Recovery of the Paracoccidioides brasiliensis virulence after animal passage promotes changes in the antioxidant repertoire of the fungus

Recovery of the Paracoccidioides brasiliensis virulence after animal passage promotes changes in... Abstract Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a cause of disease in healthy and immunocompromised persons in Latin America. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. The development of the disease depends on factors associated with the host immune response and the infectious agent's characteristics, especially virulence. The oxidative stress response is an important virulence attribute in several fungi. In this study, we assessed the enzymatic repertoire of responses to oxidative stress in the Pb18 isolate with different degrees of virulence. The virulence of attenuated Pb18 (aPb18) strain was recovered after several animal passages. Virulent strain (vPb18) showed an effective fungal oxidative stress response and several genes involved in response to oxidative stress were up-regulated in this isolate. These genes expressed the same profile when we recovered the phenotypic virulence in attenuated strain aPb18. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18), and this process positively modulated the fungus's antioxidant repertoire. Paracoccidioides brasiliensis, reactive oxygen species, oxidative stress, virulence INTRODUCTION The members of the genus Paracoccidioides are the aetiological agents of paracoccidioidomycosis (PCM), an important human disease in Latin America (San-Blas, Nino-Vega and Iturriaga 2002; Martinez 2015). Paracocccidioides spp are thermodimorphic and grow as yeast-like multibudding cells in cultures at 37°C and in infected tissues and as a mycelium at temperatures of 22°C–25°C, which is the infecting form (Camacho and Niño-Vega, 217). The infection begins with the inhalation of fungal propagules, and at body temperature, the fungus converts to the pathogenic yeast form (Lacaz 1994). Resident alveolar macrophages constitute one of the first defence lines against infection by Paracoccidioides (Silva et al.2011). PCM development relies on several host immune response factors and diverse critical features of Paracoccidioides, especially the virulence factors. Once inside the lungs, Paracoccidioides must address reactive oxygen species (ROS) generated by phagocytic cells, such as the superoxide radical (O2•−), hydrogen peroxide (H2O2), and hydroxyl (•OH), peroxyl (RO2•) and alkoxyl (RO•) radicals (Nicola, Casadevall and Goldman 2008). Thus, infection success is related to the fungal resistance to oxidative stress (Brown, Haynes and Quinn 2009). Indeed, Paracoccidioides possess an effective antioxidant defence system that provides protection against host-derived ROS (Camacho and Ninõ-Vega 2017). This system includes specialised enzymes such as superoxide dismutases (SODs; Tamayo et al.2016) and catalases (Cat) (Chagas et al.2008) and other enzymatic systems to detoxify hydroperoxide radicals such as the glutathione and thioredoxin systems (Parente-Rocha et al.2015) and cytochrome C peroxidase (Parente-Rocha et al.2015). Once they help the fungus to survive and cause disease, the antioxidant enzymes can be considered virulence factors (Camacho and Ninõ-Vega 2017). Using molecular and biochemical approaches, several genes and proteins participating in the oxidative stress response in the genus Paracoccidioides have been described (Campos et al.2005; Chagas et al.2008; Teixeira et al.2009; Martins et al.2011; Parente-Rocha et al.2015). Hence, our group has employed proteomic approaches with Paracoccidioides brasiliensis and identified potential virulence regulators, such as mitochondrial peroxiredoxin and vacuolar protease A (Castilho et al.2014). Several proteins involved in the response to oxidative stress were differentially expressed in virulent P. brasiliensis; thus, in the present study, we searched for the differences in antioxidants and oxidative stress responses of virulent and attenuated isolates. Here, we used the same P. brasiliensis strain (Pb18) with distinct degrees of virulence, one maintained in constant animal infections (virulent Pb18—vPb18) and other maintained for several years in culture (attenuated Pb18—aPb18). This strategy allows excluding genomic differences and pointing out which proteins were up-regulated in the virulent fungus compared to its attenuated counterpart as potential virulence factors. Our results suggest that P. brasiliensis possess an effective response to oxidative stress, and this feature is closely related to its virulence status. MATERIAL AND METHODS Fungal strain and growth conditions We used Paracoccidioides brasiliensis, Pb18 isolates, with different virulence profile in our experiments, as previously shown by Castilho et al. (2014). Long-term in vitro cultivated P. brasiliensis as a low virulent strain (attenuated Pb18—aPb18), which had been maintained for more than 5 years in our laboratory (Castilho et al.2014). In the present work, the virulence of aPb18 was restored after serial passages in Balb/c mice (4 passages). Briefly, 5 Balb/c mice (6–8 weeks of age) were intratracheally (i.t.) infected with 1 × 105 viable yeast cells from aPb18 in 30 μL of phosphate-buffered saline (PBS). After 30 days of infection, mice were euthanatized and lung, liver and spleen were aseptically removed. The organs were individually homogenized in 3 mL of PBS, and 100 μL of this suspension was plated in supplemented brain heart infusion agar (Singer-Vermes et al.1992). After 7 days, the fungal was recovered and other group of 5 Balb/c mice was re-infected using the recent isolated Pb18. This procedure was repeated at least 4 times (vPb18). Some experiments were performed with virulent Pb18 recovered after 2 passages in animals (aPb18-2x). These experiments are indicated in the legend of the figures. All mice were housed in pathogen-free conditions. The animals were handled according to National Institutes of Health—USA (NIH) (http://oacu.od.nih.gov/ARAC/index.htm). This study was approved by the Research Ethics Committee (CEP) of Federal University of São Paulo, Brazil under protocol number CEP 0349/12. Spot test Paracoccidioides brasiliensis (Pb18) oxidative stress sensitivity was investigated in yeast cells. Briefly, a total of 106 yeast cells/mL was exposed to different H2O2 concentrations (0.1, 1.0 or 10 mM) at 37°C for 5 h with constant shaking. After 5 h, each yeast culture was serially diluted (1:10 at each step) in modified YPD medium, mYPD (0.5% yeast extract, 1% casein peptone and 0.5% glucose, pH 6.5), and 10 μL of each suspension was spotted onto solid mYPD medium. Plates were photographed after 7 days of growth at 37°C. Experiments were performed in biological triplicates. Fungal viability To test the viability of P. brasiliensis yeast cells after treatment with H2O2, the cells (1 × 105) were seeded in 6-well culture plates, cultured with mYPD in the presence (0.1 or 1 mM) or absence of H2O2. Yeast cells were maintained at 37°C with constant stirring for 4 days. Cells were counted using a Neubauer chamber after staining with trypan blue. The experiment was performed in quadruplicate and repeated three times. Antioxidant enzymes activity assays To perform the enzymatic activities, protein extracts were prepared according the protocol of Villén, Beausoleil and Gygi (2008), with some modifications. Briefly, vPb18 and aPb18 isolates were grown in mYPD medium for 5 days at 37°C with constant agitation. Yeast cells were collected by centrifugation and 700 μL of cold lysis buffer (50 mM Tris-HCl [pH 7.5], 2 mM EDTA, 50 mM KCl, 0.2% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin and 10 mg/mL leupeptin) and glass beads (600 μm—Sigma, St. Louis, MO, USA) were added to the pellets. The yeast cells were disrupted mechanically using a vortex. Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the protein extracts according to the manufacturer's protocol. The fresh extracts were used to assess the enzymatic assay of both isolates. Total catalase activity was assayed using a spectrophotometer to measure the decrease in absorbance at 240 nm (Katsuwon, Zdor and Anderson 1993) immediately after preparing the extracts. One unit of catalase decomposed 1 millimole of H2O2 in 1 min at 25°C. Experiments were performed in biological triplicate, and catalase-specific activities were calculated using the extinction coefficient of 0.0394 mM−1 cm−1 (Aebi 1984). SOD enzymatic activity was measured according to Ewing and Janero (1995) using nitrotetrazolium blue chloride (Sigma-Aldrich), which produces a formazan dye upon reduction with O2•−, and the product can be determined by spectrophotometer at 560 nm. The Cu/ZnSOD activities from the cell lysates are expressed in units of SOD/mg, where 1 unit is the amount of SOD required to give 50% maximum inhibition of the initial rate of nitroblue tetrazolium reduction (Ewing and Janero 1995). Total intracellular glutathione was assessed as described by Rahman, Kode and Biswas (2006). The GSSG reductase recycling method was used to measured glutathione disulphide, the oxidised form (GSSG), by monitoring nicotinamide adenine dinucleotide phosphate spectrophometrically, and the glutathione (GSHtotal) assay was measured by the rate of formation of the 5΄-thio-2-nitrobenzoic acid chromophore at 412 nm. The amount of glutathione measured represents both forms in the sample, reduced and oxidised glutathione, and the amount of reduced glutathione (GSH) was measured using the expression [GSH]total = [GSH] + 2 × [GSSG]. The ratios of GSH/GSSG were used as reference values for the cellular redox environment. All enzymatic assays were performed in biological triplicate. RNA extraction and real-time quantitative RT-PCR analysis Total RNA was extracted from vPb18 and aPb18 yeast cells using the TRIzol reagent (Invitrogen), as described by Batista et al. (2007). All quantitative reverse transcription PCR (qRT-PCR) procedures were performed following the minimum information for publication of quantitative real-time PCR experiments guidelines (Bustin et al.2009). The quality of RNA was evaluated by electrophoresis in agarose gels. Next, 2.5 μg of total RNA was treated with DNAse (RQ1RNase-free DNase; Promega, Madison, WI, USA). Equal amounts of RNA were used for cDNA synthesis achieved with RevertAid Premium Reverse Transcriptase (Thermo Scientific, Bremen, GA, USA) according to the manufacturer's instructions. cDNA was used in a qRT-PCR reaction to measure the expression of genes related to the oxidative response; this reaction was performed using SYBR Green/ROX qPCR Master Mix 2X (Fermentas) according to the manufacturer's instructions. Briefly, a 10-μL total volume was used for each PCR reaction, which consisted of 1 × SYBR GreenPCR Master Mix, 250 nmol of the reverse primer, 250 nmol of the forward primer and 2 μL of cDNA. The cycling parameters were 50°C for 10 min, 95°C for 5 min and 40 cycles at 95°C for 30 s and 60°C for 1 min. A non-template control was used to detect any contamination. The quality of the reactions was based on the dissociation curves. The results obtained were then analysed for baseline and threshold cycle values (Ct) using the Step OnePlus software (Applied Biosystems). The baseline was adjusted to three or two cycles prior to the detection of the fluorescent signal, and the threshold cycle (Ct) was defined in the region of exponential amplification across all plots. A dilution series of reference cDNA template was prepared to determine the amplification efficiency. The relative expression ratio (experimental/control) was determined by the 2−ΔΔCt method (Schmittgen and Livak 2008) after normalisation to the level of the α-tubulin (α-TUB) and 18S transcript. The primers used in this work are summarised in Table 1. Experiments were performed in biological triplicate. Table 1. Sequence of qRT-PCR primers. IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  aAccession numbers from P. brasiliensis genes http://www.fungidb.org/. View Large Table 1. Sequence of qRT-PCR primers. IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  aAccession numbers from P. brasiliensis genes http://www.fungidb.org/. View Large Statistical analysis Data are expressed as the means ± SD. Significance was assessed by one-way analysis of variance with Student's t-test used for comparisons. The results with P < 0.05 were considered statistically significant. Prism 5 software (GraphPad Software Inc.) was used for the analyses and for graph construction. RESULTS Sensibility of P. brasiliensis Isolates (aPb18 and vPb18) to H2O2 treatment In a previous study, we performed a quantitative proteomic analysis of Paracoccidioides brasiliensis in the yeast (pathogenic) phase using a Pb18 strain with distinct infection profiles in Balb/c mice (vPb18 and aPb18). We demonstrated that some proteins involved in the response to oxidative stress were differentially expressed in the virulent isolate (Castilho et al.2014). Thus, to observe the sensitivity to oxidative stress of both isolates, P. brasiliensis yeast cells were exposed to different concentrations of H2O2 for 5 h at 37°C and then grown in solid YPD (mod). We observed the growth of the isolates under different stress conditions. The survival rate of vPb18 did not change when stimulated with different concentrations of H2O2 compared to the control (0 mM H2O2, Fig. 1A). In contrast, aPb18 was more sensitive to oxidative stress (Fig. 1A). Similar results were observed after culturing vPb18 and aPb18 under nitrosative stress (see Fig. S1, Supporting Information). In addition, the number of vPb18 yeast cells incubated with 0.1 or 1 mM of H2O2 responded with significant cell proliferation when compared to unstimulated control (0 mM H2O2, Fig. 1B), as previously described by Haniu et al. (2013). However, aPb18 yeast cells incubated with H2O2 (0.1 or 1 mM) showed a typical dose-response curve with decreased in cell viability (Fig. 1B). This shows that the vPb18 isolate presented an effective adaptive response to oxidative stress (ROS) compared with aPb18 (Fig. 1). Figure 1. View largeDownload slide Evaluation of the sensitivity to oxidative stress of P. brasiliensis isolates of varying virulence. (A) 2 × 106P. brasiliensis yeast cells (vPb18 or aPb18) were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. (B) Cultures of 1 × 104 viable yeast from P. brasiliensis were grown for 4 days in modYPD in presence H2O2 (0.1 and 1 mM), and viable cell counts using trypan blue were performed at specific points during cultivation. Error bars correspond to the standard deviations (SD) of measurements performed in triplicate. *P < 0.05; **P < 0.01 and ***P < 0.001. Figure 1. View largeDownload slide Evaluation of the sensitivity to oxidative stress of P. brasiliensis isolates of varying virulence. (A) 2 × 106P. brasiliensis yeast cells (vPb18 or aPb18) were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. (B) Cultures of 1 × 104 viable yeast from P. brasiliensis were grown for 4 days in modYPD in presence H2O2 (0.1 and 1 mM), and viable cell counts using trypan blue were performed at specific points during cultivation. Error bars correspond to the standard deviations (SD) of measurements performed in triplicate. *P < 0.05; **P < 0.01 and ***P < 0.001. The response to oxidative stress involves the participation of several proteins that modulate the redox environment, such as catalase, SOD and the glutathione system (Campos et al.2005). Thus, we measured the activities of antioxidant enzymes and the GSH and GSSG levels in both P. brasiliensis isolates. Catalase activity was assessed from the total protein extract and culture supernatant for both isolates. A high level of catalase-specific activity, both in the total extract (Fig. 2A) and in the culture supernatant (Fig. 2B), was detected from vPb18 grown in mYPD cultures from 5 days after inoculation. SOD activity was also increased in vPb18 compared to aPb18 (Fig. 2C). These results are consistent with the proteomic data previously obtained by Castilho et al. (2014), in which the enzymes peroxisomal catalase (PADG_00324) and SOD (PADG_04718) were up-regulated in vPb18 compared to aPb18. The increase in catalase activity in vPb18 is correlated with the resistance of this isolate to H2O2 treatment, suggesting that this enzyme plays a crucial role in the defence of this fungus against oxidative stress. Figure 2. View largeDownload slide Enzymatic activity of catalase and SOD in P. brasiliensis isolates of varying virulence. The catalase activities were measured using the total protein extract (A) and culture supernatant (B). The results are expressed as specific activity (units of catalase/μg protein or units of catalase/g yeast dry mass, respectively). Intracellular concentrations of SOD were determined using total protein extract (C). The results are expressed as specific activity (units of SOD/μg protein). All of the data shown in this figure were analysed using Student's t-test. Error bars correspond to the standard deviations of the measurements made in triplicate. *P < 0.05 and **P < 0.01. Figure 2. View largeDownload slide Enzymatic activity of catalase and SOD in P. brasiliensis isolates of varying virulence. The catalase activities were measured using the total protein extract (A) and culture supernatant (B). The results are expressed as specific activity (units of catalase/μg protein or units of catalase/g yeast dry mass, respectively). Intracellular concentrations of SOD were determined using total protein extract (C). The results are expressed as specific activity (units of SOD/μg protein). All of the data shown in this figure were analysed using Student's t-test. Error bars correspond to the standard deviations of the measurements made in triplicate. *P < 0.05 and **P < 0.01. Effect of the glutathione redox status in P. brasiliensis isolates (vP18 and aP18) The glutathione system is an important cellular buffering mechanism in response to changes in the cellular redox state. We evaluated the intracellular glutathione levels by analysing both reduced (GSH) and oxidised (GSSG) glutathione over 5 days of growth in vPb18 and aPb18 isolates (Fig. 3). GSH levels were significantly increased in vPb18 (approximately 2.5-fold) compared to its attenuated counterpart (aPb18) (Fig. 3A). On the other hand, GSSG levels were similar between the two isolates (Fig. 3B). The GSH/GSSG ratio is commonly used to estimate the redox state of biological systems. This ratio must be high for the maintenance of intracellular reducing power (Kappus 1987). The GSH/GSSG ratio was higher in the vPb18 isolate than in aPb18 (Fig. 3C). Analysis of the ratio GSH/GSSG suggested the presence of a reducing intracellular redox environment in vPb18 yeast cells. Protection against oxidative stress is related to the cellular capability of maintaining the cytosol in a more reduced state (Campos et al.2005). The enzymes participating in the glutathione system (glutathione reductase and glutathione peroxidase) did not show statistical significance in the activities of these enzymes between the isolates (see Fig. S2, Supporting Information); however, the glutathione reductase (GR) levels showed a moderate increase in vPb18. Figure 3. View largeDownload slide Activity assay results of the glutathione system assessed for yeast protein extracts. GSH (A) and GSSG levels (B) were run as described in Materials and Methods. The GSH/GSSG ratios (C) are the GSH levels divided by the GSSG levels. Student's t-test was used for statistical comparisons, and the observed differences were statistically significant (*P < 0.05). Error bars represent ±SD of n = 3. Figure 3. View largeDownload slide Activity assay results of the glutathione system assessed for yeast protein extracts. GSH (A) and GSSG levels (B) were run as described in Materials and Methods. The GSH/GSSG ratios (C) are the GSH levels divided by the GSSG levels. Student's t-test was used for statistical comparisons, and the observed differences were statistically significant (*P < 0.05). Error bars represent ±SD of n = 3. Expression levels of genes related to the antioxidant response in P. brasiliensis isolates of varying virulence Quantitative PCR was used for the transcriptional analysis of certain genes implicated in endogenous antioxidant defence, such as CATA, CATB and CATP (catalase A [PADG_ 04740], B [PADG_ 00225] and peroxisomal [PADG_ 00324], respectively); SOD1, SOD2 and SOD3 (cytoplasmic [PADG_07418 and PADG_01755] and mitochondrial SOD [PADG_01954], respectively); TRX1 and TRX2 (thioredoxin [PADG_05504 and PADG_03161], respectively); TRR (thioredoxin reductase [PADG_ 01551]); PRX1 (peroxiredoxin mitochondrial [PADG_03095]); and HYR1 (peroxiredoxine [PADG_04587]) and in glutathione metabolism, such as GS (γ-glutamylcysteine synthetase [PADG_02218]); GR (glutathione reductase [PABG_06068]); and GST1, GST2 and GST3 (glutathione transferase [PADG_01775, PADG_00697 and PAAG_03931, respectively]). At a late-exponential phase (5 days), the expression of all genes increased significantly in the vPb18 isolate, except for SOD3 (Fig. 4), with the highest levels found for CATA, HYR1, GST2 and AOX genes (12-, 7-, 7- and 15-fold, respectively) and the lowest for CATB, PRX1 and TRX1. Figure 4. View largeDownload slide Relative expression of the protein-encoding genes of vPb18 or aPb18 in P. brasiliensis yeast cells. Quantitative RT-PCR of RNA isolated from vPb18 and aPb18 grown in mYPD. The aPb18 expression was used as a calibrator. Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). Figure 4. View largeDownload slide Relative expression of the protein-encoding genes of vPb18 or aPb18 in P. brasiliensis yeast cells. Quantitative RT-PCR of RNA isolated from vPb18 and aPb18 grown in mYPD. The aPb18 expression was used as a calibrator. Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). To determine whether these genes are regulating during animal infection, we evaluated the expression of these genes before and after two passages of aPb18 in mice (Fig. 5A). This strategy had regained virulence in the Pb18 attenuated form, according to a previous study carried out by our group (Castilho et al.2014). After each animal passage, we observed an increase in the colony forming unit’s (CFU) number, and after second passage we found CFU values close to those obtained in vPb18 (data not shown). Expression analysis revealed that all antioxidant genes (except SOD3) were up-regulated in the attenuated strain that regained virulence and that was isolated after two passages in mice (aPb18-2x; Fig. 5B). We also observed the growth of the isolates (aPb18 and aPb18-2x) under different stress conditions. The survival rate of aPb18-2x did not change when stimulated with different concentrations of H2O2 in comparison to the control. In contrast, aPb18 maintained a profile of high sensitivity to oxidative stress induced by high concentrations of H2O2 (Fig. 5C). These results demonstrate that the expression of antioxidant genes can be considered potential virulence factors. Figure 5. View largeDownload slide Relative expression of the protein-encoding genes of aPb18 or aPb18–2x in P. brasiliensis yeast cells. (A) Schematic presentation of aPb18 isolate virulence recovery. Balb/c mice were intraperitoneally (i.p.) infected with 1 × 106 viable yeast cells of aPb18 in PBS. After 20 days the fungus was recovered of the mice (first passage—P1). This procedure was performed once more (second passage—P2). After the second animal passage, the gene expression levels of the isolate aPb18-2x were analysed. (B) Data represent changes in transcription between the aPb18 before and after two passages through animals (aPb18-2x). Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). (C) Evaluation of the sensitivity to oxidative stress in yeast cells of P. brasiliensis aPb18 or aPb18-2x. 2 × 106P. brasiliensis attenuated (aPb18) or aPb18-2x (aPb18 after two passages through animals to restore virulence) yeast cells were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. Figure 5. View largeDownload slide Relative expression of the protein-encoding genes of aPb18 or aPb18–2x in P. brasiliensis yeast cells. (A) Schematic presentation of aPb18 isolate virulence recovery. Balb/c mice were intraperitoneally (i.p.) infected with 1 × 106 viable yeast cells of aPb18 in PBS. After 20 days the fungus was recovered of the mice (first passage—P1). This procedure was performed once more (second passage—P2). After the second animal passage, the gene expression levels of the isolate aPb18-2x were analysed. (B) Data represent changes in transcription between the aPb18 before and after two passages through animals (aPb18-2x). Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). (C) Evaluation of the sensitivity to oxidative stress in yeast cells of P. brasiliensis aPb18 or aPb18-2x. 2 × 106P. brasiliensis attenuated (aPb18) or aPb18-2x (aPb18 after two passages through animals to restore virulence) yeast cells were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. DISCUSSION In a previous work, we analysed the proteomic profile of virulent and attenuated forms of the Paracoccidioides brasiliensis Pb18 strain, and we identified that some proteins that engage in the response to oxidative stress were up-regulated in virulent Pb18 as compared with attenuated strain (Castilho et al.2014). The ability of pathogenic fungi to cause disease entails their capability to survive in the host. Once inside the host, the fungi are challenged by oxidative stress from phagocytic cells, mainly from neutrophils and macrophages (Robinson, Ohira and Badwey 2004; Gazendam et al.2016). The survival of these microorganisms in phagocytic cells is strongly associated with putative virulence factors (Shiloh and Nathan 2000). Pathogenic fungi have developed strategies to survive in hostile conditions, such as the expression of genes related to oxidative stress. Campos et al. (2005) reported genes that could be potentially involved in the fungal oxidative stress response, such as catalase, SOD, peroxiredoxins, thioredoxin and the glutathione system. Thus, in the present study, we analysed the oxidative stress response of the Pb18 strain with distinct virulence profiles. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18) and this process positively modulated the fungus's antioxidant repertoire. Herein, we demonstrated that enzymatic activity of catalase and SOD; and GSH levels were higher in vPb18 than aPb18 (Fig. 2), suggesting that this systems may be involved with the fungal virulence. Our data suggest that Pb18 adaptation to the host depend on their ability to increase the expression of antioxidant system. After two animal passages, aPb18 increased the expression of some antioxidant enzymes (Fig. 5) and the CFU’s number (data not shown). In addition, vPb18 cultured in vitro had a decrease in antioxidant enzyme expression as their ability to infect animals (data not shown). These data demonstrate that Pb18 strain is capable of rapidly adapting in host conditions by altering the expression of antioxidant enzymes. Our results are consistent with previous studies that link antioxidant enzymes and virulence (Tamayo et al.2016; Mittra et al.2017; Tamayo et al.2017; Missall and Lodge 2005). These findings reinforce the concept that is very important to maintain Paracoccidioides spp strains freshly isolated from serial passages in animals to perform in vitro or in vivo experiments (Brummer et al.1990). Recent work showed that Galleria mellonella can be an alternative model to recover Paracoccidioides spp virulence factors (Scorzoni et al.2017). Changes in morphology, biochemistry profile and expression of surface markers were reported in Paracoccidioides spp strains before and after animal passage (Svidzinski et al.1999; Kioshima et al.2011). However, this is the first work that clearly shows that the adaptation of the fungus to the oxidative stress of the host, by increasing the expression and activity of antioxidant enzymes, may also be directly related to the virulence of the pathogen. The role of antioxidant system has been studied in pathogenic fungi. Catalase is one of the enzymes responsible for consuming H2O2, which protects the cellular environment against oxidative stress by reducing the harmful effect of H2O2 (Chagas et al.2010). In P. brasiliensis, three members of the catalase family were also found, PbCatA, PbCatC and PbCatP. The PbCATP transcript was increased after co-cultivation with murine macrophages, suggesting the importance of PbCATP in the response to exogenous oxidative stress (Chagas et al.2008). Tamayo et al. (2017) demonstrated that PbCATP expression is increased in yeast cells treated with H2O2, and they proposed that PbCATP enzymatic activity promotes protection against endogenous and exogenous H2O2. The increase of catalase activity is correlated with the resistance of P. brasiliensis to H2O2 treatment, suggesting that this enzyme plays a crucial role in the defence of the fungus against oxidative stress (Dantas et al.2008). Besides catalase, SOD also presents the first line of antioxidant defence against ROS. SODs are metallo-proteins and neutralise the toxic levels of ROS generated by the host, contributing to the virulence of some pathogenic fungi (Tamayo et al.2016). In P. brasiliensis, four genes corresponding to two SOD isoenzymes were found (Campos et al.2005). Interestingly, in our data the transcript level of SOD2 was up regulated in vPb18 but to SOD3 was observed a down-regulation in their expression. SOD3 is associated with manganese ions, and MnSOD is an important isoenzyme for preventing oxidative stress in mitochondria (Luk et al.2005). Proteomic analysis revealed that aPb18 exhibited a higher aerobic metabolism compared to vPb18 (Castilho et al.2014); thus, the up-regulation of MnSOD in aPb18 could play an important role in protecting against respiration-derived ROS. The same result has been reported by Rezende et al. (2011). Paracoccidioides lutzii mycelia possessed greater aerobic metabolism compared to the yeast phase, and MnSOD was up-regulated in this phase. In addition to catalase and SOD, fungi possess alternative pathways to maintain the intracellular redox balance. AOX have been reported in human pathogenic fungi, such as Cryptococcus neoformans and Aspergillus fumigatus, as an important enzyme that contributes to the adaptation of fungal within phagocytic cells (Akhter et al.2003; Magnani et al.2008). In P. brasiliensis, yeast with decreased PbAOX expression had increased susceptibility to INF-γ-activated alveolar macrophages, which were able to produce ROS in response to fungal infection (Ruiz et al.2011). Additionally, in a mouse model of infection, the reduction in PbAOX expression increased the survival of the mice (Ruiz et al.2011). Our results demonstrated an increase in PbAOX expression in vPb18 and aPB18-2x, supporting the relevance of PbAOX in the virulence of P. brasiliensis. The antioxidant machinery involves detoxifying enzymes and metabolites. Glutathione is an essential metabolite and plays a key role in cellular resistance to oxidative damage (Penninckx 2002). The glutathione system can provide protection against oxidative stress by maintaining the cytoplasm of the cells in a reduced state. The GSH/GSSG ratio reflects intracellular redox changes (Guedouari et al.2014). Reduced glutathione (GSH) reduces the molecules containing an unpaired electron, resulting in the oxidised form of glutathione (GSSG; Izawa, Inoue and Kimura 1995). The enzymatic activity of the glutathione system indicates that the intracellular reducing potential of vPb18 is higher compared to its attenuated counterpart. Thus, this could be an additional mechanism for protecting vPb18 from oxidative stress conditions in host phagocytic cells. The mRNA levels for the genes encoding glutathione synthetase and glutathione S-transferases (GSTs) also showed increased expression in vPb18. Weber et al. (2012) demonstrated that GST was preferentially secreted into Paracoccidioides lutzii yeasts compared to the mycelial secretoma. GSTs represent a group of detoxifying enzymes that are involved in the protection against oxidative stress and detoxification of xenobiotics and heavy metals (Hayes and Strange 1995). In Saccharomyces cerevisiae GSH synthetase mutants were sensitive to H2O2. These mutants, when treated with 4 mM H2O2 for 1 h, had a 2-fold lower survival rate compared to the wild-type yeast (Grant, Perrone and Dawes 1998). In addition, GR is involved in the stabilisation of the pool of reduced glutathione, an important antioxidant in the cell (Lushchak 2011; Townsend, Lushchak and Cooper 2014). Thus, increased GR expression in vPb18 would explain the high GSH levels in the virulent isolate. The survival of P. brasiliensis to the host implies in cellular activity very different from that necessary for their survival in the culture medium. Evidence from this and other works suggest that this adaptation involves dramatic alteration in the expression of genes, especially enzymes involved with defence mechanisms against the oxidative stress imposed mainly by host phagocytic cells. After animal passages, aPb18 increased the virulence and the expression of several genes involved in the response to oxidative stress. Thus, although speculative, the link between virulence and the expression of such enzymes cannot be ruled out. The findings presented in this work supports the relevance of using Paracoccidioides spp isolated after serial passages in animal to study virulence, pathogenicity, biochemistry and drug discovery. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. ACKNOWLEDGEMENT We are thankful to Adriano Sartori for generous and competent technical assistance. FUNDING The authors acknowledge financial support from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo/Brazil) under grants 2014/13961-–1; 2014/08987-1 and 2017/04592-0, and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Brazil) under grant 478023/2013–8. Conflict of interest. None declared. REFERENCES Aebi H. Catalase in vitro. Methods Enzymol  1984; 105: 121– 6. Google Scholar CrossRef Search ADS PubMed  Akhter S, McDade HC, Gorlach JM et al.   Role of alternative oxidase gene in pathogenesis of Cryptococcus neoformans. Infect Immun  2003; 71: 5794– 802. Google Scholar CrossRef Search ADS PubMed  Batista WL, Barros TF, Goldman GH et al.   Identification of transcription elements in the 5΄ intergenic region shared by LON and MDJ1 heat shock genes from the human pathogen Paracoccidioides brasiliensis. Evaluation of gene expression. Fungal Genet Biol  2007; 44: 347– 56. Google Scholar CrossRef Search ADS PubMed  Brown AJ, Haynes K, Quinn J. 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Recovery of the Paracoccidioides brasiliensis virulence after animal passage promotes changes in the antioxidant repertoire of the fungus

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Abstract

