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.
Fungi of the genus Paracoccidioides are the etiological agents of Paracoccidioidomycosis (PCM), the most prevalent mycosis in Latin America. Paracoccidioidomycosis infection is acquired by inhalation ...of Paracoccidioides conidia, which have first contact with the lungs and can subsequently spread to other organs/tissues. Until now, there have been no proteomic studies focusing on this infectious particle of Paracoccidioides. In order to identify the Paracoccidioides lutzii conidia proteome, conidia were produced and purified. Proteins were characterized by use of the nanoUPLC-MS
approach. The strategy allowed us to identify a total of 242 proteins in P. lutzii conidia. In the conidia proteome, proteins were classified in functional categories such as protein synthesis, energy production, metabolism, cellular defense/virulence processes, as well as other processes that can be important for conidia survival. Through this analysis, a pool of ribosomal proteins was identified, which may be important for the initial processes of dimorphic transition. In addition, molecules related to energetic and metabolic processes were identified, suggesting a possible basal metabolism during this form of resistance of the fungus. In addition, adhesins and virulence factors were identified in the P. lutzii conidia proteome. Our results demonstrate the potential role that these molecules can play during early cell-host interaction processes, as well as the way in which these molecules are involved in environmental survival during this form of propagation.
Paracoccidioides brasiliensis is a thermo-dimorphic fungus. At room temperature it grows as a mold that produces conidia, whereas in the vertebrate host it grows as a multiple-budding yeast. The ...molecular mechanisms involved in the germination from the conidia to the mycelia process remain unknown.
The kinetics of conidia to mycelia germination process were studied in the dimorphic fungus P. brasiliensis. Gene expression during this process was evaluated by construction and analysis of an EST library.
For the germination kinetics study, P. brasiliensis conidia were isolated as single cell units. Then, they were cultured at 18 degrees C in BHI (brain-heart infusion) broth for 24, 48, 72 and 96 hr. After each perion, they were examined by light microscopy. From conidia harvested at 96 hr, an EST library was constructed; at this stage the gene expression was presumed to be maximal for the germination process.
During the conidia to the mycelia developmental process, the following germination rates were observed: at 24 hr, 11.7+/-1.2%; at 48 hr, 30+/-0.6%; at 72 hr, 43+/-1.3%; and at 96 hr, 66+/-2.4%. At the 96 hour stage, an EST library was constructed. It consisted of 129 sequences grouped in 4 contigs and 7 singlets for a total of 11 possible genes. Eight of the sequences had not been described previously in other EST libraries of this fungus.
New genes were identified that were expressed during the conidia to the mycelia germination process and may represent genes specific to the germination process.
Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen ...needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MSsup.E proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.
ABSTRACT 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. IMPORTANCE Paracoccidioides 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.
Nitrogen is a crucial nutrient for microorganisms that compose essential biomolecules. However, hosts limit this nutrient as a strategy to counter infections, therefore, pathogens use adaptive ...mechanisms to uptake nitrogen from alternative sources. In fungi, nitrogen catabolite repression (NCR) activates transcription factors to acquire nitrogen from alternative sources when preferential sources are absent. Formamidase has been related to nitrogen depletion in Aspergillus nidulans through formamide degradation to use the released ammonia as a nitrogen source. In Paracoccidioides spp., formamidase is highly expressed in transcriptomic and proteomic analyses. Here, we aim to investigate the importance of formamidase to Paracoccidioides lutzii. Thereby, we developed a P. lutzii silenced strain of fmd gene (AsFmd) by antisense RNA technology using Agrobacterium tumefaciens-mediated transformation (ATMT). The AsFmd strain led to increased urease expression, an enzyme related to nitrogen assimilation in other fungi, suggesting that P. lutzii might explore urease as an alternative route for ammonia metabolism as a nitrogen source. Moreover, formamidase was important for fungal survival inside macrophages, as fungal recovery after macrophage infection was lower in AsFmd compared to wild-type (WT) strain. Our findings suggest potential alternatives of nitrogen acquisition regulation in P. lutzii, evidencing formamidase influence in fungal virulence.
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
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