Paracoccidioides spp. catalases and their role in antioxidant defense against host defense responses.

Dimorphic human pathogenic fungi interact with host effector cells resisting their microbicidal mechanisms. Yeast cells are able of surviving within the tough environment of the phagolysosome by expressing an antioxidant defense system that provides protection against host-derived reactive oxygen species (ROS). This includes the production of catalases (CATs). Here we identified and analyzed the role of CAT isoforms in Paracoccidioides, the etiological agent of paracoccidioidomycosis. Firstly, we found that one of these isoforms was absent in the closely related dimorphic pathogen Coccidioides and dermatophytes, but all of them were conserved in Paracoccidioides, Histoplasma and Blastomyces species. We probed the contribution of CATs in Paracoccidioides by determining the gene expression levels of each isoform through quantitative RT-qPCR, in both the yeast and mycelia phases, and during the morphological switch (transition and germination), as well as in response to oxidative agents and during interaction with neutrophils. PbCATP was preferentially expressed in the pathogenic yeast phase, and was associated to the response against exogenous H2O2. Therefore, we created and analyzed the virulence defects of a knockdown strain for this isoform, and found that CATP protects yeast cells from H2O2 generated in vitro and is relevant during lung infection. On the other hand, CATA and CATB seem to contribute to ROS homeostasis in Paracoccidioides cells, during endogenous oxidative stress. CAT isoforms in Paracoccidioides might be coordinately regulated during development and dimorphism, and differentially expressed in response to different stresses to control ROS homeostasis during the infectious process, contributing to the virulence of Paracoccidioides.

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

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.

Alternative oxidase plays an important role in Paracoccidioides brasiliensis cellular homeostasis and morphological transition.

Paracoccidioides brasiliensis is the etiologic agent of one of the most common systemic mycoses in Latin America. As a dimorphic fungus, it must adapt to different environments during its life cycle, either in nature or within the host, enduring external stresses such as temperature or host-induced oxidative stress. In this study we addressed the role of alternative oxidase (PbAOX) in cellular homeostasis during batch culture growth and the morphological transition of P. brasiliensis. Using a PbAOX-antisense-RNA (PbAOX-aRNA) strain with a 70% reduction in gene expression, we show that PbAOX is crucial for maintaining cell viability and vitality during batch culture growth of yeast cells, in what appears to be a pH-dependent manner. We also show that silencing of PbAOX drastically reduced expression levels of other detoxifying enzymes (PbY20 and PbMSOD). In addition, our data indicate that PbAOX plays a role during the morphological transition, namely, during the yeast-to-mycelia germination and mycelia/conidia-to-yeast transition, essential events during the establishment of infection by dimorphic fungal pathogens. Altogether, our findings support the hypothesis that PbAOX is important for the maintenance of cellular homeostasis, possibly by assisting redox balancing during cell growth and the morphological switch of P. brasiliensis.

Involvement of the 90 kDa heat shock protein during adaptation of Paracoccidioides brasiliensis to different environmental conditions.

HSP90 is a molecular chaperone that participates in folding, stabilization, activation, and assembly of several proteins, all of which are key regulators in cell signaling. In dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, the adaptation to a higher temperature, acid pH and oxidative stress, is an essential event for fungal survival and also for the establishing of the infectious process. To further understand the role of this protein, we used antisense RNA technology to generate a P. brasiliensis isolate with reduced PbHSP90 gene expression (PbHSP90-aRNA). Reduced expression of HSP90 decreased yeast cell viability during batch culture growth and increased susceptibility to acid pH environments and imposed oxidative stress. Also, PbHSP90-aRNA yeast cells presented reduced viability upon interaction with macrophages. The findings presented here suggest a protective role for HSP90 during adaptation to hostile environments, one that promotes survival of the fungus during host-pathogen interactions.

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42.

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.

Gene knockdown in Paracoccidioides brasiliensis using antisense RNA.

Paracoccidioides brasiliensis is a thermal dimorphic fungus which in the host environment exhibits a multinucleated and multibudding yeast form. The cellular and molecular mechanisms underlying these phenotypes remain to be clarified, mostly due to the absence of efficient classical genetic and molecular techniques. Here we describe a method for gene expression knockdown in P. brasiliensis by antisense RNA (aRNA) technology taking advantage of an Agrobacterium tumefaciens-mediated transformation (ATMT) system. Together, these techniques represent a reliable toolbox that can be employed for functional genetic analysis of putative virulence factors and morphogenic regulators, aiming to the identification of new potential drug targets.

