Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells

Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Differentially expressed proteins in the interaction of Paracoccidioides lutzii with human monocytes

BackgroundFungi of the genus Paracoccidioides are the etiological agents of paracoccidioidomycosis, a highly prevalent mycosis in Latin America. Infection in humans occurs by the inhalation of conidia, which later revert to the form of yeast. In this context, macrophages are positioned as an important line of defense, assisting in the recognition and presentation of antigens, as well as producing reactive oxygen species that inhibit fungal spreading.AimsThe objective of this study was to identify differentially expressed proteins during the interaction between Paracoccidioides lutzii Pb01 strain and human U937 monocytes.MethodsTwo-dimensional electrophoresis, combined with mass spectrometry, was used to evaluate the differential proteomic profiles of the fungus P. lutzii (Pb01) interacting with U937 monocytes.ResultsIt was possible to identify 25 proteins differentially expressed by Pb01 alone and after interacting with U937 monocytes. Most of these proteins are directly associated with fungal metabolism for energy generation, such as glyceraldehyde-3-phosphate dehydrogenase, and intracellular adaptation to monocytes. Antioxidant proteins involved in the response to oxidative stress, such as peroxiredoxin, cytochrome, and peroxidase, were expressed in greater quantity in the interaction with monocytes, suggesting their association with survival mechanisms inside phagocytic cells. We also identified 12 proteins differentially expressed in monocytes before and after the interaction with the fungus; proteins involved in the reorganization of the cytoskeleton, such as vimentin, and proteins involved in the response to oxidative stress, such as glioxalase 1, were identified.ConclusionsThe results of this proteomic study of a P. lutzii isolate are novel, mimicking in vitro what occurs in human infections. In addition, the proteins identified may aid to understand fungal–monocyte interactions and the pathogenesis of paracoccidioidomycosis.     

Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE₂, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE₂ inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE₂ confirmed an association between PGE₂ inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.     

The inhibitors of antioxidant cell enzymes induce permeability transition in yeast mitochondria

In this study we investigated the effects of exogenous and endogenous oxidative stress on mitochondrial membrane permeability transition in yeast cells. E. magnusii yeast was used in the study as it is the only yeast strain possessing a natural high-capacity Са²⁺ transport system. The key reactive oxygen species (ROS) detoxifying enzymes in the yeast cells - catalases (CATs) and superoxide dismutases (SODs) - were fully characterized. At least five isoforms of SODs and only one isoform of CATs were found in the E. magnusii mitochondria. The assessment of the main properties of mitochondrial non-specific permeability under physiological conditions such as dynamics of the membrane potential (?Ψ) and swelling in mitochondria showed that under physiological conditions classical inhibitors of CATs (ATZ - 3-amino-1, 2, 4-triazole) and of SODs (DDC - diethyldithiocarbamate) caused irreversible decline in ?Ψ in the yeast mitochondria. This decline was accelerated in the presence of 500 μM Са²⁺. The combined action of the inhibitors (ATZ + DDC) promoted moderate swelling in the isotonic medium, which was confirmed by transmission electron microscopy. Mitochondrial swelling in the cells exposed to antioxidant system inhibitors was accompanied by typical signs of early apoptosis, namely by chromatin margination and condensation, vacuolization of the cytosol, and damage of the plasma membrane. Here we showed, at both cellular and mitochondrial levels, that the deregulation of oxidant-scavenging enzymes directly leads to the opening of the mPTP, followed by induction of apoptotic markers in the whole yeast cells. Our studies are the first to clarify the highly contradictory data in the literature on mPTP in yeast mitochondria.     

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.     

Inhibition of Paracoccidioides lutzii Pb01 Isocitrate Lyase by the Natural Compound Argentilactone and Its Semi-Synthetic Derivatives

The dimorphic fungus Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments.     

Contribution of Catalase and Superoxide Dismutase to the Intracellular Survival of Clinical Isolates of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Murine Macrophages

The present study was performed in order to carefully investigate the interaction of Staphylococcus aureus with murine macrophages and the contribution of catalase and superoxide dismutase in intracellular persistence of Staphylococcus aureus within murine macrophages during in vitro infection. We have reported that Staphylococcus aureus internalized by murine macrophages did not appear to be rapidly killed. Data indicating the contribution of a single catalase and superoxide dismutase in intracellular survival of Staphylococcus aureus were provided using established biochemical assays. The results of the present experiment suggest that the survival of Staphylococcus aureus within phagocytic cells is facilitated by its ability to resist oxidative products. Organisms in the log phase of growth clearly demonstrate a resistance to oxidative products.     

scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4⁺ and CD8⁺ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4⁺ and CD8⁺ T cells.