbA</i> transcript abundance.

<p><i>PbsrbA</i> transcript levels were determined in <i>Pb</i>01 yeast cells during hypoxia (1% pO₂). The cells were incubated at 36°C for 72 h in BHI medium with agitation under normoxia (21% pO₂) and then were subjected to normoxia and hypoxia from 30 min up to 4 h. The data were normalized using the constitutive gene encoding the alpha tubulin as the endogenous control. Data are also expressed as mean ± SD (represented using error bars) of the three PCR replicates of independent experiments. *, significantly different comparison with normoxia condition, at a P value of ≤ 0.01.</p

Analysis of resistance of the wild type (CEA10), <i>ΔsrbA</i> and the Rec1 strains of <i>A</i>. <i>fumigatus</i> to fluconazole and voriconaloze.

<p>The susceptibility of the Rec1 (<i>PbsrbA</i>), wild type (CEA10) and <i>ΔsrbA</i> of <i>A</i>. <i>fumigatus</i> were analyzed to both drugs, fluconazole and voriconazole. The concentrations of each drug were choose based on previously published data [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004282#pntd.0004282.ref037" target="_blank">37</a>]. The fluconazole and voriconazol have no effect on wild type and Rec 1 strains showing that <i>PbsrbA</i> could restore the phenotype of wild type. In contrast, the mutant was susceptible for both drugs, from 1 and 0.012 ug/ ml of fluconazole and voriconazol, respectively.</p

Kinetic of expression of a representative gene of ergosterol pathway in <i>Paracoccidioides</i> submitted to hypoxia.

<p>The kinetic of expression of <i>Pberg3</i>, representative gene of ergosterol pathway, was analysed using qRT-PCR. <i>Pb</i>01 yeast cells were submitted to normoxia and hypoxia for 2, 4 and 6 h at 37°C in BHI medium and total RNAs were extracted. Molecules of cDNA were synthesized and used for qRT-PCR. The data were normalized using the constitutive gene encoding the alpha tubulin as the endogenous control. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments. *, significantly different from the normoxic condition (experimental control), at a p value of P ≤ 0.01. The accession number to <i>erg3</i> is PAAG_03651, 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>).</p

Conserved SREBP pathway in fungi, including <i>Paracoccidioides Pb</i>01, <i>Pb</i>03 and <i>Pb</i>18.

<p>^a NCBI (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>) reference sequence accession number (s).</p><p>^b Sterol regulatory element binding proteins.</p><p>^c<i>S</i>REBP <i>c</i>leavage <i>a</i>ctivating <i>p</i>rotein.</p><p>Conserved SREBP pathway in fungi, including <i>Paracoccidioides Pb</i>01, <i>Pb</i>03 and <i>Pb</i>18.</p

Functional classification of proteins regulated in <i>Paracoccidioides</i> upon hypoxia obtained by NanoUPLC-MS^E data.

<p>Biological processes of differentially expressed proteins (up- and down-regulated) in <i>Paracoccidioides</i>, <i>Pb</i>01, submitted to 12 and 24 h of hypoxia are shown. The biological processes were obtained using the Pedant on 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 databases (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). One hundred and thirty four and 154 proteins were differentially expressed in 12 and 24 h under hypoxia, respectively, compared with normoxia. In 12 h, 67 proteins were induced and the same number repressed while in 24 h, 102 proteins were induced and 52 were repressed under the same conditions.</p

Mitochondrial activity of <i>Paracoccidioides</i> submitted to normoxic and hypoxic stress.

<p><b>(A)</b><i>Pb</i>01 yeast cells were grown in BHI medium in normoxia (N), 21% pO₂, and hypoxia (H), 1% pO₂, for 12 and 24 h. The mitochondrial activity was evaluated by using rhodamine as a dye for mitochondrial membrane potential. The lower intensity of mitochondria activity was detected in 12 h of hypoxia. The white arrows indicates representative cells from population. <b>(B)</b> The rhodamine fluorescence intensity of cells grown in normoxia (N) and hypoxia (H) for 12 and 24 h was measured using the AxioVision Software (Carl Zeiss). The values of fluorescence intensity (in pixels) and the standard deviation of each analysis were used to plot the graph. Data are expressed as mean ± standard deviation (represented using error bars) of the minimum of 100 cells for each microscope slide, in triplicates, for each condition. *, significantly different comparison with normoxia condition, at a P value of ≤ 0.05.</p