Characterization of the APSES-family transcriptional regulators of Histoplasma capsulatum.

The fungal APSES protein family of transcription factors is characterized by a conserved DNA-binding motif facilitating regulation of gene expression in fungal development and other biological processes. However, their functions in the thermally dimorphic fungal pathogen Histoplasma capsulatum are unexplored. Histoplasma capsulatum switches between avirulent hyphae in the environment and virulent yeasts in mammalian hosts. We identified five APSES domain-containing proteins in H. capsulatum homologous to Swi6, Mbp1, Stu1 and Xbp1 proteins and one protein found in related Ascomycetes (APSES-family protein 1; Afp1). Through transcriptional analyses and RNA interference-based functional tests we explored their roles in fungal biology and virulence. Mbp1 serves an essential role and Swi6 contributes to full yeast cell growth. Stu1 is primarily expressed in mycelia and is necessary for aerial hyphae development and conidiation. Xbp1 is the only factor enriched specifically in yeast cells. The APSES proteins do not regulate conversion of conidia into yeast and hyphal morphologies. The APSES-family transcription factors are not individually required for H. capsulatum infection of cultured macrophages or murine infection, nor do any contribute significantly to resistance to cellular stresses including cell wall perturbation, osmotic stress, oxidative stress or antifungal treatment. Further studies of the downstream genes regulated by the individual APSES factors will be helpful in revealing their functional roles in H. capsulatum biology.

Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67.

Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal-host relationship is discussed.

Dual localization of Mdj1 in pathogenic fungi varies with growth temperature.

Paracoccidioides brasiliensis and P. lutzii are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis (PCM). Previously, we characterized the PbMDJ1 gene. This gene encodes P. brasiliensis chaperone Mdj1, which in yeast is a mitochondrial member of the J-domain family, whose main function is to regulate cognate Hsp70 activities. We produced rabbit polyclonal antibody antirecombinant PbMdj1 (rPbMdj1), which labeled the protein not only in mitochondria but also at the cell wall of P. brasiliensis yeasts of isolate Pb18. Here we used anti-rPbMdj1 in confocal microscopy to localize Mdj1 in Pb18 and other fungal isolates grown at different temperatures. Dual intracellular and cell surface pattern were initially seen in yeast-phase P. brasiliensis Pb3, Pb18 (control), P. lutzii Pb01, and Histoplasma capsulatum. Pb18 and Aspergillus fumigatus hyphae as well as Pb3 pseudo hyphae formed at 36°C were labeled predominantly along the cell surface. Preferential surface localization was observed by 72 h of yeast-mycelium thermotransition. It was interesting to observe that anti-rPbMdj1 concentrated at the surface tip and branching points of A. fumigatus hyphae grown at 36°C, suggesting a role in growth, whereas at 23°C, anti-rPbMdj1 was distributed along the hyphal surface. In Pb3, Pb18, and Pb01 mitochondrial extracts, the antibodies revealed a specific 55-kDa band, which corresponds to the processed Mdj1 size. The presence of Mdj1 on the fungal cell wall suggests that this protein could also play a role in the interaction with the host.

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

Morphological and ultrastructural changes in fungal agents after LASER application.

BACKGROUND: Onychomycosis is a fungal nail infection of difficult treatment due to the fungal survival capacity and reduced number of effective therapies. The present study aimed to isolate fungal agents that cause onychomycosis in immunocompetent patients and evaluate how LASER treatments affect the growth and ultrastructure of isolates. METHODS: In total, 21 patients with positive direct microscopic examination (DME) for onychomycosis had nail samples collected for cultivation and phenotypic identification of microorganisms. From these patients, 12 underwent LASER treatment, divided in Group 1 (n = 5) treated with Nd: YAG 1,064 nm, and Group 2 (n = 7) treated with Nd: YAG 1,064 nm + Er: YAG 2,940 nm + topical isoconazole. Transmission Electron Microscopy (TEM) was performed to evaluate ultrastructural changes after treatment. RESULTS: DME, cultivation, and phenotypic identification showed that the most identified fungus was Trichophyton rubrum spp. After LASER therapy, sample cultivation showed alterations in the fungal morphology with reduction of hyphae, conidia, and reproductive structures. Alterations in fungal cell wall structure, cytoplasm density, and organelles were observed by TEM. CONCLUSION: LASER irradiation causes changes in the fungal cells, especially in the number of hyphae and the presence of conidia. In addition, it affects fungal growth and reproduction capacity, which interferes with their infection ability and virulence.

Vesicle and vesicle-free extracellular proteome of Paracoccidioides brasiliensis: comparative analysis with other pathogenic fungi.

Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. In vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes.

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis.

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.

The Cell Wall-Associated Proteins in the Dimorphic Pathogenic Species of Paracoccidioides.

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis (PCM). They are dimorphic ascomycetes that grow as filaments at mild temperatures up to 28oC and as multibudding pathogenic yeast cells at 37oC. Components of the fungal cell wall have an important role in the interaction with the host because they compose the cell outermost layer. The Paracoccidioides cell wall is composed mainly of polysaccharides, but it also contains proportionally smaller rates of proteins, lipids, and melanin. The polysaccharide cell wall composition and structure of Paracoccidioides yeast cells, filamentous and transition phases were studied in detail in the past. Other cell wall components have been better analyzed in the last decades. The present work gives to the readers a detailed updated view of cell wall-associated proteins. Proteins that have been localized at the cell wall compartment using antibodies are individually addressed. We also make an overview about PCM, the Paracoccidioides cell wall structure, secretion mechanisms, and fungal extracellular vesicles.

Characterization of cell wall lipids from the pathogenic phase of Paracoccidioides brasiliensis cultivated in the presence or absence of human plasma.

BACKGROUND: The fungal cell wall is a complex and dynamic outer structure. In pathogenic fungi its components interact with the host, determining the infection fate. The present work aimed to characterize cell wall lipids from P. brasiliensis grown in the presence and absence of human plasma. We compared the results from isolates Pb3 and Pb18, which represent different phylogenetic species that evoke distinct patterns of experimental paracoccidioidomycosis. METHODOLOGY/PRINCIPAL FINDINGS: We comparatively characterized cell wall phospholipids, fatty acids, sterols, and neutral glycolipids by using both electrospray ionization- and gas chromatography-mass spectrometry analyses of lipids extracted with organic solvents followed by fractionation in silica-gel-60. We detected 49 phospholipid species in Pb3 and 38 in Pb18, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. In both Pb3 and Pb18, PC and PE had the most numerous species. Among the fatty acids, C18:1 and C18:2 were the most abundant species in both isolates, although C18:2 was more abundant in Pb18. There was a different effect of plasma supplementation on fatty acids depending on the fungal isolate. The prevalent glycolipid species was Hex-C18:0-OH/d19:2-Cer, although other four minor species were also detected. The most abundant sterol in all samples was brassicasterol. Distinct profiles of cell wall and total yeast sterols suggested that the preparations were enriched for cell wall components. The presence of plasma in the culture medium specially increased cell wall brassicasterol abundance and also other lipids. CONCLUSIONS/SIGNIFICANCE: We here report an original comparative lipidomic analysis of P. brasiliensis cell wall. Our results open doors to understanding the role of cell wall lipids in fungal biology, and interaction with anti-fungal drugs and the host.

Identification of human plasma proteins associated with the cell wall of the pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermodimorphic species that cause paracoccidioidomycosis. The cell wall is the outermost fungal organelle to form an interface with the host. A number of host effector compounds, including immunologically active molecules, circulate in the plasma. In the present work, we extracted cell-wall-associated proteins from the yeast pathogenic phase of P. brasiliensis, isolate Pb3, grown in the presence of human plasma and analyzed bound plasma proteins by liquid chromatography-tandem mass spectrometry. Transport, complement activation/regulation, and coagulation pathway were the most abundant functional groups identified. Proteins related to iron/copper acquisition, immunoglobulins, and protease inhibitors were also detected. Several human plasma proteins described here have not been previously reported as interacting with fungal components, specifically, clusterin, hemopexin, transthyretin, ceruloplasmin, alpha-1-antitrypsin, apolipoprotein A-I, and apolipoprotein B-100. Additionally, we observed increased phagocytosis by J774.16 macrophages of Pb3 grown in plasma, suggesting that plasma proteins interacting with P. brasiliensis cell wall might be interfering in the fungal relationship with the host.

Lipidomic analysis of extracellular vesicles from the pathogenic phase of Paracoccidioides brasiliensis.

BACKGROUND: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. The prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells. CONCLUSIONS/SIGNIFICANCE: The extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.

Heteroplasmy in hair: study of mitochondrial DNA third hypervariable region in hair and blood samples.

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA "alleles" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate.

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.