Abstract Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a cause of disease in healthy and immunocompromised persons in Latin America. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. The development of the disease depends on factors associated with the host immune response and the infectious agent's characteristics, especially virulence. The oxidative stress response is an important virulence attribute in several fungi. In this study, we assessed the enzymatic repertoire of responses to oxidative stress in the Pb18 isolate with different degrees of virulence. The virulence of attenuated Pb18 (aPb18) strain was recovered after several animal passages. Virulent strain (vPb18) showed an effective fungal oxidative stress response and several genes involved in response to oxidative stress were up-regulated in this isolate. These genes expressed the same profile when we recovered the phenotypic virulence in attenuated strain aPb18. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18), and this process positively modulated the fungus's antioxidant repertoire. Paracoccidioides brasiliensis, reactive oxygen species, oxidative stress, virulence INTRODUCTION The members of the genus Paracoccidioides are the aetiological agents of paracoccidioidomycosis (PCM), an important human disease in Latin America (San-Blas, Nino-Vega and Iturriaga 2002; Martinez 2015). Paracocccidioides spp are thermodimorphic and grow as yeast-like multibudding cells in cultures at 37°C and in infected tissues and as a mycelium at temperatures of 22°C–25°C, which is the infecting form (Camacho and Niño-Vega, 217). The infection begins with the inhalation of fungal propagules, and at body temperature, the fungus converts to the pathogenic yeast form (Lacaz 1994). Resident alveolar macrophages constitute one of the first defence lines against infection by Paracoccidioides (Silva et al.2011). PCM development relies on several host immune response factors and diverse critical features of Paracoccidioides, especially the virulence factors. Once inside the lungs, Paracoccidioides must address reactive oxygen species (ROS) generated by phagocytic cells, such as the superoxide radical (O2•−), hydrogen peroxide (H2O2), and hydroxyl (•OH), peroxyl (RO2•) and alkoxyl (RO•) radicals (Nicola, Casadevall and Goldman 2008). Thus, infection success is related to the fungal resistance to oxidative stress (Brown, Haynes and Quinn 2009). Indeed, Paracoccidioides possess an effective antioxidant defence system that provides protection against host-derived ROS (Camacho and Ninõ-Vega 2017). This system includes specialised enzymes such as superoxide dismutases (SODs; Tamayo et al.2016) and catalases (Cat) (Chagas et al.2008) and other enzymatic systems to detoxify hydroperoxide radicals such as the glutathione and thioredoxin systems (Parente-Rocha et al.2015) and cytochrome C peroxidase (Parente-Rocha et al.2015). Once they help the fungus to survive and cause disease, the antioxidant enzymes can be considered virulence factors (Camacho and Ninõ-Vega 2017). Using molecular and biochemical approaches, several genes and proteins participating in the oxidative stress response in the genus Paracoccidioides have been described (Campos et al.2005; Chagas et al.2008; Teixeira et al.2009; Martins et al.2011; Parente-Rocha et al.2015). Hence, our group has employed proteomic approaches with Paracoccidioides brasiliensis and identified potential virulence regulators, such as mitochondrial peroxiredoxin and vacuolar protease A (Castilho et al.2014). Several proteins involved in the response to oxidative stress were differentially expressed in virulent P. brasiliensis; thus, in the present study, we searched for the differences in antioxidants and oxidative stress responses of virulent and attenuated isolates. Here, we used the same P. brasiliensis strain (Pb18) with distinct degrees of virulence, one maintained in constant animal infections (virulent Pb18—vPb18) and other maintained for several years in culture (attenuated Pb18—aPb18). This strategy allows excluding genomic differences and pointing out which proteins were up-regulated in the virulent fungus compared to its attenuated counterpart as potential virulence factors. Our results suggest that P. brasiliensis possess an effective response to oxidative stress, and this feature is closely related to its virulence status. MATERIAL AND METHODS Fungal strain and growth conditions We used Paracoccidioides brasiliensis, Pb18 isolates, with different virulence profile in our experiments, as previously shown by Castilho et al. (2014). Long-term in vitro cultivated P. brasiliensis as a low virulent strain (attenuated Pb18—aPb18), which had been maintained for more than 5 years in our laboratory (Castilho et al.2014). In the present work, the virulence of aPb18 was restored after serial passages in Balb/c mice (4 passages). Briefly, 5 Balb/c mice (6–8 weeks of age) were intratracheally (i.t.) infected with 1 × 105 viable yeast cells from aPb18 in 30 μL of phosphate-buffered saline (PBS). After 30 days of infection, mice were euthanatized and lung, liver and spleen were aseptically removed. The organs were individually homogenized in 3 mL of PBS, and 100 μL of this suspension was plated in supplemented brain heart infusion agar (Singer-Vermes et al.1992). After 7 days, the fungal was recovered and other group of 5 Balb/c mice was re-infected using the recent isolated Pb18. This procedure was repeated at least 4 times (vPb18). Some experiments were performed with virulent Pb18 recovered after 2 passages in animals (aPb18-2x). These experiments are indicated in the legend of the figures. All mice were housed in pathogen-free conditions. The animals were handled according to National Institutes of Health—USA (NIH) (http://oacu.od.nih.gov/ARAC/index.htm). This study was approved by the Research Ethics Committee (CEP) of Federal University of São Paulo, Brazil under protocol number CEP 0349/12. Spot test Paracoccidioides brasiliensis (Pb18) oxidative stress sensitivity was investigated in yeast cells. Briefly, a total of 106 yeast cells/mL was exposed to different H2O2 concentrations (0.1, 1.0 or 10 mM) at 37°C for 5 h with constant shaking. After 5 h, each yeast culture was serially diluted (1:10 at each step) in modified YPD medium, mYPD (0.5% yeast extract, 1% casein peptone and 0.5% glucose, pH 6.5), and 10 μL of each suspension was spotted onto solid mYPD medium. Plates were photographed after 7 days of growth at 37°C. Experiments were performed in biological triplicates. Fungal viability To test the viability of P. brasiliensis yeast cells after treatment with H2O2, the cells (1 × 105) were seeded in 6-well culture plates, cultured with mYPD in the presence (0.1 or 1 mM) or absence of H2O2. Yeast cells were maintained at 37°C with constant stirring for 4 days. Cells were counted using a Neubauer chamber after staining with trypan blue. The experiment was performed in quadruplicate and repeated three times. Antioxidant enzymes activity assays To perform the enzymatic activities, protein extracts were prepared according the protocol of Villén, Beausoleil and Gygi (2008), with some modifications. Briefly, vPb18 and aPb18 isolates were grown in mYPD medium for 5 days at 37°C with constant agitation. Yeast cells were collected by centrifugation and 700 μL of cold lysis buffer (50 mM Tris-HCl [pH 7.5], 2 mM EDTA, 50 mM KCl, 0.2% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin and 10 mg/mL leupeptin) and glass beads (600 μm—Sigma, St. Louis, MO, USA) were added to the pellets. The yeast cells were disrupted mechanically using a vortex. Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the protein extracts according to the manufacturer's protocol. The fresh extracts were used to assess the enzymatic assay of both isolates. Total catalase activity was assayed using a spectrophotometer to measure the decrease in absorbance at 240 nm (Katsuwon, Zdor and Anderson 1993) immediately after preparing the extracts. One unit of catalase decomposed 1 millimole of H2O2 in 1 min at 25°C. Experiments were performed in biological triplicate, and catalase-specific activities were calculated using the extinction coefficient of 0.0394 mM−1 cm−1 (Aebi 1984). SOD enzymatic activity was measured according to Ewing and Janero (1995) using nitrotetrazolium blue chloride (Sigma-Aldrich), which produces a formazan dye upon reduction with O2•−, and the product can be determined by spectrophotometer at 560 nm. The Cu/ZnSOD activities from the cell lysates are expressed in units of SOD/mg, where 1 unit is the amount of SOD required to give 50% maximum inhibition of the initial rate of nitroblue tetrazolium reduction (Ewing and Janero 1995). Total intracellular glutathione was assessed as described by Rahman, Kode and Biswas (2006). The GSSG reductase recycling method was used to measured glutathione disulphide, the oxidised form (GSSG), by monitoring nicotinamide adenine dinucleotide phosphate spectrophometrically, and the glutathione (GSHtotal) assay was measured by the rate of formation of the 