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.
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.
Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of ...treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.
Fungi of the
genus are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis restricted to the countries of Latin America. Currently, the
complex is represented by
,
,
,
, and
. ...Even with advances in techniques used for diagnosing fungal diseases, high rates of false-positive results for PCM are still presented. Additionally, there is no efficient antigen that can be used to follow up the efficiency of patient treatment. The immunoproteomic is considered a powerful tool for the identification of antigens. In addition, antigens are molecules recognized by the immune system, which make them excellent targets for diagnostic testing of diseases caused by microorganisms. In this vein, we investigated which antigens are secreted by species representing
complex to increase the spectrum of molecules that could be used for future diagnostic tests, patient follow-up, or PCM therapy. To identify the profile of antigens secreted by
spp., immunoproteomic approaches were used combining immunoprecipitation, followed by antigen identification by nanoUPLC-MS
-based proteomics. Consequently, it was possible to verify differences in the exoantigen profiles present among the studied species. Through a mass spectrometry approach, it was possible to identify 79 exoantigens in
species. Using bioinformatics tools, two unique exoantigens in
species were identified, as well as 44 epitopes exclusive to the
complex and 12 unique antigenic sequences that can differentiate between
species. Therefore, these results demonstrate that
species have a range of B-cell epitopes exclusive to the complex as well as specific to each
species. In addition, these analyses allowed us the identification of excellent biomarker candidates for epidemiology screening, diagnosis, patient follow-up, as well as new candidates for PCM therapy.
Although members of the
complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the ...spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MS
technology, we analyzed the proteomic response of
during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of
. The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite-host relationship and pathogenesis of PCM.
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.
Human Adenoviruses (HAdVs) are etiological agents of different syndromes such as gastroenteritis, cystitis, ocular, and respiratory diseases, and infection by these viruses may cause alterations in ...cellular homeostasis. The objective of the study was the proteomic analysis of A-549 cells infected with HAdV-40 using LC–MS. At 30 h of infection, the quantitative analysis revealed 336 differentially expressed proteins. From them, 206 were induced (up-regulated) and 130 were suppressed (down-regulated). The majority of up-regulated proteins were related to energy, cellular organization, stress response, and apoptosis pathways. It was observed alteration of cell metabolism with increase of the glycolytic pathway, β-oxidation, and respiratory chain. Also, the results suggest cytoskeleton reorganization and apoptosis induction. The data can improve knowledge about the replication of HAdV-40 in cell culture considering the proteins related to distinct metabolic pathways induced by viral infection in A-549 cells.
Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the ...infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment.
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-MSE 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.
is a Gram-positive and coagulase negative cocci that composes the skin microbiota and can act as an opportunistic agent causing urinary tract infections, being more frequent in sexually active young ...women. The ability of a pathogen to cause infection in the host is associated to its ability to adhere to host cells and to survive host immune defenses. In this work, we presented the comparative proteomic profile of three
strains. It was possible to characterize differences in the proteome content, specially related to expression of virulence factors. We compiled this data and previous data and we detected one strain (9325) possessing higher production and secretion of proteins related to virulence. Our results show that phenotypic, genotypic, and proteomic differences reflect in the ability to survive during interaction with host cells, since the 9325 strain presented a higher survival rate after macrophage interaction. In counterpart, the 7108 strain that possesses lower content of proteins related to virulence presented higher ability to form biofilm suggesting that this strain can be better adapted to persist in the host and in the environment. Our work describes, for the first time, proteomic flexibility among
strains, reflecting in virulence and persistence.
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