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

Alternative Oxidase Mediates Pathogen Resistance in Paracoccidioides brasiliensis Infection

Thermally dimorphic pathogenic fungi are responsible for potentially life-threatening diseases of immunocompetent and immunocompromised individuals. These microorganisms grow as conidia-producing mycelia in the environment, which when inhaled by the host convert to the pathogenic yeast form at 37°C. During adaptation and growth, fungi interact with host immune cells and must cope with defense mechanisms such as imposed-oxidative stress (e.g., reactive oxygen species; ROS). Alternative oxidase (AOX) is an enzyme recently implicated in the reduction of ROS production by the mitochondria when triggered by external stimuli, such as temperature and ROS. During this work we have evaluated the relevance of AOX during infection with Paracoccidioides brasiliensis, the etiological agent of one of the most prevalent mycoses in Latin America, paracoccidioidomycosis. We show that PbAOX gene expression is stimulated after interaction with alveolar macrophages or in the presence of H2O2 and is essential for survival against fungicidal activity of both the immune cells and the ROS compound. Moreover, decreasing PbAOX gene expression in P. brasiliensis led to increased survival of infected mice. Altogether, our data supports a relevant role for AOX in the virulence of P. brasiliensis

<i>PbAOX</i> plays an important role in the detoxification of H₂O₂.

<p>(A) Cell viability of PbWt and <i>PbAOX-aRNA</i> after incubation for 1 hour at different concentrations of H₂O₂. (B) <i>PbAOX</i> gene expression after incubation for 1 hour at different concentrations of H₂O₂ (gene expression levels obtained by RT-PCR were normalized with the internal reference, TUB2; <i>*</i> indicates <i>P</i><0.05 when compared with <i>PbAOX-aRNA</i> strains).</p

<i>PbAOX</i> expression during infection of alveolar macrophages is essential to maintain fungal viability.

<p><i>PbAOX</i> expression of cells growing in culture medium (w/o MH-S) and in the presence of non-activated (MH-S-na) or IFN-g-activated (MH-S-a) alveolar macrophages: A. PbWt yeast cells; B. PbWt conidia; C. <i>PbAOX</i>-aRNA yeast cells; D. <i>PbAOX</i>-aRNA conidia. <i>* P</i><0.05 when compared with non-activated macrophages.</p

<i>PbAOX</i> expression during infection of alveolar macrophages is essential to maintain fungal viability.

<p>Fungal viability of cells growing in culture medium (w/o MH-S) and in the presence of non-activated (MH-S-na) or IFN-g-activated (MH-S-a) alveolar macrophages: A. PbWt yeast cells; B. PbWt conidia; C. <i>PbAOX</i>-aRNA yeast cells; D. <i>PbAOX</i>-aRNA conidia. <i>* P</i><0.05 when compared with non-activated macrophages.</p

Generation of a <i>P. brasiliensis PbAOX-</i>aRNA strain.

<p>(A) T-DNA construct for aRNA silencing of <i>PbAOX</i> in <i>P. brasiliensis</i> via ATMT. <i>PbAOX-</i>aRNA oligonucleotides AS1 (base pairs 1 to 100 of <i>PbAOX</i>; exon 1), AS2 (base pairs 153 to 260 of <i>PbAOX</i>; exon 2), and AS3 (base pairs 99 to 260 of <i>PbAOX</i>; exon 3) were amplified, individually placed under the control of the calcium-binding protein (<i>CBP1</i>) promoter, and later on inserted into the T-DNA of the binary vector pUR5750 for ATMT of <i>P. brasiliensis</i>. (B) Gene expression levels of <i>PbAOX</i> in PbWt, PbWt transformed with the empty vector (PbWt+EV), and <i>PbAOX</i>-aRNA yeast cells after subculture for 15, 30, 60 and 120 days (gene expression levels obtained by RT-PCR were normalized with the internal reference, TUB2; <b>*</b>: <i>p</i><0.05 when compared with PbWt and PbWt+EV).</p

Silencing of <i>PbAOX</i> decreases virulence of <i>P. brasiliensis</i> in a murine model of infection.

<p>A. <i>P. brasiliensis</i> colony-forming units (CFU) recovered from pulmonary tissues (n = 8 mice per periods). CFU are expressed as log₁₀ per g. of lung. H: hours; w: weeks. <i>* P</i><0.05 when compared with 3 days after infection. B. Silencing of <i>PbAOX</i> decreases virulence of <i>P. brasiliensis</i> in a murine model of infection. Representative survival plot of an experimental infection carried out in BALB/c mice (n = 10 mice) challenged i.n. with 4×10⁶ PbWt or <i>PbAOX</i>-aRNA conidia cells (<i>P</i><0.0001 when compared with mice infected with PbWt conidia).</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