5΄-thio-2-nitrobenzoic acid chromophore at 412 nm. The amount of glutathione measured represents both forms in the sample, reduced and oxidised glutathione, and the amount of reduced glutathione (GSH) was measured using the expression [GSH]total = [GSH] + 2 × [GSSG]. The ratios of GSH/GSSG were used as reference values for the cellular redox environment. All enzymatic assays were performed in biological triplicate. RNA extraction and real-time quantitative RT-PCR analysis Total RNA was extracted from vPb18 and aPb18 yeast cells using the TRIzol reagent (Invitrogen), as described by Batista et al. (2007). All quantitative reverse transcription PCR (qRT-PCR) procedures were performed following the minimum information for publication of quantitative real-time PCR experiments guidelines (Bustin et al.2009). The quality of RNA was evaluated by electrophoresis in agarose gels. Next, 2.5 μg of total RNA was treated with DNAse (RQ1RNase-free DNase; Promega, Madison, WI, USA). Equal amounts of RNA were used for cDNA synthesis achieved with RevertAid Premium Reverse Transcriptase (Thermo Scientific, Bremen, GA, USA) according to the manufacturer's instructions. cDNA was used in a qRT-PCR reaction to measure the expression of genes related to the oxidative response; this reaction was performed using SYBR Green/ROX qPCR Master Mix 2X (Fermentas) according to the manufacturer's instructions. Briefly, a 10-μL total volume was used for each PCR reaction, which consisted of 1 × SYBR GreenPCR Master Mix, 250 nmol of the reverse primer, 250 nmol of the forward primer and 2 μL of cDNA. The cycling parameters were 50°C for 10 min, 95°C for 5 min and 40 cycles at 95°C for 30 s and 60°C for 1 min. A non-template control was used to detect any contamination. The quality of the reactions was based on the dissociation curves. The results obtained were then analysed for baseline and threshold cycle values (Ct) using the Step OnePlus software (Applied Biosystems). The baseline was adjusted to three or two cycles prior to the detection of the fluorescent signal, and the threshold cycle (Ct) was defined in the region of exponential amplification across all plots. A dilution series of reference cDNA template was prepared to determine the amplification efficiency. The relative expression ratio (experimental/control) was determined by the 2−ΔΔCt method (Schmittgen and Livak 2008) after normalisation to the level of the α-tubulin (α-TUB) and 18S transcript. The primers used in this work are summarised in Table 1. Experiments were performed in biological triplicate. Table 1. Sequence of qRT-PCR primers. IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  aAccession numbers from P. brasiliensis genes http://www.fungidb.org/. View Large Table 1. Sequence of qRT-PCR primers. IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  IDa  Gene  Primer sequence (5'→3')  PADG_04740  CATA  GCA CAA CGA AGT CCC TCA A CCG ACA TGG CCC ACA TAA A  PADG_00225  CATB  CTA CCA CGA CAA GAA GAC TAC CGG ATA TGA GAA CAG CGA CTT  PADG_00324  CATP  CCA GGG CAA CAA GAC CTT TA CGC TCG ATG GCT TTG AAT AGA  PADG_03095  PRX1  CCT GCA GAC AAC CGA TAA GA TCT TCA CAG CAG GAG GAA TG  PADG_04587  HYR1  AAG GCA AAG TCG TCC TCA TC GTA GTC GAG AGG GAG GTG TAG  PADG_05504  TRX1  AGC CAT CCC TAG TCG TCA TA GTT CGG GTA GGA TTC GGA AA  PADG_03161  TRX2  GAC ACG GAC GAA CAG CTA AT GGA GGT GAT CTG TAG CGA ATG  PADG_01551  TRR  GTG TTG GTG AGA CGG GAT AAA TCC ACC GCA ACC GTA TTG  PADG_07418  SOD1  ACC ACC AAA CCT ACG TAA ACT C GGG ATT TGA TAT CGG CCT TCT C  PADG_01755  SOD2  TCA ACC CGT TCG GCA AAT CCT GAG CGT CAG TGG TAA TG  PADG_01954  SOD3  GCT CCA GAG ACG AAG ATT GAG CAA TAG GGC CTC GTT GAA TTT G  PADG_06068  GR  GTT GAG ACG CAC ATG TTT ATC C GTG ACG CCC ATA TCC TCA TAC  PADG_01775  GST1  ATA ATG GCC AGG TCG ATA TGC GGC GAT TGG TCT GAG TGA TT  PADG_00697  GST2  GAA CCG CAA ACC CTA ATC CT GAG GAA GGT TGC GTA GTC TTT AT  PADG_03931  GST3  CGC AGT CTA CGT ACA GCA TTT AGC GGG AAT ATC CCA GTA CA  PADG_02218  GS  CAC ACG GAT CTC TTG TCA TCT C GAT GTG ACG GCG CTA TTC T  PADG_00128  α-TUB  CGG CAT ATG GAA AAT ACA TGG C GTC TTG GCC TTG AGA GAT GCA  PADG_12090  18S  CGG AGA GAG GGA GCC TGA GAA GGG ATT GGG TAA TTT GCG C  aAccession numbers from P. brasiliensis genes http://www.fungidb.org/. View Large Statistical analysis Data are expressed as the means ± SD. Significance was assessed by one-way analysis of variance with Student's t-test used for comparisons. The results with P < 0.05 were considered statistically significant. Prism 5 software (GraphPad Software Inc.) was used for the analyses and for graph construction. RESULTS Sensibility of P. brasiliensis Isolates (aPb18 and vPb18) to H2O2 treatment In a previous study, we performed a quantitative proteomic analysis of Paracoccidioides brasiliensis in the yeast (pathogenic) phase using a Pb18 strain with distinct infection profiles in Balb/c mice (vPb18 and aPb18). We demonstrated that some proteins involved in the response to oxidative stress were differentially expressed in the virulent isolate (Castilho et al.2014). Thus, to observe the sensitivity to oxidative stress of both isolates, P. brasiliensis yeast cells were exposed to different concentrations of H2O2 for 5 h at 37°C and then grown in solid YPD (mod). We observed the growth of the isolates under different stress conditions. The survival rate of vPb18 did not change when stimulated with different concentrations of H2O2 compared to the control (0 mM H2O2, Fig. 1A). In contrast, aPb18 was more sensitive to oxidative stress (Fig. 1A). Similar results were observed after culturing vPb18 and aPb18 under nitrosative stress (see Fig. S1, Supporting Information). In addition, the number of vPb18 yeast cells incubated with 0.1 or 1 mM of H2O2 responded with significant cell proliferation when compared to unstimulated control (0 mM H2O2, Fig. 1B), as previously described by Haniu et al. (2013). However, aPb18 yeast cells incubated with H2O2 (0.1 or 1 mM) showed a typical dose-response curve with decreased in cell viability (Fig. 1B). This shows that the vPb18 isolate presented an effective adaptive response to oxidative stress (ROS) compared with aPb18 (Fig. 1). Figure 1. View largeDownload slide Evaluation of the sensitivity to oxidative stress of P. brasiliensis isolates of varying virulence. (A) 2 × 106P. brasiliensis yeast cells (vPb18 or aPb18) were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. (B) Cultures of 1 × 104 viable yeast from P. brasiliensis were grown for 4 days in modYPD in presence H2O2 (0.1 and 1 mM), and viable cell counts using trypan blue were performed at specific points during cultivation. Error bars correspond to the standard deviations (SD) of measurements performed in triplicate. *P < 0.05; **P < 0.01 and ***P < 0.001. Figure 1. View largeDownload slide Evaluation of the sensitivity to oxidative stress of P. brasiliensis isolates of varying virulence. (A) 2 × 106P. brasiliensis yeast cells (vPb18 or aPb18) were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. (B) Cultures of 1 × 104 viable yeast from P. brasiliensis were grown for 4 days in modYPD in presence H2O2 (0.1 and 1 mM), and viable cell counts using trypan blue were performed at specific points during cultivation. Error bars correspond to the standard deviations (SD) of measurements performed in triplicate. *P < 0.05; **P < 0.01 and ***P < 0.001. The response to oxidative stress involves the participation of several proteins that modulate the redox environment, such as catalase, SOD and the glutathione system (Campos et al.2005). Thus, we measured the activities of antioxidant enzymes and the GSH and GSSG levels in both P. brasiliensis isolates. Catalase activity was assessed from the total protein extract and culture supernatant for both isolates. A high level of catalase-specific activity, both in the total extract (Fig. 2A) and in the culture supernatant (Fig. 2B), was detected from vPb18 grown in mYPD cultures from 5 days after inoculation. SOD activity was also increased in vPb18 compared to aPb18 (Fig. 2C). These results are consistent with the proteomic data previously obtained by Castilho et al. (2014), in which the enzymes peroxisomal catalase (PADG_00324) and SOD (PADG_04718) were up-regulated in vPb18 compared to aPb18. The increase in catalase activity in vPb18 is correlated with the resistance of this isolate to H2O2 treatment, suggesting that this enzyme plays a crucial role in the defence of this fungus against oxidative stress. Figure 2. View largeDownload slide Enzymatic activity of catalase and SOD in P. brasiliensis isolates of varying virulence. The catalase activities were measured using the total protein extract (A) and culture supernatant (B). The results are expressed as specific activity (units of catalase/μg protein or units of catalase/g yeast dry mass, respectively). Intracellular concentrations of SOD were determined using total protein extract (C). The results are expressed as specific activity (units of SOD/μg protein). All of the data shown in this figure were analysed using Student's t-test. Error bars correspond to the standard deviations of the measurements made in triplicate. *P < 0.05 and **P < 0.01. Figure 2. View largeDownload slide Enzymatic activity of catalase and SOD in P. brasiliensis isolates of varying virulence. The catalase activities were measured using the total protein extract (A) and culture supernatant (B). The results are expressed as specific activity (units of catalase/μg protein or units of catalase/g yeast dry mass, respectively). Intracellular concentrations of SOD were determined using total protein extract (C). The results are expressed as specific activity (units of SOD/μg protein). All of the data shown in this figure were analysed using Student's t-test. Error bars correspond to the standard deviations of the measurements made in triplicate. *P < 0.05 and **P < 0.01. Effect of the glutathione redox status in P. brasiliensis isolates (vP18 and aP18) The glutathione system is an important cellular buffering mechanism in response to changes in the cellular redox state. We evaluated the intracellular glutathione levels by analysing both reduced (GSH) and oxidised (GSSG) glutathione over 5 days of growth in vPb18 and aPb18 isolates (Fig. 3). GSH levels were significantly increased in vPb18 (approximately 2.5-fold) compared to its attenuated counterpart (aPb18) (Fig. 3A). On the other hand, GSSG levels were similar between the two isolates (Fig. 3B). The GSH/GSSG ratio is commonly used to estimate the redox state of biological systems. This ratio must be high for the maintenance of intracellular reducing power (Kappus 1987). The GSH/GSSG ratio was higher in the vPb18 isolate than in aPb18 (Fig. 3C). Analysis of the ratio GSH/GSSG suggested the presence of a reducing intracellular redox environment in vPb18 yeast cells. Protection against oxidative stress is related to the cellular capability of maintaining the cytosol in a more reduced state (Campos et al.2005). The enzymes participating in the glutathione system (glutathione reductase and glutathione peroxidase) did not show statistical significance in the activities of these enzymes between the isolates (see Fig. S2, Supporting Information); however, the glutathione reductase (GR) levels showed a moderate increase in vPb18. Figure 3. View largeDownload slide Activity assay results of the glutathione system assessed for yeast protein extracts. GSH (A) and GSSG levels (B) were run as described in Materials and Methods. The GSH/GSSG ratios (C) are the GSH levels divided by the GSSG levels. Student's t-test was used for statistical comparisons, and the observed differences were statistically significant (*P < 0.05). Error bars represent ±SD of n = 3. Figure 3. View largeDownload slide Activity assay results of the glutathione system assessed for yeast protein extracts. GSH (A) and GSSG levels (B) were run as described in Materials and Methods. The GSH/GSSG ratios (C) are the GSH levels divided by the GSSG levels. Student's t-test was used for statistical comparisons, and the observed differences were statistically significant (*P < 0.05). Error bars represent ±SD of n = 3. Expression levels of genes related to the antioxidant response in P. brasiliensis isolates of varying virulence Quantitative PCR was used for the transcriptional analysis of certain genes implicated in endogenous antioxidant defence, such as CATA, CATB and CATP (catalase A [PADG_ 04740], B [PADG_ 00225] and peroxisomal [PADG_ 00324], respectively); SOD1, SOD2 and SOD3 (cytoplasmic [PADG_07418 and PADG_01755] and mitochondrial SOD [PADG_01954], respectively); TRX1 and TRX2 (thioredoxin [PADG_05504 and PADG_03161], respectively); TRR (thioredoxin reductase [PADG_ 01551]); PRX1 (peroxiredoxin mitochondrial [PADG_03095]); and HYR1 (peroxiredoxine [PADG_04587]) and in glutathione metabolism, such as GS (γ-glutamylcysteine synthetase [PADG_02218]); GR (glutathione reductase [PABG_06068]); and GST1, GST2 and GST3 (glutathione transferase [PADG_01775, PADG_00697 and PAAG_03931, respectively]). At a late-exponential phase (5 days), the expression of all genes increased significantly in the vPb18 isolate, except for SOD3 (Fig. 4), with the highest levels found for CATA, HYR1, GST2 and AOX genes (12-, 7-, 7- and 15-fold, respectively) and the lowest for CATB, PRX1 and TRX1. Figure 4. View largeDownload slide Relative expression of the protein-encoding genes of vPb18 or aPb18 in P. brasiliensis yeast cells. Quantitative RT-PCR of RNA isolated from vPb18 and aPb18 grown in mYPD. The aPb18 expression was used as a calibrator. Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). Figure 4. View largeDownload slide Relative expression of the protein-encoding genes of vPb18 or aPb18 in P. brasiliensis yeast cells. Quantitative RT-PCR of RNA isolated from vPb18 and aPb18 grown in mYPD. The aPb18 expression was used as a calibrator. Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). To determine whether these genes are regulating during animal infection, we evaluated the expression of these genes before and after two passages of aPb18 in mice (Fig. 5A). This strategy had regained virulence in the Pb18 attenuated form, according to a previous study carried out by our group (Castilho et al.2014). After each animal passage, we observed an increase in the colony forming unit’s (CFU) number, and after second passage we found CFU values close to those obtained in vPb18 (data not shown). Expression analysis revealed that all antioxidant genes (except SOD3) were up-regulated in the attenuated strain that regained virulence and that was isolated after two passages in mice (aPb18-2x; Fig. 5B). We also observed the growth of the isolates (aPb18 and aPb18-2x) under different stress conditions. The survival rate of aPb18-2x did not change when stimulated with different concentrations of H2O2 in comparison to the control. In contrast, aPb18 maintained a profile of high sensitivity to oxidative stress induced by high concentrations of H2O2 (Fig. 5C). These results demonstrate that the expression of antioxidant genes can be considered potential virulence factors. Figure 5. View largeDownload slide Relative expression of the protein-encoding genes of aPb18 or aPb18–2x in P. brasiliensis yeast cells. (A) Schematic presentation of aPb18 isolate virulence recovery. Balb/c mice were intraperitoneally (i.p.) infected with 1 × 106 viable yeast cells of aPb18 in PBS. After 20 days the fungus was recovered of the mice (first passage—P1). This procedure was performed once more (second passage—P2). After the second animal passage, the gene expression levels of the isolate aPb18-2x were analysed. (B) Data represent changes in transcription between the aPb18 before and after two passages through animals (aPb18-2x). Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). (C) Evaluation of the sensitivity to oxidative stress in yeast cells of P. brasiliensis aPb18 or aPb18-2x. 2 × 106P. brasiliensis attenuated (aPb18) or aPb18-2x (aPb18 after two passages through animals to restore virulence) yeast cells were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. Figure 5. View largeDownload slide Relative expression of the protein-encoding genes of aPb18 or aPb18–2x in P. brasiliensis yeast cells. (A) Schematic presentation of aPb18 isolate virulence recovery. Balb/c mice were intraperitoneally (i.p.) infected with 1 × 106 viable yeast cells of aPb18 in PBS. After 20 days the fungus was recovered of the mice (first passage—P1). This procedure was performed once more (second passage—P2). After the second animal passage, the gene expression levels of the isolate aPb18-2x were analysed. (B) Data represent changes in transcription between the aPb18 before and after two passages through animals (aPb18-2x). Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated by the ΔΔCt method with two housekeepers (α-TUB and 18S). Fold change values (2ΔΔCT) were logarithmised to the base 2 (log2). Data are presented as fold changes in gene expression levels in the sample of interest normalised to the housekeepers and relative to the aPb18 sample. Error bars represent ±SD of n = 3 by Student´s t-test (*P < 0.05; **P < 0.01 and ***P < 0.001). (C) Evaluation of the sensitivity to oxidative stress in yeast cells of P. brasiliensis aPb18 or aPb18-2x. 2 × 106P. brasiliensis attenuated (aPb18) or aPb18-2x (aPb18 after two passages through animals to restore virulence) yeast cells were spotted onto mYPD supplemented with different H2O2 concentrations (0.1, 1 or 10 mM) and allowed to grow for 7 days at 37°C. DISCUSSION In a previous work, we analysed the proteomic profile of virulent and attenuated forms of the Paracoccidioides brasiliensis Pb18 strain, and we identified that some proteins that engage in the response to oxidative stress were up-regulated in virulent Pb18 as compared with attenuated strain (Castilho et al.2014). The ability of pathogenic fungi to cause disease entails their capability to survive in the host. Once inside the host, the fungi are challenged by oxidative stress from phagocytic cells, mainly from neutrophils and macrophages (Robinson, Ohira and Badwey 2004; Gazendam et al.2016). The survival of these microorganisms in phagocytic cells is strongly associated with putative virulence factors (Shiloh and Nathan 2000). Pathogenic fungi have developed strategies to survive in hostile conditions, such as the expression of genes related to oxidative stress. Campos et al. (2005) reported genes that could be potentially involved in the fungal oxidative stress response, such as catalase, SOD, peroxiredoxins, thioredoxin and the glutathione system. Thus, in the present study, we analysed the oxidative stress response of the Pb18 strain with distinct virulence profiles. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18) and this process positively modulated the fungus's antioxidant repertoire. Herein, we demonstrated that enzymatic activity of catalase and SOD; and GSH levels were higher in vPb18 than aPb18 (Fig. 2), suggesting that this systems may be involved with the fungal virulence. Our data suggest that Pb18 adaptation to the host depend on their ability to increase the expression of antioxidant system. After two animal passages, aPb18 increased the expression of some antioxidant enzymes (Fig. 5) and the CFU’s number (data not shown). In addition, vPb18 cultured in vitro had a decrease in antioxidant enzyme expression as their ability to infect animals (data not shown). These data demonstrate that Pb18 strain is capable of rapidly adapting in host conditions by altering the expression of antioxidant enzymes. Our results are consistent with previous studies that link antioxidant enzymes and virulence (Tamayo et al.2016; Mittra et al.2017; Tamayo et al.2017; Missall and Lodge 2005). These findings reinforce the concept that is very important to maintain Paracoccidioides spp strains freshly isolated from serial passages in animals to perform in vitro or in vivo experiments (Brummer et al.1990). Recent work showed that Galleria mellonella can be an alternative model to recover Paracoccidioides spp virulence factors (Scorzoni et al.2017). Changes in morphology, biochemistry profile and expression of surface markers were reported in Paracoccidioides spp strains before and after animal passage (Svidzinski et al.1999; Kioshima et al.2011). However, this is the first work that clearly shows that the adaptation of the fungus to the oxidative stress of the host, by increasing the expression and activity of antioxidant enzymes, may also be directly related to the virulence of the pathogen. The role of antioxidant system has been studied in pathogenic fungi. Catalase is one of the enzymes responsible for consuming H2O2, which protects the cellular environment against oxidative stress by reducing the harmful effect of H2O2 (Chagas et al.2010). In P. brasiliensis, three members of the catalase family were also found, PbCatA, PbCatC and PbCatP. The PbCATP transcript was increased after co-cultivation with murine macrophages, suggesting the importance of PbCATP in the response to exogenous oxidative stress (Chagas et al.2008). Tamayo et al. (2017) demonstrated that PbCATP expression is increased in yeast cells treated with H2O2, and they proposed that PbCATP enzymatic activity promotes protection against endogenous and exogenous H2O2. The increase of catalase activity is correlated with the resistance of P. brasiliensis to H2O2 treatment, suggesting that this enzyme plays a crucial role in the defence of the fungus against oxidative stress (Dantas et al.2008). Besides catalase, SOD also presents the first line of antioxidant defence against ROS. SODs are metallo-proteins and neutralise the toxic levels of ROS generated by the host, contributing to the virulence of some pathogenic fungi (Tamayo et al.2016). In P. brasiliensis, four genes corresponding to two SOD isoenzymes were found (Campos et al.2005). Interestingly, in our data the transcript level of SOD2 was up regulated in vPb18 but to SOD3 was observed a down-regulation in their expression. SOD3 is associated with manganese ions, and MnSOD is an important isoenzyme for preventing oxidative stress in mitochondria (Luk et al.2005). Proteomic analysis revealed that aPb18 exhibited a higher aerobic metabolism compared to vPb18 (Castilho et al.2014); thus, the up-regulation of MnSOD in aPb18 could play an important role in protecting against respiration-derived ROS. The same result has been reported by Rezende et al. (2011). Paracoccidioides lutzii mycelia possessed greater aerobic metabolism compared to the yeast phase, and MnSOD was up-regulated in this phase. In addition to catalase and SOD, fungi possess alternative pathways to maintain the intracellular redox balance. AOX have been reported in human pathogenic fungi, such as Cryptococcus neoformans and Aspergillus fumigatus, as an important enzyme that contributes to the adaptation of fungal within phagocytic cells (Akhter et al.2003; Magnani et al.2008). In P. brasiliensis, yeast with decreased PbAOX expression had increased susceptibility to INF-γ-activated alveolar macrophages, which were able to produce ROS in response to fungal infection (Ruiz et al.2011). Additionally, in a mouse model of infection, the reduction in PbAOX expression increased the survival of the mice (Ruiz et al.2011). Our results demonstrated an increase in PbAOX expression in vPb18 and aPB18-2x, supporting the relevance of PbAOX in the virulence of P. brasiliensis. The antioxidant machinery involves detoxifying enzymes and metabolites. Glutathione is an essential metabolite and plays a key role in cellular resistance to oxidative damage (Penninckx 2002). The glutathione system can provide protection against oxidative stress by maintaining the cytoplasm of the cells in a reduced state. The GSH/GSSG ratio reflects intracellular redox changes (Guedouari et al.2014). Reduced glutathione (GSH) reduces the molecules containing an unpaired electron, resulting in the oxidised form of glutathione (GSSG; Izawa, Inoue and Kimura 1995). The enzymatic activity of the glutathione system indicates that the intracellular reducing potential of vPb18 is higher compared to its attenuated counterpart. Thus, this could be an additional mechanism for protecting vPb18 from oxidative stress conditions in host phagocytic cells. The mRNA levels for the genes encoding glutathione synthetase and glutathione S-transferases (GSTs) also showed increased expression in vPb18. Weber et al. (2012) demonstrated that GST was preferentially secreted into Paracoccidioides lutzii yeasts compared to the mycelial secretoma. GSTs represent a group of detoxifying enzymes that are involved in the protection against oxidative stress and detoxification of xenobiotics and heavy metals (Hayes and Strange 1995). In Saccharomyces cerevisiae GSH synthetase mutants were sensitive to H2O2. These mutants, when treated with 4 mM H2O2 for 1 h, had a 2-fold lower survival rate compared to the wild-type yeast (Grant, Perrone and Dawes 1998). In addition, GR is involved in the stabilisation of the pool of reduced glutathione, an important antioxidant in the cell (Lushchak 2011; Townsend, Lushchak and Cooper 2014). Thus, increased GR expression in vPb18 would explain the high GSH levels in the virulent isolate. The survival of P. brasiliensis to the host implies in cellular activity very different from that necessary for their survival in the culture medium. Evidence from this and other works suggest that this adaptation involves dramatic alteration in the expression of genes, especially enzymes involved with defence mechanisms against the oxidative stress imposed mainly by host phagocytic cells. After animal passages, aPb18 increased the virulence and the expression of several genes involved in the response to oxidative stress. Thus, although speculative, the link between virulence and the expression of such enzymes cannot be ruled out. The findings presented in this work supports the relevance of using Paracoccidioides spp isolated after serial passages in animal to study virulence, pathogenicity, biochemistry and drug discovery. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. ACKNOWLEDGEMENT We are thankful to Adriano Sartori for generous and competent technical assistance. 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FEMS Yeast ResearchOxford University Press

Published: Mar 1, 2018

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