Therapeutic treatment of PCM.

<p>Gene immunization started 30 days after infection. CFUs are from lungs of BALB/c (□) and B10.A(▪) mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) or IL-12 (pIL-12) DNA inserts. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 60 days after infection. Each bar represents the average counts and standard deviations of CFU in lungs from 10 animals in each group. Experiments were performed three times and similar results were achieved. <b>*</b><i>p</i>≤0.05, <b>**</b><i>p</i>≤0.005, comparing vector with and without insert; ^##<i>p</i>≤0.005, comparing untreated and other groups.</p

Summary of the treatment protocols used.

<p><b>First protocol</b>: BALB/c mice received 4 weekly injections. Animals were infected and sacrificed 30 or 60 days later. <b>Second protocol</b>: BALB/c and B10.A mice were infected intratracheally. Mice received 4 weekly vaccine doses and animals were sacrificed 1 week after the last injection. <b>Third protocol</b>: B10.A mice were infected i.t. and one month after infection, they were immunized with the DNA vaccine. The animals were sacrificed one month after the last injection, six months after infection.</p

Cytokines in lung homogenates of B10.A mice infected i.t. with <i>P. brasiliensis</i> Pb 18 yeasts and submitted to gene immunization for 5 months.

<p>Mice were sacrificed 1 month after the last dose.</p

Immunoprophylactic treatment of PCM.

<p>Gene immunization was initiated 30 days before fungal challenge. CFUs are from lungs of BALB/c mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) and/or IL-12 (pIL-12) DNA insert. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 30 (▪) and 60 (□) days after infection. Each bar represents the average counts and standard deviations of CFUs in lungs from 10 animals in each group. Experiments were carried out in triplicate with similar results. <b>**</b><i>p</i>≤0.0001, comparing vector with and without insert; ^##<i>p</i>≤0.0001, .comparing untreated and other groups.</p

Histopathology of lungs from intratracheally infected B10.A mice.

<p>Animals were infected with <i>P. brasiliensis</i> for one month, treated with or without vectors carrying P10 or IL-12 DNA inserts according to protocol 3, and sacrificed 6 months after the initial infection. Infected mice treated with (<b>A</b>) control pcDNA3, (<b>B</b>) pP10, (<b>C</b>) pIL-12 DNA, and (<b>D</b>) P10 and IL-12 DNA. Gomori staining; original magnification, 40×.</p

Long term therapeutic treatment of experimental PCM.

<p>Gene immunization started 30 days after infection and mice were sacrificed 6 months after infection. CFUs were counted in lungs of B10.A mice infected intratracheally with 3×10⁵ yeast cells and immunized with vectors containing the insert encoding P10 (pP10) or IL-12 (pIL-12). Control mice were inoculated with PBS or with vector without insert. Each bar represents the average counts and standard deviations of CFU in lungs from 5 to 10 animals in each group. Experiments were carried out in triplicate, with similar results. <b>**</b><i>p</i>≤0.001, comparing vector with and without insert; ^##<i>p</i>≤0.001, comparing untreated and other groups.</p

Exposure of SOD knockdown strains to ROS-inducing agents.

<p><b>(A)</b> Quantification of the sensitivity to H₂O₂ in <i>P</i>. <i>brasiliensis</i> yeast cells. The zone of inhibition was determined as the area lacking yeast cell growth. Data presented are the average for three replicate tests, with error bars representing standard deviations. Asterisks represent significant differences from the wild-type strain as determined by Student’s <i>t</i> test. <b>(B)</b> Images of <i>P</i>. <i>brasiliensis</i> growth around H₂O₂ saturated disks. Saturated filter disks containing 1M H₂O₂<b>. (C)</b> Sensitivity to menadione of <i>PbSOD1</i> and <i>PbSOD3</i> mutants. We used a 96-well plate in which a total of 1X10⁴ yeast cells of each isolate were inoculated on BHI containing increased amounts of menadione (0.5, 5, 10, 20, 40, 80 and 160 μM). The plates were incubated at 36°C, with 5% of CO₂ for 8 days. The red box indicates the concentration at which no growth in <i>PbSOD1</i>-aRNA and <i>PbSOD3</i>-aRNA occurred, contrary wise to PbWT60855 in which we observed growth. <b>(D)</b> Survival of <i>P</i>. <i>brasiliensis</i> yeast cells following challenge with xanthine oxidase. Yeast cells were incubated for 4h at 36°C in the presence of hypoxanthine 100 μM and xanthine oxidase 5 mU/ml in order to generate superoxide anions. After incubation time passed, viable CFUs were determined. Results are the mean of three individual experiments. Asterisk denotes <i>P</i> ≤0.05 compared to PbWT and PbEV as determined by Student’s <i>t</i> test.</p

SODs of <i>Paracoccidioides</i>.

<p>SODs of <i>Paracoccidioides</i>.</p

Silencing of <i>SOD1</i> and <i>SOD3</i> in <i>P</i>. <i>brasiliensis</i>.

<p><b>(A)</b> Transfer DNA (T-DNA) inserted into the genome of <i>P</i>. <i>brasiliensis</i> yeast cells via ATMT in order to silence <i>PbSOD3</i> gene/protein. The antisense oligonucleotide was directed to exon 1 (dashed box), with a length of 104 bp. This AS oligonucleotide was placed under control of the calcium binding protein (<i>CBP</i>-1), the terminator (<i>CAT</i>-B) and harbored hygromycin B phosphotransferase (<i>HPH</i>) under control of glyceraldehyde 3-phosphate of <i>Aspergillus nidulans</i> (<i>PGPDA</i>) and with the terminator (<i>TTRCP</i>). <b>(B)</b> Gene expression levels of <i>PbSOD3</i> obtained by RT-qPCR assay. The measurement was normalized with the housekeeping gene β-tubulin in PbWT, PbEV and Pb<i>SOD3</i>-aRNA yeast cells grown at exponential phase. Mitotic stability was confirmed by sub-culturing <i>P</i>. <i>brasiliensis PbSOD3</i>-aRNA yeast cells, checking for low expression levels in this isolate after successive sub-cultures. Results are the mean of three individual experiments. Asterisk represent significant differences compared to PbWT and PbEV as determined by Student’s <i>t</i> test. <b>(C)</b> Growth curve in PbWT60855, PbEV60855, <i>PbSOD1</i>-aRNA and <i>PbSOD3</i>-aRNA. Yeast cells were grown in BHI liquid medium at 36°C, OD600 nm was determined along each time point. <b>(D)</b> Vitality in PbWT60855, PbEV60855, <i>PbSOD1</i>-aRNA and <i>PbSOD3</i>-aRNA. The pH in yeast suspensions was monitored at three minutes intervals for 30 minutes.</p

Isolates used in this study.

<p>Isolates used in this study.</p