Genomic diversity of the human pathogen Paracoccidioides across the South American continent.

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis widely reported in the Gran Chaco ecosystem. The disease is caused by different species from the genus Paracoccidioides, which are all endemic to South and Central America. Here, we sequenced and analyzed 31 isolates of Paracoccidioides across South America, with particular focus on isolates from Argentina and Paraguay. The de novo sequenced isolates were compared with publicly available genomes. Phylogenetics and population genomics revealed that PCM in Argentina and Paraguay is caused by three distinct Paracoccidioides genotypes, P. brasiliensis (S1a and S1b) and P. restrepiensis (PS3). P. brasiliensis S1a isolates from Argentina are frequently associated with chronic forms of the disease. Our results suggest the existence of extensive molecular polymorphism among Paracoccidioides species, and provide a framework to begin to dissect the connection between genotypic differences in the pathogen and the clinical outcomes of the disease.

Sporotrichosis between 1898 and 2017: The evolution of knowledge on a changeable disease and on emerging etiological agents.

The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.

Paracoccidioides Spp.: Virulence Factors and Immune-Evasion Strategies.

Paracoccidioides spp. are dimorphic fungal pathogens responsible for one of the most relevant systemic mycoses in Latin America, paracoccidioidomycosis (PCM). Their exact ecological niche remains unknown; however, they have been isolated from soil samples and armadillos (Dasypus novemcinctus), which have been proposed as animal reservoir for these fungi. Human infection occurs by inhalation of conidia or mycelia fragments and is mostly associated with immunocompetent hosts inhabiting and/or working in endemic rural areas. In this review focusing on the pathogen perspective, we will discuss some of the microbial attributes and molecular mechanisms that enable Paracoccidioides spp. to tolerate, adapt, and ultimately avoid the host immune response, establishing infection.

Differential identification of Sporothrix spp. and Leishmania spp. by conventional PCR and qPCR in multiplex format.

Sporotrichosis and cutaneous leishmaniasis are skin infections with similar clinical manifestations but different treatment methods. The present study aimed to evaluate qPCR and conventional PCR for differential detection of the etiological agents of both infections in multiplex format. Assays were designed using two sets of reported primers: SS1/SS2, designed on the 18S ribosomal RNA gene from Sporothrix spp., and JW11/JW12, designed on the kinetoplast DNA (kDNA) minicircles of Leishmania spp. qPCR detected 200 fg of DNA per reaction for both Sporothrix and Leishmania. Melting curve analysis revealed two distinctive Tm peaks for Sporothrix spp. (85.5°C), and Leishmania spp. (82.6°C). A detection limit of 20 pg was determined for the diagnosis of both with conventional PCR. No other clinically important organisms were detected by either PCR or qPCR. However, a Blast analysis on GenBank databases, using as query the sequence of the PCR fragment obtained with primers SS1/SS2, showed 100% identity to environmental fungi of the Ophiostomales order. Lower percentages of identity (≤80%), with mismatches at primers' sequence regions were obtained for other environmental or clinically important fungi. Proper handling of clinical samples is required to avoid false negatives due to contamination with environmental fungi of the Ophiostomales order.

Molecular epidemiology of human sporotrichosis in Venezuela reveals high frequency of Sporothrix globosa.

BACKGROUND: Sporotrichosis is a cutaneous and subcutaneous fungal disease of humans and other mammals, known to be caused by the Sporothrix schenckii species complex, which comprises four species of clinical importance: S. brasiliensis, S. globosa, S. luriei, and S. schenckii sensu stricto. Of them, S. globosa and S. schenckii s. str. show global distribution and differences in global frequency as causal agents of the disease. In the Americas, only three species are present: S. schenckii s. str., S. brasiliensis (so far, only reported in Brazil), and S. globosa. In Venezuela, since the first case of sporotrichosis reported in 1935, S. schenckii have been considered its unique etiological agent. In the present work, the presence of more than one species in the country was evaluated. METHODS: By phenotypic key features and molecular phylogeny analyses, we re-examined 30 isolates from diverse Venezuelan regions belonging to the fungi collection of Instituto de Biomedicina, Caracas, Venezuela, and national reference center for skin diseases. All isolates were collected between 1973 and 2013, and maintained in distilled water. RESULTS: Sporotrichosis in Venezuela is mainly caused by S. schenckii s. str. (70%). However, a significant proportion (30%) of sporotrichosis cases in the country can be attributable to S. globosa. A correlation between intraspecific genotypes and clinical presentation is proposed. CONCLUSIONS: Our data suggest that sporotrichosis various clinical forms might be related to genetic diversity of isolates, and possibly, to diverse virulence profiles previously reported in the S. schenckii species complex. Sporothrix globosa was found to be the causative agent of 30% of sporotrichosis for the Venezuelan cases re-examined, the highest frequency of this species so far reported in the Americas. The high genetic variability presented by S. schenckii s. str. indicates that species distinction based on phenotypic key features could be a challenging and uncertain task; molecular identification should be always employed.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

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.

Geographical distribution and ecological niche modeling of the etiological agents of human sporotrichosis in Venezuela.

