Proteomics of Paracoccidioides lutzii: Overview of Changes Triggered by Nitrogen Catabolite Repression.

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, ß-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.

Paracoccidioides lutzii Formamidase Contributes to Fungal Survival in Macrophages.

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.

Staphylococcus saprophyticus Proteomic Analyses Elucidate Differences in the Protein Repertories among Clinical Strains Related to Virulence and Persistence.

Staphylococcus saprophyticus 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 S. saprophyticus 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 S. saprophyticus strains, reflecting in virulence and persistence.

Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma.

Although members of the Paracoccidioides 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 E technology, we analyzed the proteomic response of Paracoccidioides brasiliensis 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 P. brasiliensis. 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.

Immunoproteomic Approach of Extracellular Antigens From Paracoccidioides Species Reveals Exclusive B-Cell Epitopes.

Fungi of the Paracoccidioides genus are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis restricted to the countries of Latin America. Currently, the Paracoccidioides complex is represented by Paracoccidioides lutzii, Paracoccidioides americana, Paracoccidioides brasiliensis, Paracoccidioides restrepiensis, and Paracoccidioides venezuelensis. 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 Paracoccidioides 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 Paracoccidioides spp., immunoproteomic approaches were used combining immunoprecipitation, followed by antigen identification by nanoUPLC-MSE-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 Paracoccidioides species. Using bioinformatics tools, two unique exoantigens in P. lutzii species were identified, as well as 44 epitopes exclusive to the Paracoccidioides complex and 12 unique antigenic sequences that can differentiate between Paracoccidioides species. Therefore, these results demonstrate that Paracoccidioides species have a range of B-cell epitopes exclusive to the complex as well as specific to each Paracoccidioides 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.

A proteomic dataset of secreted proteins by three Staphylococcus saprophyticus strains.

This article presents a proteomic dataset generated from a comparative analysis of the exoproteome of Staphylococcus saprophyticus, ATCC 15305, 7108 and 9325 strains. The extract of secreted proteins were obtained after incubation of stationary phase cells in BHI medium. All samples were submitted to nano-ESI-UPLC-MSE, and the spectrum obtained was processed and analyzed by ProteinLynx Global Server (PLGS), Uniprot and Pedant databases, for identification, annotation and functional classification of proteins. Fold changes and protein relative abundances were properly reported. This report is related to the research article entitled "The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents" (Oliveira et al., 2018). The proteomic data generated have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with a project number PXD008643, https://www.ebi.ac.uk/pride/archive/projects/PXD008643.

The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents.

Staphylococcus saprophyticus is a gram-positive microorganism responsible for urinary tract infections (UTIs). Although some virulence factors are characterized, such as urease, autolysins, adhesins and hemagglutinins, large-scale proteomic studies have not been performed within this species. We performed the characterization of the exoproteome from three S. saprophyticus strains: the reference strain ATCC 15,305, a non-capsular strain 7108 and the 9325 strain containing a thick capsule which were cultured in BHI medium and culture supernatants were analysed by using mass spectrometry approach. We observed a core of 72 secreted proteins. In addition, it was possible to detect diversity in the protein profiles of the exoproteomes. Interestingly, strain 7108 presented no secretion of three antigenic proteins, including the classical SsaA antigen. In addition, the level of antigenic proteins secreted by strain 9325 was higher than in ATCC 15,305. This result was confirmed by Western blot analysis using anti-SsaA polyclonal antibodies, and no production/ secretion of SsaA was detected in strain 7108. Transcriptional data shows that 7108 strain produces transcripts encoding SsaA, suggesting post-transcriptional regulation occurs in this strain. Moreover, when compared with the other strains that were analyzed, it was possible to detect higher levels of proteases secreted by strain 7108 and higher levels of antigenic proteins and transglycosylases secreted by 9325 strain. The results reveal diversity in protein secretion among strains. This research is an important first step towards understanding the variability in S. saprophyticus exoproteome profile and could be significant in explaining differences among strains.

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-MSE) 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.

Antifungal Resistance, Metabolic Routes as Drug Targets, and New Antifungal Agents: An Overview about Endemic Dimorphic Fungi.

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.

Effects of Argentilactone on the Transcriptional Profile, Cell Wall and Oxidative Stress of Paracoccidioides spp.

Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy.

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.

Transcriptome Profile of the Response of Paracoccidioides spp. to a Camphene Thiosemicarbazide Derivative.

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.