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.

New insights into a complex fungal pathogen: the case of Paracoccidioides spp.

Paracoccidioidomycosis is a systemic mycosis endemic to Latin America, with Paracoccidioides brasiliensis and P. lutzii being the causal agents of this disorder. Several issues have been raised in the 100 years since its discovery and in this article we discuss features of this fascinating fungal pathogen, including its biology, eco-epidemiology and aspects of its pathogenicity. We also consider some of its virulence determinants, the most recent advances in the study of its metabolic pathways and the molecular and genetic research tools developed for this research. We also review the animal models used to study host-fungal interactions and how the host defence mechanisms against this pathogen work.

Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis.

Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

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.

Paracoccidioides brasiliensis PbP27 gene: knockdown procedures and functional characterization.

Paracoccidioides brasiliensis PbP27 gene encodes a protein localized in both the fungal cytoplasm and cell wall. The parasitic infectious form produces this protein preferentially with the gene's expression varying between the fungus phylogenetic species. The biological function of the native p27 has yet to be determined during either growth of the yeast or host infection. Therefore, in this study, through the use of antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated mitotically stable PbP27 mutants (PbP27 aRNA) with the goal to evaluate the role of p27 in the biology and virulence of this fungus. PbP27 expression was reduced 60-75% in mutants, as determined by real-time PCR in correlation with a decrease in p27 expression. No alterations in the growth curve or in the ability to shift from mycelia to yeast or from yeast to mycelia were observed in PbP27 aRNA strains; however, we did observe a reduction in cell vitality. Moreover, a decrease in cell viability of PbP27 aRNA yeast cells after interaction with IFN-γ-stimulated macrophages was detected. Based on these results, we propose that p27 plays a role in yeast cell architecture and represents one of the mechanisms employed by this fungus for its interaction with the monocyte/macrophage system.

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.

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen.

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.

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.

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.

Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell.

Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence factors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40/79 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P. brasiliensis conidia induced infection.

Paracoccidioides Genomes Reflect High Levels of Species Divergence and Little Interspecific Gene Flow.

The fungus Paracoccidioides is a prevalent human pathogen endemic to South America. The genus is composed of five species. In this report, we use 37 whole-genome sequences to study the allocation of genetic variation in Paracoccidioides We tested three genome-wide predictions of advanced speciation, namely, that all species should be reciprocally monophyletic, that species pairs should be highly differentiated along the whole genome, and that there should be low rates of interspecific gene exchange. We find support for these three hypotheses. Species pairs with older divergences show no evidence of gene exchange, while more recently diverged species pairs show evidence of modest rates of introgression. Our results indicate that as divergence progresses, species boundaries become less porous among Paracoccidioides species. Our results suggest that species in Paracoccidioides are at different stages along the divergence continuum.IMPORTANCEParacoccidioides is the causal agent of a systemic mycosis in Latin America. Most of the inference of the evolutionary history of Paracoccidioides has used only a few molecular markers. In this report, we evaluate the extent of genome divergence among Paracoccidioides species and study the possibility of interspecific gene exchange. We find that all species are highly differentiated. We also find that the amount of gene flow between species is low and in some cases is even completely absent in spite of geographic overlap. Our study constitutes a systematic effort to identify species boundaries in fungal pathogens and to determine the extent of gene exchange among fungal species.

Draft Genome Sequences of Clinical and Environmental Isolates of Aspergillus tamarii from Colombia.

Aspergillus is a very diverse genus of fungi that are common in the environment and can affect human health. Here, we report the draft genome sequences of two Colombian isolates of Aspergillus tamarii, an emerging pathogenic species. One isolate was obtained from an infected patient and the other from the environment in a hospital.

Draft Genome Sequences of Two Sporothrix schenckii Clinical Isolates Associated with Human Sporotrichosis in Colombia.

Sporothrix schenckii is a thermodimorphic fungal pathogen with a high genetic diversity. In this work, we present the assembly and similarity analysis of the whole-genome sequences of two clinical isolates from Colombia of S. schenckiisensu stricto.

Response of Paracoccidioides lutzii to the antifungal camphene thiosemicarbazide determined by proteomic analysis.

AIM: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action. METHODS: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound. RESULTS: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors. CONCLUSION: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.

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.

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

Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy.

Caracterización de Candida spp. aisladas a partir de urocultivos en la ciudad de Medellín / Characterization of Candida spp isolated from urine cultures of Medellín

Resumen Candida spp. es un agente etiológico importante en infecciones del tracto urinario, principalmente en población con terapia antimicótica de amplio espectro y con catéteres urinarios. Candida albicans es la especie más frecuente, pero otras especies han surgido como patógenos emergentes. En este trabajo se recolectaron aislamientos de Candida spp. de urocultivos de pacientes que consultaron en Dinamica IPS entre enero 2016 y noviembre 2017. Para estimar la frecuencia de las especies y observar los patrones de sensibilidad, se realizó la identificación fenotípica y su perfil de sensibilidad con el sistema comercial Vitek 2® (BioMérieux, Inc.), adicionalmente se evaluaron mediante análisis de las secuencia y filogenética ITS1-5.8S-ITS2. En el estudio se incluyeron 78 aislamientos de Candida spp. Las frecuencias de especies de Candida identificadas empleando las herramientas moleculares fueron: C. albicans (38,5%), C. tropicalis (23,1%), C. glabrata (21,8%), C. parapsilosis (10,3%), C. metapsilosis y C. krusei (2,5%) y C. guillermondi (1,3%). La identificación por métodos moleculares y por el sistema Vitek 2 fue: C. albicans (93,3%), C. glabrata (94,1%), C. tropicalis (83,3%), C. parapsilosis (75%) C. guilliermondii y C. krusei (100%). La sensibilidad de todos los aislamientos al fluconazol fue 93,6%.

Abstract Candida spp is an important etiologic agent in urinary tract infections, mainly in patients in broad-spectrum antifungal therapy, with urinary catheters. Candida albicans is the most frequent specie; but other species have arised as emerging pathogens. In this study, isolates of Candida spp. of urine cultures from patients who consulted in Dinamica IPS between January 2016 and November 2017 were evaluated. To estimate the frequency of the species and to observe the sensitivity patterns, the phenotypic identification and its sensitivity profile was performed employed the Vitek 2® commercial system. (BioMérieux, Inc) In addition the isolates were evaluated by sequence analysis and phylogenetics ITS1-5.8S-ITS2. This study included 78 isolates of Candida spp. The frequencies of Candida species identified using the molecular tools were: C. albicans (38.5%), C. tropicalis (23.1%), C. glabrata (21.8%), C. parapsilosis (10.3%), C. guillermondi (1.3%) and C. metapsilosis and C. krusei (2.5%). The identification by molecular methods and by Vitek 2 system were: C. albicans (93.3%), for C. glabrata (94.1%), C. tropicalis (83.3%), C. parapsilosis (75%) and 100% for C. guilliermondii and C. krusei.. fluconazole sensitivity of all isolates was 93.6%

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress.

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.

Hemoglobin uptake by Paracoccidioides spp. is receptor-mediated.

Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms.

Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis.

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.