The geographical distribution and ecological niche of the two circulating species of the Sporothrix genus in Venezuela was established. For this, 68 isolates of Sporothrix spp. from patients of different regions of the country were analyzed. A molecular taxonomy analysis was conducted using a fragment of the calmodulin gene (CAL), and ITS regions, confirming the presence of S. schenckii (62%) and S. globosa (38%). Computational models of ecological niche for each species were obtained by the maximum entropy method using the MaxEnt software, which predicted the best environmental conditions for the presence of the two species. These models predict that the main variables influencing the presence of S. schenckii were altitude and annual mean temperature, while for S. globosa, the more influent variable was the land use, with 82% of S. globosa located at urban areas vs 56% for S. schenckii. The results here presented could contribute to understand the specific environmental factors that might modulate the occurrence of Sporothrix spp. as well as its transmission. To our knowledge, our analyses show for the first time Sporothrix spp.-specific ecological niche data, a valuable tool to promote evidence-based public health policymaking within endemic areas of sporotrichosis.

Comparison of Cell Wall Polysaccharide Composition and Structure Between Strains of Sporothrix schenckii and Sporothrix brasiliensis.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-ß-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-ß-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.

The Heat Shock Protein 60 and Pap1 Participate in the Sporothrixschenckii-Host Interaction.

Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 µg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen-host interaction.

Caspofungin affects growth of Paracoccidioides brasiliensis in both morphological phases.

Five Paracoccidioides brasiliensis isolates were grown in the presence of caspofungin (0 to 1 µg/ml). Inhibition of the yeast phase ranged from 20 to 65%, while in the mycelial form it ranged from 75% to 82%. Such variability was loosely related to the amount of cell wall ß-1,3-glucan. No association with point mutations in the ß-1,3-glucan synthase was detected. Caspofungin induced physical changes and cytoplasmic deterioration in both fungal phases.

Expression of Paracoccidioides brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant demonstrates its functionality as a chitin synthase gene.

We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall.

Cell wall glucan synthases and GTPases in Paracoccidioides brasiliensis.

In this report we identified orthologues of fungal AGS1, RHO1, RHO2, RAC1 and CDC42 genes in the dimorphic fungus Paracoccidioides brasiliensis. Based on its homology to known fungal sequences, P. brasiliensis Ags1 was identified as an alpha-1,3-glucan synthase, while Rho1, Rho2, Rac1 and Cdc42 proteins were classified into the Rho1, Rho2, Rac1 and Cdc42 subgroups of fungal Rho GTPases, respectively. Of them, Rho1 is one of two subunits of a putative beta-1,3-glucan synthase complex, the other being the synthase itself (Fks1), while Rho2 has been associated to the alpha-1,3-glucan synthase (Ags1). Expression studies showed that mRNAs levels of RHO2 and AGS1 kept a direct relationship but the levels of RHO1 and FKS1 did not. P. brasiliensis RHO1 successfully restored growth of Saccharomyces cerevisiae rho1 mutant under restrictive temperature conditions. Chemical analyses of P. brasiliensis alpha-1,3-glucan, synthesized by Ags1p, indicated that it is essentially a linear polysaccharide, with <3% of alpha-1,4-linked glucose branches, occasionally attached as single units to the alpha-1,3-backbone.

Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells.

The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host-fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and ß-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.

Influences of the Culturing Media in the Virulence and Cell Wall of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are etiological agents of sporotrichosis, a human subcutaneous mycosis. Although the protocols to evaluate Sporothrix virulence in animal models are well described, the cell preparation before inoculation is not standardized, and several culturing media are used to grow yeast-like cells. Here, we found that carbon or nitrogen limitation during fungal cell preparation negatively impacted the ability of S. schenckii and S. brasiliensis to kill Galleria mellonella larvae, but not S. globosa. The fungal growth conditions associated with the short median survival of animals were accompanied by increased hemocyte countings, phenoloxidase activity, and cytotoxicity. The fungal growth under carbon or nitrogen limitation also affected the cell wall composition of both S. schenckii and S. brasiliensis and showed increased exposure of ß-1,3-glucan at the cell surface, while those growing conditions had a minimal impact on the S.globosa wall, which had higher levels of this polysaccharide exposed on the wall regardless of the culture condition. This polysaccharide exposure was linked to the increased ability of insect hemocytes to uptake fungal cells, suggesting that this is one of the mechanisms behind the lower virulence of S.globosa or cells from the other species grown in carbon or nitrogen limitation.

Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration.

The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583nt open reading frame, interrupted by three introns of 82, 87 and 97bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4kb from the 5' UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxidative stress, alteration of external osmolarity and under glucose-repressible conditions, suggesting a common regulatory mechanism of transcription.

New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen.

By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the genomic variation known in this genus.

Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.

Fungal Strategies to Evade the Host Immune Recognition.

The recognition of fungal cells by the host immune system is key during the establishment of a protective anti-fungal response. Even though the immune system has evolved a vast number of processes to control these organisms, they have developed strategies to fight back, avoiding the proper recognition by immune components and thus interfering with the host protective mechanisms. Therefore, the strategies to evade the immune system are as important as the virulence factors and attributes that damage the host tissues and cells. Here, we performed a thorough revision of the main fungal tactics to escape from the host immunosurveillance processes. These include the composition and organization of the cell wall, the fungal capsule, the formation of titan cells, biofilms, and asteroid bodies; the ability to undergo dimorphism; and the escape from nutritional immunity, extracellular traps, phagocytosis, and the action of humoral immune effectors.