Transcriptome profile of the response of paracoccidioides spp. to a camphene thiosemicarbazide derivative

ABSTARCT: Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment

Functional classification of up and down-regulated genes from <i>P</i>. <i>lutzii</i> yeast cells in the presence TSC-C.

<p>Genes in bold correspond to single genes in condition TSC-C. The signs + and - represent induced and repressed genes, respectively.</p><p>Functional classification of up and down-regulated genes from <i>P</i>. <i>lutzii</i> yeast cells in the presence TSC-C.</p

Formation of ROS by TSC-C.

<p>(A) Fluorescence microscopy of <i>P</i>. <i>lutzii</i> yeast cells stained with 2`,7`-dichlorofluorescein diacetate. Yeast cells were grown in the absence of TSC-C for <b>i)</b> 4 h, <b>ii)</b> 8 h and <b>iii)</b> 12 h and in the presence of TSC-C for <b>iv)</b> 4 h, <b>v)</b> 8 h and <b>vi)</b> 12 h. <b>(B)</b> Flow cytometry analysis of yeast cells grown in the absence or in the presence of TSC-C. The cells were monitored for <b>i)</b> 4 h, <b>ii)</b> 8 h and <b>iii)</b> 12 h stained with 2`,7`-dichlorofluorescein diacetate. Black histograms represent control yeast cells, and green histograms represent yeast cells treated with TSC-C.</p

Susceptibility of <i>P</i>. <i>brasiliensis SOD1</i>-aRNA to TSC-C.

<p>1x10⁶ yeast cells of <i>P</i>. <i>brasiliensis</i> WT60855, EV60855 and <i>SOD1</i>-aRNA were spotted on solid BHI supplemented with 39.5, 79 and 158 μM TSC-C. Control cells were spotted on BHI without TSC-C or with 39.5, 79 and 158 μM TSC-C and ascorbic acid. The plates were incubated for 7 days at 36°C before photo documentation.</p

Effect of TSC-C on <i>P</i>. <i>lutzii</i> yeast cell growth.

<p><b>(A)</b> Inhibition of <i>Paracoccidioide</i>s cell growth after treatment with TSC-C. The inhibition was visualized by addition of resazurin reagent to culture and measuring the absorbance at 600 nm. To calculate the IC₅₀ value, two absorbance readings were performed; ‘1° day’ refers to reading at the beginning of the experiment, ‘3° days’ refers to reading after 3 days of incubation with 316 μM, 158 μM, 79 μM and 39.5 μM TSC-C. The positive control was performed in the absence of the compound. <b>(B</b>) Cell viability after 1, 2, 3, 4, 8 and 24 h exposure to TSC-C. The data are presented as percentage of cell viability. The Student’s <i>t</i>-test was used for statistical comparisons, and the observed differences were statistically significant (<i>p</i> ≤ 0.05). The error bars represent the standard deviation of three biological replicates.</p

Susceptibility of <i>P</i>. <i>lutzii</i> yeast cells exposed to TSC-C.

<p>Samples containing 1x10⁷, 1x10⁶ and 1x10⁵ yeast cells were spotted on Fava-Netto plates supplemented with TSC-C at the concentrations indicated above. The plates were incubated for 7 days at 36°C before photo documentation.</p

Oligonucleotide primers used in qRT-PCR.

<p>Oligonucleotide primers used in qRT-PCR.</p

Effect of TSC-C on the mitochondrial membrane potential of <i>P</i>. <i>lutzii</i>.

<p>The mitochondrial membrane potential (ΔΨm) was determined by flow cytometry analysis of yeast cells treated with TSC-C for <b>A)</b> 4 h, <b>B)</b> 8 h and <b>C)</b> 12 h and stained with rodhamine123. Histograms in black represent the controls, and red histograms represent cells treated with TSC-C.</p

Effect of TSC-C on the <i>P</i>. <i>lutzii</i> cell cycle.

<p>The DNA content of yeast in each cell cycle phase was analyzed by flow cytometry in the absence of TSC-C for <b>A)</b> 4 h, <b>B)</b> 8 h and <b>C)</b> 12 h or in the presence of TSC-C for <b>D)</b> 4 h, <b>E)</b> 8 h and <b>F)</b> 12 h and subsequently stained with ethidium iodide as represented by histograms.</p

Statistically enriched MIPS functions.

<p><b>(A)</b> Total ESTs represented by classified and unclassified categories. <b>(B)</b> Genes expressed differentially in the presence of camphene thiosemicarbazide derivate. Up- <b>(C)</b> or down- <b>(D)</b> regulated <i>P</i>. <i>lutzii</i> genes after exposure of yeast cells to TSC-C. The functional classification was based on the MIPS functional annotation scheme. Each functional class is represented as a color-coded segment and expressed as a percentage of the total number of ESTs.</p