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.
Fungi of the
Paracoccidioides
genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients ...and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe
3+
chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe
3+
assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of
Paracoccidioides brasiliensis
(
Pb
18) undergoing Fe deprivation and by nanoUPLC-MS
E
. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of
Paracoccidioides
in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of
Saccharomyces cerevisiae
and
Aspergillus fumigatus.
Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions
in vitro
, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in
P. brasiliensis
.
The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a ...morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.
spp. are endemic fungi from Latin America that cause Paracoccidioidomycosis, a systemic disease. These fungi present systems for high-affinity metal uptake, storage, and mobilization, which ...counteract host nutritional immunity and mitigate the toxic effects of metals. Regarding Cu mobilization, the metallochaperone Atx1 is regulated according to Cu bioavailability in
spp., contributing to metal homeostasis. However, additional information in the literature on
Atx1 is scarce. Therefore, in the present work, we aimed to study the
Atx1 protein-protein interaction networks. Heterologous expressed
Atx1 was used in a pull-down assay with
cytoplasmic extract. Nineteen proteins that interacted with
Atx1 were identified by HPLC-MS
. Among them, a relevant finding was a Cytochrome
(
Cyb5), regulated by Fe bioavailability in
and highly secreted by
in Fe deprivation. We validated the interaction between
Atx1-
Cyb5 through molecular modeling and far-Western analyses. It is known that there is a relationship between Fe homeostasis and Cu homeostasis in organisms. In this sense, would
Atx1-
Cyb5 interaction be a new metal-sensor system? Would it be supported by the presence/absence of metals? We intend to answer those questions in future works to contribute to the understanding of the strategies employed by
spp. to overcome host defenses.
Paracoccidoides brasiliensis and Paracoccidioides lutzii, the etiologic agents of paracoccidioidomycosis, cause disease in healthy and immunocompromised persons in Latin America. We developed a ...method for harvesting P. brasiliensis yeast cells from infected murine lung to facilitate in vivo transcriptional and proteomic profiling. P. brasiliensis harvested at 6 h post-infection were analyzed using RNAseq and LC-MS
E
. In vivo yeast cells had 594 differentially expressed transcripts and 350 differentially expressed proteins. Integration of transcriptional and proteomic data indicated that early in infection (6 h), P. brasiliensis yeast cells underwent a shift in metabolism from glycolysis to β-oxidation, upregulated detoxifying enzymes to defend against oxidative stress, and repressed cell wall biosynthesis. Bioinformatics and functional analyses also demonstrated that a serine proteinase was upregulated and secreted in vivo. To our knowledge this is the first study depicting transcriptional and proteomic data of P. brasiliensis yeast cells upon 6 h post-infection of mouse lung.
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-
dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell ...wall is a dynamic outer structure,
composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These
glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited
N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell
wall remodeling. Our results suggest that cell wall synthesis related genes, such as β-1,3-glucanosyltransferase (PbGEL3),
1,3-β-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as Nacetyl-
β-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and β-1,3-glucanase (PbBGN1 and PbBGN2), have
their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression
levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation
caused by TM.
The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a ...morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.
Pneumococcal nasopharyngeal carriage isolates recovered from Brazilian children attending day-care centres in 2005 were assessed for serotype, genotype and penicillin susceptibility phenotype. As 124 ...of the 253 isolates (49 %) were characterized previously with respect to serotype and penicillin susceptibility, the primary objectives were to examine clonal associations and penicillin susceptibility within major serotypes and to assess the suitability of conventional multiplex PCR for deducing carriage serotypes within this population. Using a combination of PCR-based serotyping and the Quellung reaction, serotypes were identified for 81 % (205/253) of the isolates, with serogroups or types 14, 6, 23F, 19F and 18 being predominant. Included within the 205 isolates successfully serotyped by PCR were 28 isolates that had become non-viable. Forty-eight isolates were non-typable using both the PCR method and the Quellung reaction. Penicillin non-susceptibility was observed within 16 of the 18 multilocus sequence types detected. Thus, this study provides further evidence from a diverse collection of pneumococcal clones that PCR-based serotype deduction is useful for providing supportive evidence for pneumococcal conjugate vaccine implementation.
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.
•Comparative analysis of Paracoccidioides under in vitro, ex vivo and in vivo model.•Identification of metabolic pathways regulated in the conditions analyzed.•Update of the strategies used by the pathogen to establish infection.
Paracoccidioides spp. are endemic fungi from Latin America that cause Paracoccidioidomycosis, a systemic disease. These fungi present systems for high-affinity metal uptake, storage, and ...mobilization, which counteract host nutritional immunity and mitigate the toxic effects of metals. Regarding Cu mobilization, the metallochaperone Atx1 is regulated according to Cu bioavailability in Paracoccidioides spp., contributing to metal homeostasis. However, additional information in the literature on PbAtx1 is scarce. Therefore, in the present work, we aimed to study the PbAtx1 protein–protein interaction networks. Heterologous expressed PbAtx1 was used in a pull-down assay with Paracoccidioides brasiliensis cytoplasmic extract. Nineteen proteins that interacted with PbAtx1 were identified by HPLC-MSsup.E. Among them, a relevant finding was a Cytochrome bsub.5 (PbCyb5), regulated by Fe bioavailability in Aspergillus fumigatus and highly secreted by P. brasiliensis in Fe deprivation. We validated the interaction between PbAtx1-PbCyb5 through molecular modeling and far-Western analyses. It is known that there is a relationship between Fe homeostasis and Cu homeostasis in organisms. In this sense, would PbAtx1-PbCyb5 interaction be a new metal-sensor system? Would it be supported by the presence/absence of metals? We intend to answer those questions in future works to contribute to the understanding of the strategies employed by Paracoccidioides spp. to overcome host defenses.
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