Larissa V. G. Longo

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.

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.

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.

The Paracoccidioides cell wall: past and present layers toward understanding interaction with the host

The cell wall of pathogenic fungi plays import roles in the interaction with the host, so that its composition and structure may determine the course of infection. Here we present an overview of the current and past knowledge on the cell wall constituents of Paracoccidioides brasiliensis and P. lutzii. These are temperature-dependent dimorphic fungi that cause paracoccidioidomycosis, a systemic granulomatous, and debilitating disease. Focus is given on cell wall carbohydrate and protein contents, their immune-stimulatory features, adhesion properties, drug target characteristics, and morphological phase specificity. We offer a journey toward the future understanding of the dynamic nature of the cell wall and of the changes that may occur when the fungus infects the human host.

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.

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.

Cyclopalladated compound 7a induces apoptosis- and autophagy-like mechanisms in Paracoccidioides and is a candidate for paracoccidioidomycosis treatment

ABSTRACT 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.

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.

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.