Transcriptome Profile of the Response of Paracoccidioides spp. to a Camphene Thiosemicarbazide Derivative

Abstract

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.

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

“…To further analyze the function of SOD1 and SOD3 in P . brasiliensis , we used an antisense-RNA (aRNA) knockdown strain for SOD1 ( PbSOD1 -aRNA), which was previously constructed [ 51 ] ( S4 Fig ) and a PbSOD3 -aRNA knockdown strain constructed in this study using ATMT. Briefly, one region of the gene was selected in order to design two different aRNA oligonucleotides and generate knockdown strains.…”

“…DNA from Pb60855 was extracted from yeast cells cultures during exponential growth. We employed a Platinum high-fidelity Taq DNA polymerase (Invitrogen) to amplify aRNA oligonucleotides, designed on the sequences identified as PADG_07418 for PbSOD1 gene [ 51 ] and PADG_02842 for PbSOD3 gene. Gene sequences of the strain Pb03 were obtained from the Paracoccidioides genome database [ 30 , 31 ].…”

Argentilactone Molecular Targets in Paracoccidioides brasiliensis Identified by Chemoproteomics

“…Studies have shown the adaptation of Paracoccidioides spp. to stresses such as osmotic stress (31), oxidative stress (32), nutrient deprivation (33,34), cell wall stressor agents (35), and drugs (36)(37)(38)(39), suggesting prospects for the research and development of new antifungals.…”

Effects of Argentilactone on the Transcriptional Profile, Cell Wall and Oxidative Stress of Paracoccidioides spp.

“…The argentilactone sensitivity assay was performed from three independent experiments using P . brasiliensis , Pb 339, Pb 60855, Pb 60855EV, Pb 339EV and the silenced mutants for superoxide dismutase (SOD) protein ( Pbsod -aRNA) [ 14 ], heat shock protein HSP90 ( Pbhsp90 -aRNA) [ 24 ], cytochrome C peroxidase protein ( Pbccp -aRNA) [ 25 ] and hemoglobin ligand protein RBT5 ( Pbrbt5 -aRNA) [ 26 ] were grown in liquid Fava-Netto for 72 h with agitation at 150 rpm at 36°C, and then transferred to MMcM liquid medium, where they remained overnight. The cells were then washed with phosphate buffered saline (PBS 1X) and diluted to concentrations of 10 4 , 10 5 and 10 6 .…”

“…In this way, our group has identified new antifungal targets, such as the enzymes 1,3-β-D-glucan synthase [ 8 , 9 ], malate synthase [ 10 , 11 ], isocitrate lyase [ 12 ] and (S)-adenosyl-L-methionine: C24 sterol methyl transferase [ 13 ], from Paracoccidioides spp. In addition, we have investigated new antifungal compounds, including thiosemicarbazide [ 14 ] and oenothein [ 15 , 16 ]. Here, as part of a continuing search for diverse chemicals from plants, we have examined argentilactone, a bioactive metabolite isolated from Hyptis ovalifolia , which is renowned for its wide range of anticancer, insecticidal and antimicrobial activities [ 17 , 18 ], including those against P .…”