Characterization of extracellular proteins in members of the Paracoccidioides complex

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) – an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MS^E) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes.     

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.     

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.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex

Background

Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes.

Results

We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR.

Conclusion

Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1564-7) contains supplementary material, which is available to authorized users.     

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.     

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.     

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.     

Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.     

Paracoccidioidomicosis en pediatría: descripción de 4 casos

BackgroundParacoccidioidomycosis is the most frequent systemic mycosis in Latin America, caused by the dimorphic fungus Paracoccidioides. Paracoccidioidomycosis in children is uncommon. Our aim is to describe clinical features of patients who had a confirmed diagnosis of paracoccidioidomycosis in our hospital in the last 10 years.Case reportsWe describe 4 cases of paracoccidioidomycosis in previously healthy children from the north of our country. Diagnoses were made by biopsy or culture.ConclusionsThe diagnosis of paracoccidioidomycosis should be considered in a patient coming from regions where Paracoccidioides is endemic, and presenting with a lymphoproliferative syndrome, anemia, eosinophilia and hypergammaglobulinemia.     

Induction of defense response against Colletotrichum capsici in chili fruit by the yeast Pichia guilliermondii strain R13

Pichia guilliermondii strain R13, a yeast isolated from Thai rambutan, has been shown to suppress the fungal pathogen Colletotrichum capsici in harvested chili. Its multiple modes of action include nutrient competition, tight attachment to the fungus, and hydrolytic enzyme secretion. This study investigated the ability of the P. guilliermondii strain R13 to induce resistance against C. capsici in chili fruit. The pretreatment of chili with the yeast antagonist, physically separated from the fungus by known distances, significantly reduced disease incidence and lesion diameter caused by C. capsici. Compared to the controls, the yeast treatment also significantly enhanced the activities of phenylalanine ammonia-lyase, chitinase, and β-1,3-glucanase, and the accumulation of capsidiol phytoalexin in chili tissue. Scanning electron micrographs showed that the morphology of C. capsici spores and hyphae were abnormal and that the pathogen had restricted growth on chili tissue adjacent to the yeast-inoculated sites. The results indicate that the induction of resistance may be another mechanism by which the yeast antagonist suppresses C. capsici.     

Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental Paracoccidioides lutzii

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 before infection reduced the pulmonary fungal burden as compared to mice treated with irrelevant mAb. Hence, mAbs raised to H. capsulatum Hsp60 are protective against P. lutzii, including mAb 7B6 which was non-protective against H. capsulatum, suggesting differences in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful.