Clayton Luiz Borges

The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process

BackgroundParacoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The fungus is thermally dimorphic with two distinct forms corresponding to completely different lifestyles. Upon elevation of the temperature to that of the mammalian body, the fungus adopts a yeast-like form that is exclusively associated with its pathogenic lifestyle. We describe expressed sequence tags (ESTs) analysis to assess the expression profile of the mycelium to yeast transition. To identify P. brasiliensis differentially expressed sequences during conversion we performed a large-scale comparative analysis between P. brasiliensis ESTs identified in the transition transcriptome and databases.ResultsOur analysis was based on 1107 ESTs from a transition cDNA library of P. brasiliensis. A total of 639 consensus sequences were assembled. Genes of primary metabolism, energy, protein synthesis and fate, cellular transport, biogenesis of cellular components were represented in the transition cDNA library. A considerable number of genes (7.51%) had not been previously reported for P. brasiliensis in public databases. Gene expression analysis using in silico EST subtraction revealed that numerous genes were more expressed during the transition phase when compared to the mycelial ESTs [1]. Classes of differentially expressed sequences were selected for further analysis including: genes related to the synthesis/remodeling of the cell wall/membrane. Thirty four genes from this family were induced. Ten genes related to signal transduction were increased. Twelve genes encoding putative virulence factors manifested increased expression. The in silico approach was validated by northern blot and semi-quantitative RT-PCR.ConclusionThe developmental program of P. brasiliensis is characterized by significant differential positive modulation of the cell wall/membrane related transcripts, and signal transduction proteins, suggesting the related processes important contributors to dimorphism. Also, putative virulence factors are more expressed in the transition process suggesting adaptation to the host of the yeast incoming parasitic phase. Those genes provide ideal candidates for further studies directed at understanding fungal morphogenesis and its regulation.

Angiotensin-Converting Enzyme 2 Activation Improves Endothelial Function

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin–angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1–7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1–7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II–induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Proteomic Analysis Reveals That Iron Availability Alters the Metabolic Status of the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways.

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.

The Homeostasis of Iron, Copper, and Zinc in Paracoccidioides Brasiliensis, Cryptococcus Neoformans Var. Grubii, and Cryptococcus Gattii: A Comparative Analysis

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.

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.

A quantitative view of the morphological phases of Paracoccidioides brasiliensis using proteomics

Paracoccidioides brasiliensis is a fungal pathogen with a broad distribution in Latin American countries. The mycelia-to-yeast morphological transition of P. brasiliensis is involved in the virulence of this pathogen, and this event is essential to the establishment of infection. Here, we report the first proteomic comparison between the mycelia, the mycelia-to-yeast transition and the yeast cells. Changes in the relative abundance of the components of the proteome during phase conversion of P. brasiliensis were analyzed by two-dimensional gel electrophoresis coupled to mass spectrometry. Using MALDI-TOF-MS, we identified 100 total proteins/isoforms. We show that 18, 30 and 33 proteins/isoforms in our map are overexpressed in the mycelia, the mycelia-to-yeast transition and in yeast cells, respectively. Nineteen proteins/isoforms did not present significant differences in the volume spots in the three analyzed conditions. The differential expression was confirmed for six different proteins by Western blot analysis. The quantitative differences observed by the proteomic analysis were correlated with the transcript levels, as determined by quantitative RT-PCR of the analyzed conditions, including conidial formation and the transition from conidia-to-yeast cells. The analysis of the functional categories to which these proteins belong provided an integrated view of the metabolic reorganization during the morphogenesis of P. brasiliensis.

A proteomic view of the response of Paracoccidioides yeast cells to zinc deprivation

Zinc plays a critical role in a diverse array of biochemical processes. However, an excess of zinc is deleterious to cells; therefore, cells require finely tuned homeostatic mechanisms to balance the uptake and the storage of zinc. There is also increasing evidence supporting the importance of zinc during infection. To understand better how Paracoccidioides adapts to zinc deprivation, we compared the two-dimensional (2D) gel protein profile of yeast cells during zinc starvation to yeast cells grown in a zinc rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity, as determined by image analysis. In response to zinc deprivation, a total of 423 out of 845 protein spots showed a significant change in abundance. Quantitative RT-qPCR analysis of RNA from Paracoccidioides grown under zinc restricted conditions validated the correlation between the differentially regulated proteins and transcripts. According to the proteomic data, zinc deficiency may be a stressor to Paracoccidioides, as suggested by the upregulation of a number of proteins related to stress response, cell rescue, and virulence. Other process induced by zinc deprivation included gluconeogenesis. Conversely, the methylcitrate cycle was downregulated. Overall, the results indicate a remodelling of the Paracoccidioides response to the probable oxidative stress induced during zinc deprivation.

A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins

BackgroundParacoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.ResultsA cDNA (Pb sp) encoding a secreted serine protease (Pb SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant Pb SP was produced in Escherichia coli, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the P. brasiliensis proteome. In vitro deglycosylation assays with endoglycosidase H demonstrated that Pb SP is a N-glycosylated molecule. The Pb sp transcript and the protein were induced during nitrogen starvation. The Pb sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of Pb SP with P. brasiliensis proteins were evaluated by two-hybrid assay in the yeast Saccharomyces cerevisiae. Pb SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2.ConclusionsA secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. Pb SP interactions with other P. brasiliensis proteins were reported. Proteins interacting with Pb SP are related to folding process, protein trafficking and cytoskeleton reorganization.

Analysis of Paracoccidioides secreted proteins reveals fructose 1,6-bisphosphate aldolase as a plasminogen-binding protein

BackgroundDespite being important thermal dimorphic fungi causing Paracoccidioidomycosis, the pathogenic mechanisms that underlie the genus Paracoccidioides remain largely unknown. Microbial pathogens express molecules that can interact with human plasminogen, a protein from blood plasma, which presents fibrinolytic activity when activated into plasmin. Additionally, plasmin exhibits the ability of degrading extracellular matrix components, favoring the pathogen spread to deeper tissues. Previous work from our group demonstrated that Paracoccidioides presents enolase, as a protein able to bind and activate plasminogen, increasing the fibrinolytic activity of the pathogen, and the potential for adhesion and invasion of the fungus to host cells. By using proteomic analysis, we aimed to identify other proteins of Paracoccidioides with the ability of binding to plasminogen.ResultsIn the present study, we employed proteomic analysis of the secretome, in order to identify plasminogen-binding proteins of Paracoccidioides, Pb01. Fifteen proteins were present in the fungal secretome, presenting the ability to bind to plasminogen. Those proteins are probable targets of the fungus interaction with the host; thus, they could contribute to the invasiveness of the fungus. For validation tests, we selected the protein fructose 1,6-bisphosphate aldolase (FBA), described in other pathogens as a plasminogen-binding protein. The protein FBA at the fungus surface and the recombinant FBA (rFBA) bound human plasminogen and promoted its conversion to plasmin, potentially increasing the fibrinolytic capacity of the fungus, as demonstrated in fibrin degradation assays. The addition of rFBA or anti-rFBA antibodies was capable of reducing the interaction between macrophages and Paracoccidioides, possibly by blocking the binding sites for FBA. These data reveal the possible participation of the FBA in the processes of cell adhesion and tissue invasion/dissemination of Paracoccidioides.ConclusionsThese data indicate that Paracoccidioides is a pathogen that has several plasminogen-binding proteins that likely play important roles in pathogen-host interaction. In this context, FBA is a protein that might be involved somehow in the processes of invasion and spread of the fungus during infection.