The hydrolase PbHAD32 participates in the adherence of Paracoccidioides brasiliensis conidia to epithelial lung cells.

Adherence of the dimorphic pathogenic fungus Paracoccidioides brasiliensis to lung epithelial cells is considered an essential event for the establishment of infection. We have previously shown that the PbHAD32 hydrolase is important in this early stage of the host-P. brasiliensis yeast cells interaction. The aim of this study was to further elucidate the role of PbHAD32 in conidial thermodimorphism and their interaction with lung epithelial cells. Analysis of the PbHAD32 gene expression revealed higher mRNA levels during the conidia to mycelia (C-M) germination when compared to the conidia to yeast (C-Y) transition. Moreover, PbHAD32 was consistently expressed at higher levels upon infection of lung epithelial cells, but to a greater extent when conidia germinated to produce mycelia. Interestingly, at this particular transitional stage, more conidia adhered to epithelial cells than when they were transiting to the yeast form. Altogether our data further corroborates the importance of PbHAD32 during initial adherence to host cells and suggest that the 32-KDa hydrolase may also participate at different stages of the C-M and C-Y conversions.

The C allele of rs5743836 polymorphism in the human TLR9 promoter links IL-6 and TLR9 up-regulation and confers increased B-cell proliferation.

In humans, allelic variants in Toll-like receptors (TLRs) associate with several pathologies. However, the underlying cellular and molecular mechanisms of this association remain largely unknown. Analysis of the human TLR9 promoter revealed that the C allele of the rs5743836 polymorphism generates several regulatory sites, including an IL-6-responding element. Here, we show that, in mononuclear cells carrying the TC genotype of rs5743836, IL-6 up-regulates TLR9 expression, leading to exacerbated cellular responses to CpG, including IL-6 production and B-cell proliferation. Our study uncovers a role for the rs5743836 polymorphism in B-cell biology with implications on TLR9-mediated diseases and on the therapeutic usage of TLR9 agonists/antagonists.

Alternative oxidase mediates pathogen resistance in Paracoccidioides brasiliensis infection.

BACKGROUND: Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells. AIMS AND METHODOLOGY: In this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell's viability and PbAOX in the presence of H2O2. RESULTS: Interaction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H2O2, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells. CONCLUSIONS: These data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis.

A 32-kilodalton hydrolase plays an important role in Paracoccidioides brasiliensis adherence to host cells and influences pathogenicity.

One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.

Yeast protein expression profile during acetic acid-induced apoptosis indicates causal involvement of the TOR pathway.

Although acetic acid has been shown to induce apoptosis in yeast, the exact apoptotic mechanisms remain unknown. Here, we studied the effects of acetic acid treatment on yeast cells by 2-DE, revealing alterations in the levels of proteins directly or indirectly linked with the target of rapamycin (TOR) pathway: amino-acid biosynthesis, transcription/translation machinery, carbohydrate metabolism, nucleotide biosynthesis, stress response, protein turnover and cell cycle. The increased levels of proteins involved in amino-acid biosynthesis presented a counteracting response to a severe intracellular amino-acid starvation induced by acetic acid. Deletion of GCN4 and GCN2 encoding key players of general amino-acid control (GAAC) system caused a higher resistance to acetic acid indicating an involvement of Gcn4p/Gcn2p in the apoptotic signaling. Involvement of the TOR pathway in acetic acid-induced apoptosis was also reflected by the higher survival rates associated to a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-negative phenotype and lower reactive oxygen species levels of Deltator1 cells. In addition, deletion mutants for several downstream mediators of the TOR pathway revealed that apoptotic signaling involves the phosphatases Pph21p and Pph22p but not Sit4p. Altogether, our results indicate that GAAC and TOR pathways (Tor1p) are involved in the signaling of acetic acid-induced apoptosis.

Nitric oxide signaling is disrupted in the yeast model for Batten disease.

The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-Delta) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1-Delta phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1-Delta limit the synthesis of nitric oxide (.NO) in both physiological and oxidative stress conditions. This defect in .NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1-Delta phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize .NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease.