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

Sporotrichosis between 1898 and 2017: The evolution of knowledge on a changeable disease and on... Abstract 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. Sporothrix schenckii, Sporothrix brasiliensis, Sporothrix globosa, genome, sporotrichosis Etiology of sporotrichosis: historical aspects The description of the first clinical case of cutaneous sporotrichosis was published in 1898 by Benjamin Schenck.1 The yeast-like pathogenic phase and the lymphocutaneous / extracutaneous forms of this disease were observed in humans and in rats in 1907 by Lutz and Splendore.2 In the century that followed, but particularly during the last decades, an enormous expansion took place in the knowledge of this mycosis on humans and animals. Now that genetic and molecular tools have become available,3 some classical viewpoints deserve renewed attention. Particularly, the description of cryptic species with pathogenic potential has changed the visions on this disease.4 The biology and genomic data of these fungal pathogens, cell wall antigens, the host immune response, as well as some epidemiological and diagnostic aspects associated with emerging etiological agents of sporotrichosis, will be the focus of this review. Today, nearly all research in fungal pathogens has become multidisciplinary. Since 2011 a Working Group on Sporotrichosis is active under the umbrella of ISHAM, where clinicians cooperate with basic scientists, bioinformaticians, and epidemiologists; the present review is one of the product of this broad cooperation. The recently described species S. brasiliensis and S. globosa, which are prevalent in South America and East Asia, respectively,5–7 together with S. schenckii (sensu stricto) and S. luriei make up the ‘pathogenic clade’ of the genus Sporothrix.8 It is important to notice that S. luriei has a low clinical-epidemiological impact within this genus. This revision will cover the clinically relevant species: S. schenckii, S. brasiliensis and S. globosa. Sporothrix species are dimorphic fungi that present a saprophytic mycelium phase at room temperature (25–28°C) and a yeast-like pathogenic phase at 36–37°C. Sporothrix propagules usually entry the warm-blooded host through minor cutaneous trauma from contaminated plant debris or through scratches or bites from animals (mostly felines) carrying the fungus. Often multiple infections arise from a single source, leading to outbreaks of the disease, potentially with thousands of patients.6 The classical species S. schenckii is related to a benign chronic subcutaneous mycosis, and in its restricted sense the species exhibits a moderate virulence profile in animal models.9,10 In contrast, S. brasiliensis is highly virulent in animal models and is associated with severe clinical forms of sporotrichosis.9–16 Interestingly, both S. brasiliensis and S. globosa are less susceptible to itraconazole, judging from clinical reports and animal model studies.17,18 Of the recently described siblings, S. globosa (formerly reported as S. schenckii) is a well-established agent of prevalently plant-transmitted infection,19 but S. brasiliensis is emerging only since the nineties of the previous century and shows preponderance of cat-transmission. Both routes of infection are common in the ancestral species S. schenckii. The differential biology of these newly emerging species may trigger different host recognition and immune response.3 Clinical-epidemiological profiles and the evolutionary processes of S. brasiliensis and S. globosa differ from each other and from S. schenckii in adaptation to mammal tissue.5,19–21 A recent evolutionary specialization seems to have taken place in the two siblings, which has led to these remarkably deviating sources of infection and transmission routes between these closely related species. There is a consensus that sporotrichosis basically is an implantation disease. However, some authors refer to inhalation as a possible route of infection,22 but not proved experimentally. Although the most common types of sporotrichosis are the lympho-cutaneous and fixed cutaneous forms, atypical, extracutaneous forms and mucosal sporotrichosis have also been reported.23 Cat-transmitted S. brasiliensis infection was observed for the first time around 2000, in Rio de Janeiro, Brazil,20,21 showing exponential expansion, even though a close interaction of stray cats and susceptible hosts existed already for hundreds of years. How does feline sporotrichosis arise? How Sporothrix did become adapted to animal infection? These are key questions to be answered that can help us to understand the change in sporotrichosis from an uncommon, benign disease to an important emerging mycosis with severe clinical forms in immunocompromised as well as in immunocompetent hosts. The work published by Lutz and Splendore in 1907,2 who described the yeast parasitic phase of S. schenckii, can bring some light to cat infection. They also studied Sporothrix infection in Rattus novergicus (brown rats) and part of their findings must be highlighted here: “One of us (Lutz), from many years had been aware of special lesions that manifest themselves in the common rat … we were able to collect a total of more than 40 cases of infected animals. The most common form of this mycosis, which is spontaneously observed in the rat, consists of lesions located at the extremities and the tail. It usually appears in the tarsal region of one or more extremities, or at any point of the tail, a local swelling, remembering the affections produced in the man by the articular tuberculosis …. There are also internal locations in the form of isolated and few numerous miliary tubers that are seen in the spleen, liver, lungs, kidneys, genital glands and internal serosa ….” - translated from the original manuscript of Lutz and Splendore, published in Portuguese. In summary, Lutz and Splendore had observed sporotrichosis in wild rats and experimental animals, describing osteo-articular and cutaneous lesions. They also hypothesized that sporotrichosis can be acquired by ingestion based on their experimental model.2 This can represent a host shift in S. brasiliensis infection (unknow species until 2007) from rats to cats in the last century. One of the major predators of rats are cats. Furthermore, Sporothrix infection by ingestion was proposed by other authors.23 Some aspects of the possible adaptation of S. brasiliensis to cat saliva will be discussed later. Another important aspect is the fact that the feline sporotrichosis epidemic has started in Rio de Janeiro State, Brazil, in areas of low socioeconomic status and, precarious sanitary and health system, with high demographic density of both the human and feline population.24,25 There is no information collected from rats in these endemic areas. The skin disease that had long been considered as a benign occupational disease of horticulturalists, acquired mainly by the manipulation of contaminated soil or plant material (such as Sphagnum moss), has in fact become an urban problem with a peri-domicile character. The high zoonotic potential of S. brasiliensis compared to S. schenckii21 has led to an increasing number of severe and /or systemic reported clinical cases.12–16,26,27 In parallel with the development of molecular tools, cell biological models are now available to understand pathogenesis and the balance between virulence factors of ancestral and evolved Sporothrix species, as well as the host's immune status. Global distribution patterns of S. brasiliensis and S. globosa Sporotrichosis occurs worldwide, but the infectious agents are not evenly distributed since the main pathogenic species of Sporothrix have high degrees of endemicity. Sporothrix is unique in the fungal kingdom by its prevalent occurrence in the form of outbreaks and in that these outbreaks differ fundamentally from each other between closely related species. In S. brasiliensis a huge zoonosis is taking place today, while the contemporary outbreak of similar dimensions in China is a sapronosis.19S. globosa exhibits a global distribution pattern and is involved in an expanding sapronosis with preponderant plant transmission.5,28 The contemporary zoonotic outbreak with similar dimensions in Brazil is caused by S. brasiliensis and geographically limited to Brazil.20,21 In China, sporotrichosis exists nationwide but is mainly observed in the Northeastern provinces. S. globosa is the overwhelming pathogen, with several thousands of cases having been reported from China,19,28–30 while there are only four known isolates of S. schenckii from Jiangxi province and Hunan province, respectively.19 With lower endemicity outside Asia, S. globosa exhibits a global distribution. It mainly causes benign, fixed or lymphocutaneous infections and is never vectored by animals.19,31 The species is hypothesised to grow exponentially in rotten, self-heated plant debris like corn or reed that serves as a potential inoculum for plant harvesters in northeast China, where the prevalent clinical type is facial.32 Upon removal of the infectious material, the human sapronosis will die out with some delay, matching with the observation that most cases in China's Jilin Province become apparent during winter following harvest. Several studies showed a relationship between geographical distribution and genotypes among species of Sporothrix.4,19,33 With the exception of Africa and Australia, S. globosa has been reported worldwide, similar to its hypothesized ancestral species S. schenckii, but at different prevalence: the latter species is preponderant in eastern South America, Africa, and Australia.19 Epidemiological studies proved that strains of S. globosa from Brazil, China, Japan, Spain, and the United Kingdom had nearly identical genotypes.19,30 An as yet unknown mechanism seems to be involved in the rapid dispersal of S. globosa. Given the large geographical distances between the localities harboring genetically indistinguishable isolates, airborne distribution seems to be the most plausible explanation. The absence of reported S. globosa infections and environmental isolates from Africa and Australia may perhaps be explained by sampling effects. Notably, S. globosa infections are derived from plant debris and this infection is classically known as ‘reed toxin’.30 Sporothrix globosa strains analysed thus far were nearly identical. Isolates studied by Yu et al.30 were divided into two highly supported subclades (S. globosa I and S. globosa II). Group I comprised the majority of Chinese clinical isolates, three Chinese environmental isolates from reed, corn stalks, and soil, the type strain of S. globosa, and some isolates from the United States, India, Japan, Brazil, and the United Kingdom. Sporothrix globosa II included some Chinese clinical isolates and a single isolate from Italy. More material is needed to ascertain whether S. globosa is preponderantly clonal. Interestingly, cats have never been observed as sources of infection in endemic areas of S. globosa, in contrast to the large-scale, expanding outbreak of S. brasiliensis among humans and cats in South America. Sporothrix brasiliensis also shows low degrees of variability and has been suggested to be clonal.7 The low variability of this taxon is supported by low chromosomal polymorphisms and homogeneous susceptibility profiles to antifungal agents. In Southeast Brazil, transmission nearly always occurs by cats.20 Cat saliva is a stable environment, and despite the presence of antibodies—which generally have a low impact on fungal infections—repeated colonization by Sporothrix once it has adapted to these conditions may be expected. The large outbreak in this area suggests that the number of cases increases relative to the number of patients and cat vectors. Several peculiarities of cats may facilitate the dispersal of the fungus in the environment within limited endemic areas. First, they are the most common pet animals with close contact to humans. Second, given the hypothesized origin of Sporothrix in cat saliva and its transmission to claws during licking, cat mobility and clawing enable them to take up and transmit the fungus, either to each other during play or fight with house cats or stray cats, or to human hosts via scratches or bites.20 Thus, despite the preponderance of cat vectors, the animal host species may vary, just as the plant host species did. This leads us to a hypothesis of wild animals occasionally providing conditions similar to those in fermented plant material. Cats take up propagules from the soil and easily transmit them to their mouth by licking. Conditions in animal saliva at the feline body temperature (normal range 37.7−39.1°C) might be a stimulating factor for the production of the Sporothrix yeast phase.20 Cat saliva has a pH of 7.5−8.0, which is similar to that of self-heating bulk corn debris (around 8.0) and optimal for the mould-to-yeast conversion.20 We evaluated the impact of the feline host on the epidemiology, spatial distribution, prevalence and genetic diversity of human sporotrichosis. Nuclear and mitochondrial markers revealed large genetic differences between S. brasiliensis and ancestral S. schenckii populations, suggesting that the interplay of host, pathogen and environment has a structuring effect on the diversity, frequency, and distribution of Sporothrix species. Phylogenetic data support a recent habitat shift within S. brasiliensis from plant to cat. According to the above it should be plant to rat and then rat to cat that seems to have occurred in Southeastern Brazil and is responsible for its emergence. A clonal structure was found in the early expanding phase of the cat–human epidemic.34 However, the preponderant recombination structure in the prevalently plant-associated pathogen S. schenckii generates a diversity of genotypes that did not show any significant increase in frequency as pathogens of human infection over time.7,21 These characters suggest that closely related causative agents can follow different strategies in epidemics. Thus, species-specific types of transmission may require public health strategies to consider these distinctions. Sapronoses, providing very special conditions promoting fungal growth, basically can be controlled by removal of the plant biomass allowing this contamination. In contrast, the zoonoses of cats compose a much more diffuse source of infection, which is difficult to control. With a hypothesis of conditional similarities between fermenting plant material and animal digestive saliva, the unique host shift of Sporothrix from plant to animal becomes understandable. Therefore, we hypothesise that Sporothrix species are not plant pathogens but require particular conditions in decaying plant material, which are reached only occasionally during advanced decomposition. We postulate that a particular state of decay and fermentation of the plant material promotes excessive growth of Sporothrix. High temperature and humidity, associated with metabolic changes (induction of respiratory system) and oxidative stress during decay and fermentation may shift the morphology, favoring the invasive yeast growth form. What does the genome tell us? To date, the genomes of S. schenckii, S. brasiliensis, and S. globosa, for the clinically important species, as well as the nonvirulent environmental species, Sporothrix pallida have been sequenced, although only annotated for S. schenckii and S. brasiliensis.35,36,37 The genome is similar in size for S. schenckii, S. brasiliensis, and S. globosa, although the number of predicted protein encoding genes are notably variable for the three species, with a larger number of putative genes in S. schenckii and the lowest number present in S. globosa (Table 1). However, when compared with the environmental, nonvirulent species S. pallida, some outstanding differences are apparent. S. pallida genome is almost 5 Mb larger than the medically important species and shows higher number of predicted protein encoding genes. It has been proposed that pathogenic fungi with an environmental phase in their life cycle, have evolve to survive in the mammalian host through processes of genetic expansion and contraction of gene families, which ultimately allows the fungus to colonize and survive the environmental hazards of the mammalian immune system.35,38,39 Since the genomes of S. globosa and S. pallida are only present as draft genomes in the public databases, it is not possible to produce a comparative analysis of the four genomes above mentioned, to address the question of adaptation to the mammalian host. However, recently Teixeira et al.35 published a thorough comparative analysis of the S. schenckii and S. brasiliensis genomes with 14 fungi, either dimorphic or plant-associated, addressing that same question. In Coccidioides spp., a reduction of plant cell wall degrading enzymes and an expansion of peptidase genes has been related to adaptation to the mammal host and lead to the suggestion that Coccidioides spp. are not soil saprophytes but have evolved to remain associated to their dead host in soil.38 On the other hand, dimorphic pathogens with a saprophyte stage in their life cycle, such as Paracoccidioides spp. present expansion of peptidase gene families, related to their adaptation to the mammal host, but keep the plant cell wall degrading enzymes genes, related to their saprophyte stage.39 In their work, Teixeira et al.35 did not find an expansion of peptidase genes in the S. schenckii and S. brasiliensis genomes; however, they found lack of plant decay-associated polysaccharide lyase genes, present in other soil related Sordariomycetes, which lead the authors to suggest evolutionary adaptation of S. schenckii and S. brasiliensis from plant pathogens or saprobes to an animal pathogenic life style.35 Table 1. Genomes size and number of predicted coding genes for Sporothrix spp.* Organism  Genome size (Mb)  Predicted protein coding genes  G + C content  tRNA  S. schenckii  32.4  10 293  62%  139  S. brasiliensis  33.2  9091  62%  140  S. globosa  33.5  7719–7760  54.37%  126  S. pallida  37.8  11 356  52.8%  151  Organism  Genome size (Mb)  Predicted protein coding genes  G + C content  tRNA  S. schenckii  32.4  10 293  62%  139  S. brasiliensis  33.2  9091  62%  140  S. globosa  33.5  7719–7760  54.37%  126  S. pallida  37.8  11 356  52.8%  151  *Adapted from Teixeira et al.35; D’Alessandro et al.36; and Huang et al.37 View Large Fungal dimorphism The dimorphism exhibited by S. schenckii sensu lato is also found in other human pathogenic fungi, such as Coccidioides immitis, C. posadasi, Blastomyces dermatitidis, B. gilchristii, Talaromyces marneffei, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, and Emmonsia spp., and is essential for the establishment of the infection. Interestingly, Sporothrix belongs to a different taxonomic order than the above listed organisms, indicating that this phenotypical trait is a result of convergent evolution.35 Even though there is genetic distance between Onygenales and Ophiostomatales, it is likely that dimorphic members of both orders share similar molecular mechanisms controlling the dimorphic process. The histidine kinase drk1 participates controlling the mycelium to yeast transition in both B. dermatitidis and H. capsulatum.40,41 The putative ortholog to this gene has been identified in S. schenckii and is likely to be involved in regulating the dimorphic process.42 Another important player in regulating the dimorphic process in H. capsulatum is the gene required for yeast phase growth (RYP1), which is a transcriptional regulator of yeast-specific genes and a functional ortholog of Candida albicans WOR1, the master regulator not only of the yeast-to-hypha transition but the mating and the phenotypical switch.40 Bioinformatics analyses found the putative RYP1 ortholog as part of both S. schenckii and S. brasiliensis genomes.35 In addition, RYP2, RYP3, and VEA1 have been involved in the formation of H. capsulatum yeast-like cells,43 and the putative orthologs are found within the Sporothrix genome (GenBank accession codes: XP_016590923.1, ERS98419.1, and XP_016588232.1, respectively). In T. marneffei, the p21-activated kinase pakB is required to avoid yeast-like cell formation at 25°C, temperature where the fungus grows like hyphae.44 The putative orthologs are found in the genome of both S. schenckii sensu stricto and S. brasiliensis (GenBank accession codes: XP_016583587.1 and KIH88219.1, respectively). The transcription factor hgrA has a hypha-specific expression responsible of the hyphal growth program in T. marneffei, and its expression must be repressed to undergo dimorphism.45 Interestingly, the putative ortholog is found within the S. brasiliensis genome (GenBank accession code KIH95137.1) but not as part of S. schenckii. Cell wall: a glycobiology overview The fungal cell wall is an essential structure for cell viability and interaction with the environment, and contains macromolecules involved in the virulence of pathogenic fungi.46 Since it is composed of compounds that are not synthesized by the human and animal hosts, the study of the biosynthetic pathways involved could potentially lead to the discovery of additional molecules with antifungal properties. The sequencing and gene annotation of the S. schenckii and S. brasiliensis genomes have provided a closer view of the synthesis of glycoproteins, β-glucans, and chitin in these organisms.35 Protein glycosylation Glycoproteins are key components of the Sporothrix cell wall and among the first aspects studied in this organism.47 In the fungal cell, as in other kind of eukaryotic cells, there are three main types of protein glycosylation: addition of oligosaccharides to residues of Asn (N-linked glycosylation), to Ser/Thr (O-linked glycosylation) or incorporation of the glycolipid glycosylphosphatidylinositol (GPI) at the C-terminal end of the polypeptide. The first steps in the elaboration of N-linked oligosaccharides are extremely conserved in eukaryotic cells, and as expected, the bioinformatic analysis of both S. schenckii and S. brasiliensis genomes revealed the presence of all the elements required for synthesis and processing of the N-linked glycan core (see supplementary Table 1S). The first step during elaboration of the N-linked oligosaccharides is the synthesis of a precursor attached to dolichol phosphate (Dol-P), which is within the endoplasmic reticulum (ER) membrane. This stepwise process is highly conserved in all eukaryotes and is performed in two stages: first at the ER cytosolic face, where the glycolipid Dol-P-Man5GlcNAc2 is synthesized, followed by a luminal stage where the glycolipid is further elaborated to generate Dol-P-Glc3Man9GlcNAc2, also named N-linked glycan precursor.48,49 The N-linked glycan precursor is then transferred in bloc to an Asn residue within the N-linked glycosylation sequon Asn-X-Ser/Thr (where X can be any amino acid except Pro) by the oligosaccharyl transferase complex.50 The N-linked glycan on the glycoprotein surface then undergoes processing by ER α-glucosidases I and II that remove the outermost α-1,2-glucose unit and the two remaining glucose residues, respectively,51–53 and the ER α-1,2-mannosidase that trims one α-1,2-mannose residue generating Man8GlcNAc2 isomer B.52–54 The N-linked glycan biosynthesis has been thoroughly studied in Saccharomyces cerevisiae and in a lesser extent in Aspergillus fumigatus. Table 2 shows a comparative analysis of these organisms with S. schenckii and S. brasiliensis, indicating that Sporothrix spp. contain all the genes involved in the synthesis of Glc3Man9GlcNAc2 and transfer to nascent proteins and processing by glycosidases. Table 2. Genes involved in O-linked glycosylation in S. cerevisiae, A. fumigatus, S. schenckii, and S. brasiliensis. Protein function  S. cerevisiae1  A. fumigatus2  S. schenckii3  S. brasiliensis3  ER mannosyltransferases          Dol-P-Man:protein-O-D-mannosyltransferase  PMT1  pmt1  SS05892  SB04624  Dol-P-Man:protein-O-D-mannosyltransferase  PMT2  pmt2  SS08548  SB01344  Dol-P-Man:protein-O-D-mannosyltransferase  PMT4  pmt4  SS08628  SB08186  Golgi mannosyltransferases          α1,2-Mannosyltransferase  KTR1  mnt1  SS09069  SB08384  Protein function  S. cerevisiae1  A. fumigatus2  S. schenckii3  S. brasiliensis3  ER mannosyltransferases          Dol-P-Man:protein-O-D-mannosyltransferase  PMT1  pmt1  SS05892  SB04624  Dol-P-Man:protein-O-D-mannosyltransferase  PMT2  pmt2  SS08548  SB01344  Dol-P-Man:protein-O-D-mannosyltransferase  PMT4  pmt4  SS08628  SB08186  Golgi mannosyltransferases          α1,2-Mannosyltransferase  KTR1  mnt1  SS09069  SB08384  1Standard names were retrieved from http://www.yeastgenome.org/. 2Systematic names were retrieved from http://www.aspergillusgenome.org/. 3Systematic names were retrieved from Teixeira et al.35 View Large Subsequently, the glycoproteins are transported to the Golgi complex where they are further modified by glycosyltransferases. In filamentous fungi and higher eukaryotes there are Golgi mannosidases IA, IB and IC that process Man8GlcNAc2 to Man5GlcNAc2, just before action of transferases.55–57 This processing step is critical to establish the final structure of N-linked oligosaccharides, which in fungal cells can be either high mannose or hybrid N-linked glycans. Organisms lacking Golgi mannosidases, such as S. cerevisiae, exclusively elaborate high mannose N-linked glycans.48 The presence of Golgi-resident mannosidases IA, IB, and IC in S. schenckii cells,53 strongly suggest that this organism modifies the N-linked glycan core to generate hybrid N-linked glycans. This is in line with the previously described N-linked rhamnomannan structure.47 Interestingly, Sporothrix does not have any obvious ortholog for Golgi mannosidase II, a key enzyme in the elaboration of complex N-linked glycans in higher eukaryotes.58 In S. cerevisiae, there are exclusively Golgi mannosyltransferases and they are responsible of the elaboration of the N-linked glycan outer chain. The first reaction is catalyzed by the α1, 6-mannosyltransferase Och1 followed by the α1, 6-mannosyltransferase complexes MolP-I and MolP-II, generating an α-1, 6-polymannose backbone.59 Both S. schenckii and S. brasiliensis genomes have the genes encoding these enzyme activities and the OCH1-like gene family, including OCH2, OCH3 and OCH4.60 Again, this data support elaboration of the N-linked glycan outer chain reported previously.47 The S. schenckii and S. brasiliensis genomes also contain genes encoding members of the KRE2/MNT1 gene family, and at least one member is likely to participate in the modification of the outer chain backbone, with branches of α1, 2-mannose residues.61 The presence of a gene with significant similarity to those encoding the N-acetylglucosaminidase III (see supplementary Table 2S) that adds the bisecting GlcNAc residue found in both hybrid and complex N-linked glycans62 supports our hypothesis on the N-linked glycan structure in S. schenckii sensu lato. Presence of galactose-containing glycans has been reported63 and some putative galactosyltransferases have been identified during the genome analysis (see supplementary Table 1S), although it remains to be addressed whether these enzymes participate in elaboration of glycoproteins and/or glycolipids. Furthermore, S. schenckii and S. brasiliensis contain an ortholog of Aspergillus nidulans ugmA, whose products generates UDP-galactofuranose from UDP-galactopyranose, the galactomannan-building sugar donor in Aspergillus.64 Sialic acid has been reported as component of the S. schenckii cell wall glycolipids.65 So it is likely that the putative Golgi CMP-sialic acid transporter (see supplementary Table 2S) is involved in modification of such lipids. Figure 1A shows the current working model on the N-linked glycan structure based on bioinformatics. The processing of the N-linked oligosaccharide in the ER does not only have a significant role during N-linked glycan elaboration, but also in the glycoprotein ER-associated degradation, responsible of labeling misfolded glycoproteins for degradation by the cytosolic proteasome.66 The glycoproteins carrying the monoglucosylated GlcMan9GlcNAc2N-linked oligosaccharide generated by ER glucosidase II52 are recognized by the complex calnexin-Pdi1 to promote protein refolding.66 The glycoprotein-chaperone complex interaction is then broken by glucosidase II that trims the last glucose residue. The UDP-glucose:glycoprotein α-glucosyltransferase senses the surfaces of the released glycoproteins and those that are still misfolded undergo glucosylation of the Man9GlcNAc2 oligosaccharide, allowing interaction again with the chaperon complex.66 This glucosylation / deglucosylation cycle will continue until the protein is properly folded or alternatively, can be disrupted by ER α-1,2-mannosidase and EDEM proteins that demannosylate the Man9GlcNAc2 oligosaccharide53 generating glycans unable to be recognized by the UDP-glucose:glycoprotein α-glucosyltransferase. If the glycoprotein is still misfolded upon the cycle has been disrupted by the ER α-1,2-mannosidase and EDEM proteins, it suffers retrograde transport to the cytosolic compartment for degradation.67 This glycoprotein quality control system is likely to occur in both S. schenckii and S. brasiliensis, as all basic components are present in their genomes.53 Figure 1. View largeDownload slide Hypothetical structures of N-linked (panel A) and O-linked glycans (panel B) generated from the bioinformatics analysis of S. schenckii and S. brasiliensis genomes Figure 1. View largeDownload slide Hypothetical structures of N-linked (panel A) and O-linked glycans (panel B) generated from the bioinformatics analysis of S. schenckii and S. brasiliensis genomes Similar to N-linked glycosylation, elaboration of O-linked glycans starts in the ER, where a mannose residue is transferred from the Dol-P-Man donor to a Ser- or Thr-residue at the surface of a nascent protein by protein mannosyltransferases, encoded by members of the PMT family,68 which is composed of three enzymes in both S. schenckii and S. brasiliensis (Table 2). Then, the O-linked glycans are elongated by members of the KRE2/MNT1 gene family, which generate linear α1,2-mannose polymers69 that in S. schenckii may contain two mannose units.70 Since some members of the KRE2/MNT1 gene family can add mannose units to both N-linked and O-linked glycans,71–73 it is possible that the three members of this gene family found in Sporothrix can work in both pathways. Experimental evidence suggests that at least one of them can participate in both O-linked and N-linked glycosylation pathways.61 The best characterized O-linked glycans were isolated from the peptido-rhamnomannan (PRM) fraction and contains an α1, 2-mannobiose core, one α1,2-glucuronic acid unit substituted by one or two rhamnose residues.70 The S. schenckii and S. brasiliensis genomes contain three putative glucuronosyltransferases that might participate in the elaboration of this O-linked glycan (Table 2). Our bioinformatics analysis could not find any obvious ortholog for rhamnosyltransferases, but both Sporothrix species contain all the required genes for synthesis of UDP-L-rhamnose (Table 2) and the sugar donor in the enzyme reaction catalyzed by rhamnosyltransferases.73–75 Figure 1B shows the current working model of the O-linked glycan structure based on these bioinformatic analyses. Finally, synthesis of GPI is extremely conserved in eukaryotic cells and the genomes of both Sporothrix species contain all genes to elaborate this glycolipid (see supplementary Table 2S). Interestingly, the S. schenckii genome lacks a putative ortholog of S. cerevisiae GPI11 (see supplementary table 2S) that encodes for a small subunit of the luminal phosphoethanolamine transferase complex.76 Since S. brasiliensis has a putative GPI11 ortholog, it remains to be established whether this difference significantly affects the structure of GPI anchors in S. schenckii and S. brasiliensis. Cell wall polysaccharides The cell wall provides protection to the fungus, acting as an initial barrier against hostile environments, while preserving the cell's integrity against internal turgor pressure. It is a dynamic structure, presenting continuous changes in composition and structural organization as the cell grows or presents morphological changes.77 These changes are strongly regulated during the cell cycle and in response to changing environmental conditions, stress, and mutations in cell wall biosynthetic processes.78,79 Over 90% of its components are polysaccharides, which are present as alkali-insoluble and alkali-soluble polysaccharides. The alkali-insoluble polysaccharides are arranged into a fibrillar skeleton, and are mainly composed of branched β1,3-glucan cross-linked to microfibers of chitin by β-1,4-linkages.77 Their proportions change according to the fungal species, and in some cases, to the cell type in dimorphic or polymorphic fungi.80 The alkali-soluble matrix is composed of amorphous polysaccharides, whose chemical composition also changes according to the fungal species.80,81 In S. schenckii, cell wall components of the yeast (Y), mycelial (M), and conidial (C) forms have been determined.82 Chitin is a minor component (7 to 8%) of the cell wall's fibrillar skeleton in all three cell types, while β-Glucan is a major component. β-glucan is present as a linear glucan, mainly composed of β-1,3-linkages (up to 66%), with β-1,6-(26 to 29%) and β-1,4-linkages (5 to 10%) in all three cell forms.82 The alkali-soluble fraction of S. schenckii cell wall is mainly composed of β-glucans and rhamnomannans, the later also complexed as a peptido-rhamnomannan after extraction by milder methods.83 The structure of the alkali-soluble β-glucan is like the alkali-insoluble β-glucan, although with differences in the proportions of linkages present in the polysaccharide (β-1,3: 44–45%; β-1,6: 28–31%; β-1,4: 24–28%).82 No variations in β-glucan-chitin composition can be related to the morphology of the fungus, since their relative composition is similar in the yeast, mycelial and conidial forms.82 In fungi, the synthesis of cell wall β-1,3-glucan is carried out through a complex formed by the catalytic unit (β-1,3-glucan synthase; Fks1), activated by Rho1, the GTP-dependent regulatory subunit in the β-1,3-glucan complex.84,85 The genomes of S. schenckii and S. brasiliensis contain single FKS orthologues35 (Table 3), which are different from those of C. albicans and S. cerevisiae, both of which present three β-1,3-glucan synthase genes with different functions in vegetative and conidial growth.84,86 However, several dimorphic and filamentous fungi present single FKS orthologues, as in Sporothrix species.35 No genes related to the synthesis of either β-1,6- or β-1,4-glucans could be identified, although hydrolase orthologs for the three types of β-glucan linkages present in the S. schenckii cell wall are present in both genomes (Table 3).35 Table 3. Genes identified as potentially involved in glucan synthesis and hydrolysis in S. schenckii and S. brasiliensis.* Protein function  S. schenckii  S. brasiliensis  Endo-β-1,3-glucanase (Cazy 81)  SS05995  SB04729  β-1,3-glucanase (Cazy 64)  SS03158  SB00339  β-1,3(4)-glucanase (Cazy 16)  SS02566  SB03506  β-1,6-glucanase (Cazy 2)  SS06336  SB05043  β-1,3-glucan synthase  SS01365  SB04029  Protein function  S. schenckii  S. brasiliensis  Endo-β-1,3-glucanase (Cazy 81)  SS05995  SB04729  β-1,3-glucanase (Cazy 64)  SS03158  SB00339  β-1,3(4)-glucanase (Cazy 16)  SS02566  SB03506  β-1,6-glucanase (Cazy 2)  SS06336  SB05043  β-1,3-glucan synthase  SS01365  SB04029  *Modified from Teixeira et al.35 View Large Chitin synthesis in fungi is regulated by multigene families, encoding chitin synthase isoenzymes.87–89 Their activities are spatially regulated to fulfil their roles.87 Seven chitin synthase (CHS) genes are present in both S. schenckii and S. brasiliensis genomes. Each one of the translation products of the seven CHS Sporothrix genes identified can be classified into each one of the seven chitin synthase classes known (I to VII, Table 4).35 The large number of chitin synthase genes identified in S. schenckii and S. brasiliensis contrasts with the low chitin content of the Sporothrix cell wall (7 to 8% of total cell wall content). In S. cerevisiae, where only three CHS genes have been identified, chitin synthesis is regulated both temporarily and spatially in relation to the cell cycle.90 However, in this fungus, none of the CHS genes is essential, although the triple mutant is impaired. In contrast, C. albicans has four CHS genes, of which CHS1 (whose translated product, Chs1 belongs to class II) is essential for cell viability.90 The significance of each of the seven chitin synthase classes is not yet understood, as mutations affecting members of the same enzyme class, do not always result in a similar phenotype.91 This suggests that the different fungal Chs enzymes perform distinct and specific functions in every fungus, even though they have homologous sequences. Table 4. Genes identified as potentially involved in chitin synthesis in S. schenckii and S. brasiliensis. Protein function  S. schenckii  S. brasiliensis  CHS class  Proposed gene name  Chitin synthase  SPSK08492  SPBR08106  I  CHS1    SPSK06891  SPBR02298  II  CHS2    SPSK06989  SPBR02297  III  CHS3    SPSK07523  SPBR06424  IV  CHS4    SPSK06859  SPBR02173  V  CHS5    SPSK00405  SPBR08833  VI  CHS6    SPSK6887  SPBR02195  VII  CHS7  Protein function  S. schenckii  S. brasiliensis  CHS class  Proposed gene name  Chitin synthase  SPSK08492  SPBR08106  I  CHS1    SPSK06891  SPBR02298  II  CHS2    SPSK06989  SPBR02297  III  CHS3    SPSK07523  SPBR06424  IV  CHS4    SPSK06859  SPBR02173  V  CHS5    SPSK00405  SPBR08833  VI  CHS6    SPSK6887  SPBR02195  VII  CHS7  *Modified from Teixeira et al.35 View Large Large numbers of chitinases in fungi have been proposed as being directly related to the chitin content in the cell wall.92 However, this clearly does not apply to Sporothrix spp., where 10 chitinase genes have been found in the S. schenckii genome while nine are present in that of the S. brasiliensis genome.35 The functions of such large numbers of chitinases is yet to be elucidated. The polysaccharide synthesis and hydrolysis-related genes identified in S. schenckii and S. brasiliensis genomes35 correlate with the polysaccharide composition of the cell wall previously reported for S. schenckii.82 As in many fungi, there is still a long way to go in determining whether individual cell wall polysaccharides synthase and/or hydrolase-related genes here discussed, might be involved in shaping the yeast, mycelial and/or conidial cell forms of Sporothrix spp., how they could be related in host-fungal interactions, or even if any of them might have a role in the fungal survival, and therefore being targets for the development of specific antifungal drugs. Diagnosis and identification of clinically relevant Sporothrix species Direct mycological examination using potassium hydroxide (KOH) or differential staining is low sensitive for the diagnosis of human sporotrichosis due to the scarcity of fungal elements in the lesions, particularly in lymphocutaneous and fixed cutaneous forms. However, Gram, Giemsa, Periodic-Schiff (PAS), and Grocott-Gomori's staining (silver staining) can be successfully used in disseminated manifestations. On the other hand, in feline sporotrichosis there is a high fungal load in the lesions favoring direct examination for the rapid diagnosis of the disease.93 In general, round, oval, often elongated yeasts are observed, resembling a cigar shape. Although direct examination does not allow differentiation of Sporothrix species, it is important to rule out other cutaneous sporotrichoid infections.94 The gold standard for diagnosing Sporothrix is based on conventional culture of clinical specimens obtained from active lesions, pus, secretions or biopsy.95 Samples are cultured on Sabouraud agar and may be followed by antifungal susceptibility testing and additional phenotypic characterization on a case-by-case basis. Positive cultures appear in the first 2 weeks of incubation, however, in some cases it will be necessary to observe for up to 30 days before discarding them as negative. Cultures held at 25°C develop thin hyphae with erect conidiophores bearing several hyaline single-celled conidia, disposed in a flower-like arrangement. Numerous sessile, brown, (sub)globose, or triangular conidia are visible along undifferentiated hyphae. Note that this phenotypic character is shared by several environmental Sporothrix species.96 Demonstration of dimorphism is important to confirm the supposed agent.97 Phenotypic identification often requires 7 to 14 days for culture, and 10 to 21 additional days for physiological assays.96 Moreover, morphological characteristics are insufficiently diagnostic for the clinically relevant species.98 Molecular tools are required for the recognition of cryptic entities. Accurate molecular diagnostic tests have a key role in patient management guiding therapy99,100 and from an epidemiological point of view it will help to recognize and control nascent outbreaks due to distinct Sporothrix species.21 Sequencing reduces identification time to about 12 h, with an improved diagnosis. Phylogenetic analyses based on the rRNA operon supports monophyletic of human-pathogenic Sporothrix group.8,21,101 Global sampling of clinical Sporothrix does not affect the success of species recognition by ITS sequencing confirming the robustness of this marker.102 A striking phylogenetic bipartition is observed between the pathogenic species S. brasiliensis, S. schenckii, S. globosa, and S. luriei and remaining environmental Sporothrix species living in association with soil and plant debris.8 It is noteworthy that the taxonomic resolution of the ITS region (ITS1/5.8 s/ITS2) is low among agents embedded in the S. pallida and S. stenoceras complexes. In these cases, it is necessary to use protein-coding loci for the recognition of cryptic entities.33 Frequently used protein-coding loci include beta-tubulin (BT2), calmodulin (CAL), and elongation factor 1α (EF-1α). Multilocus analysis significantly increases the taxonomic resolution among species in the clinical clade and is helpful to recognize rare agents such as S. pallida, S. mexicana, and S. chilensis.33 Among the loci above, the region spanning exons 3 to 5 of the calmodulin gene is the main marker for the recognition of Sporothrix (Fig. 2).4,103,104 DNA polymorphisms can also be used to study genetic diversity, population structure, recombination analysis, and molecular epidemiology of Sporothrix species.34,105,106 Figure 2. View largeDownload slide Phylogenetic relationships among members of the clinical clade and environmental clade in Sporothrix, based on sequences of calmodulin encoding gene (exon 3–5). Method: Maximum Likelihood and Neighbor-joining. The numbers near the branches (ML / NJ) refer to re-sampling percentages (1000 bootstraps). Genbank accession number can be found close to species name. All sequences have been previously published [8, 34, 130, 138] and are available from GenBank. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Phylogenetic relationships among members of the clinical clade and environmental clade in Sporothrix, based on sequences of calmodulin encoding gene (exon 3–5). Method: Maximum Likelihood and Neighbor-joining. The numbers near the branches (ML / NJ) refer to re-sampling percentages (1000 bootstraps). Genbank accession number can be found close to species name. All sequences have been previously published [8, 34, 130, 138] and are available from GenBank. This Figure is reproduced in color in the online version of Medical Mycology. Amplification of a target sequence in the fungal genome through polymerase chain reaction (PCR) followed by digestion of the amplicon with one or a combination of restriction enzymes (RFLP) has been successfully employed for the identification of Sporothrix species of clinical interest. An 800 bp fragment of the calmodulin gene is amplified using primers CL1 and CL2A and digested with the enzyme HhaI (5΄-GCGC-3΄) to produce species-specific profiles assigned to S. brasiliensis, S. schenckii, S. globosa, and S. luriei.106 As the primers (CL1 and CL2A) are not specific for Sporothrix, it is necessary to isolate the fungus in culture, not allowing direct detection from clinical samples (Fig. 3). Figure 3. View largeDownload slide Recognition of Sporothrix species using classical and molecular approaches. Classical methods present a great phenotypic overlap among closely related species such as S. brasiliensis, S. schenckii and S. globosa, being therefore their use discouraged as the only identification method. Molecular methods based on DNA sequencing followed by phylogenetic analysis allows the identification of all 51 Sporothrix described to date. Methods such as species-specific PCR, PCR-RFLP and RCA are important tools for identifying species of clinical interest. Direct detection and simultaneous identification of Sporothrix from complex samples can be performed with species-specific PCR or RCA. Characterizing Sporothrix in molecular epidemiology studies requires the development of new markers to access genetic diversity, aiming to answer questions related to population structure, transmission routes, intra- and interspecific variability, recombination and reproduction modes, speciation, among many other biological questions. Sybr, SYBR Green dye; PCR, polymerase chain reaction; PCR-RFLP, PCR-restriction fragment length polymorphism; RCA, rolling circle amplification; MLST, multilocus sequence typing; WGS, whole genome sequencing; SNPs, single-nucleotide polymorphisms; AFLP, amplified fragment length polymorphism; RAPD, random amplification of polymorphic DNA. This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. View largeDownload slide Recognition of Sporothrix species using classical and molecular approaches. Classical methods present a great phenotypic overlap among closely related species such as S. brasiliensis, S. schenckii and S. globosa, being therefore their use discouraged as the only identification method. Molecular methods based on DNA sequencing followed by phylogenetic analysis allows the identification of all 51 Sporothrix described to date. Methods such as species-specific PCR, PCR-RFLP and RCA are important tools for identifying species of clinical interest. Direct detection and simultaneous identification of Sporothrix from complex samples can be performed with species-specific PCR or RCA. Characterizing Sporothrix in molecular epidemiology studies requires the development of new markers to access genetic diversity, aiming to answer questions related to population structure, transmission routes, intra- and interspecific variability, recombination and reproduction modes, speciation, among many other biological questions. Sybr, SYBR Green dye; PCR, polymerase chain reaction; PCR-RFLP, PCR-restriction fragment length polymorphism; RCA, rolling circle amplification; MLST, multilocus sequence typing; WGS, whole genome sequencing; SNPs, single-nucleotide polymorphisms; AFLP, amplified fragment length polymorphism; RAPD, random amplification of polymorphic DNA. This Figure is reproduced in color in the online version of Medical Mycology. Molecular assays based on DNA amplification can easily detect Sporothrix from clinical samples or small amounts of cells in culture without the need for sequencing, thereby improving sensitivity and considerably shortening the time required for identification to a few hours.107 Species-specific primers are available that selectively amplify S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and S. stenoceras from minimal amounts of target DNA (10–100 fg DNA) (Fig. 3). Rolling circle amplification (RCA) was first introduced in the mid-1990s as a simple and powerful technique capable of synthesizing large amounts of DNA from very low initial concentrations.108 The method is particularly useful for padlock probe amplification, that is, linear DNA probes become circular (via Pfu DNA ligase enzyme) when specific recognition of a given target sequence occurs. The combination of probe-padlock circularization and amplification through RCA under isothermal conditions has been shown to be useful for the sensitive detection of Sporothrix sequences (up to 3 × 106 copies of the target), although the technique is not used in routine diagnostics. RCA can be applied from pure cultures to complex environmental samples such as soil and vegetables allowing ecological studies that are still scarce in Sporothrix (Fig. 3).109 Methods independent of DNA sequencing (e.g., PCR-RFLP, species-specific PCR and RCA) are important because they provide fast, accurate results in addition to lower costs for identification. This is particularly important during epizootics or zoonotic outbreaks where hundreds to thousands of cases emerge in a short time33 and a precise diagnosis is required to assist in choosing the best therapeutic regimen for the patient.34 Future perspectives might concern the development of multiplex real-time PCR assays using fluorescent probes for simultaneous detection and identification of clinically relevant Sporothrix species. Despite the technical difficulty in standardizing such reactions (annealing temperature, specificity, interactions between multiple primers and probes, etc.), multiplexing significantly reduces the cost of the reaction, the volume of samples, increases sensitivity, and allows the detection of mixed infections. Characterizing Sporothrix in molecular epidemiology studies requires the development of new powerful markers such as those based on whole genome sequencing (WGS) or single-nucleotide polymorphisms (SNPs)-arrays panels (Fig. 3) to access genetic diversity, aiming to answer questions related to population structure, transmission routes, intra- and interspecific variability, recombination and reproduction modes, speciation, among many other biological questions. Sporothrix antigens and vaccine development Although the classical mycological test is still useful in a routine basis, the development of immunochemical tests together with molecular tools can improve the diagnostic time and help to follow-up treatment. For example, resistance to azoles is already reported in S. brasiliensis and S. globosa.17,110–113 Moreover, due to the emergence of S. brasiliensis and S. globosa and the growing incidence of sporotrichosis in humans and cats, new strategies are necessary for controlling sporotrichosis in endemic areas. Proteomics and glycosciences can help to map and identify new antigens for these purposes.47,114 Furthermore, a peptido-rhamnomannan or PRM is one of the main components of Sporothrix spp. cell wall.47 Distinct cell wall components of pathogenic fungi can be of key importance in host recognition and immune response.115 The peptido-rhamnomannan was first isolated by Lloyd and Bitoon.83 These authors showed that this peptido-polysaccharide could be recognized by human serum antibodies of patients with sporotrichosis. Travassos and coworkers,116,117 studying the sugar moieties of PRM, showed that the epitopes present on the rhamnomannans differed between the yeast parasitic phase (α-L-Rhap 1→3 α-D-Manp) and the mycelium phase (α-L-Rhap 1→2 α-L-Rhap 1→3 α-D-Manp) of S. schenckii. For a long time these N-linked epitopes were considered as the main antigenic structures present on the cell wall PRM.116 Interestingly, PRM is recognized by the lectin Concanavalin A (ConA), but the rhamnomannans isolated by hot alkali extraction (N-linked glycan chains) loose ConA reactivity.116 Years later, Lopes-Alves et al.70 determined the fine structure of the O-linked glycan chains of PRM and reported that they bear the ConA binding sites, a mannobiose core of α-D-Manp 1→2 α-D-Manp. Among the PRM O-linked glycan chains, two new epitopes were described (α-L-Rhap 1→ 2 α-D-GlcAp and α-L-Rhap 1→ 2 [α-L-Rhap 1→ 4] α-D-GlcAp) at the non-reducing ends of a tetra- and pentasaccharides.47,118 These O-glycan epitopes are not described in any other fungal pathogen being exclusive of Sporothrix spp.47 Moreover, the PRM O-linked glycan chains were the main epitopes recognized by IgG antibodies present in patient's sera.119 Based on these biochemical findings, a Con-A binding cell wall antigen was further isolated and purified from the PRM fraction.120 This antigenic fraction denominated as SsCBF (Sporothrix schenckiiConA binding fraction) was used to develop an ELISA test for the serodiagnosis of human and feline sporotrichosis.121–123 The ELISA test was clinically validated for human sporotrichosis with 90% global efficacy (sensibility and specificity)122 and is used as an “in house” test in two University Hospitals in Rio de Janeiro, Brazil. The SsCBF ELISA test can give a serodiagnostic result in few hours and is very useful for treatment monitoring.24,26,27,124 A cell wall antigenic molecule described in S. schenckii and S. brasiliensis clinical isolates is a glycoprotein of 60–70 kDa (Gp70).9,125 The 70 kDa antigen was first described as a component of the cell wall PRM fraction of S. schenckii.120 A decade after its first description, the first evidence that this cell wall antigen has protective properties came out.126 A IgG1 monoclonal antibody raised against the extracellular Gp70 (MAb P6E7) was used successfully to induce a passive immunization to mice infected with Sporothrix schenckii126 and several subsequent studies showed its therapeutic efficacy towards Sporothrix spp. infection.126–129 A proteomic study was performed and the Gp70 antigen was sequenced by MS/MS.9 In a subsequent proteomic study, a 2-D fluorescence difference gel electrophoresis (DIGE) approach was used to compare the expression of this antigen isolated from S. schenckii and S. brasiliensis yeast cell extracts.125 The authors showed that this antigen has proteoforms in both species. A 60 kDa glycoform is predominant in S. brasiliensis.125 In conclusion, both antigenic bands of 60 and 70 kDa (Gp60 and Gp70, respectively), previously described in the literature as distinct antigens,130 are actually related to the same gene sequence.125 A recent study showed that a recombinant phage displaying a Gp70 peptide (kpvqhalltplgldr) on the major coat protein (pIII) was efficiency as a vaccine for preventing S. globosa infection.131 The data described above allow us to conclude that the Gp70 antigen identified so far has a potential for the development of a vaccine to control Sporothrix infection.125–129,131 A cell wall protein extract previously described by Castro et al.9 in their work showing the cell wall expression and peptide sequence of the Gp70 antigen, was used in recent vaccination studies.132,133 This DTT protein extract, denominated ssCWP, was associated with aluminum hydroxide (AH).132 Two AH-adsorbed ssCWP based vaccine formulations were tested in mice systemically infected with S. schenckii. In this study, Portuondo et al.132 characterized in the ssCWP extract a new antigen of 47 kDa identified as enolase and a 44 kDa non-antigenic band identified as a peptide hydrolase. These authors also corroborated the presence, in the DTT extract, of an antigenic band localized at the 70 kDa gel region (71 kDa).132 The passive immunization with sera of mice treated with an AH-adsorbed ssCWP formulation (AH+CWP10) showed a reduction in the number of colony forming units (cfu) in liver and spleen. In a recent study, these authors evaluated a Montanide™ Pet Gel A (PGA) formulated with ssCWP and compared with the AH-SsCWP formulation.133 Immune response against Sporothrix The cell wall of members of the pathogenic Sporothrix clade is the main source of pathogen-associated molecular patterns (PAMPs). Thus far the best characterized component is the peptido-rhamnomannan.47,116,117 It is now clear that the cell-mediated immune response plays a key role in controlling Sporothrix dissemination, as demonstrated by protection against infection upon transference of normal immune spleen cells to athymic nude mice.134,135 The innate immunity is key during the establishment of a protective anti-Sporothrix response. Phagocytosis by neutrophils and macrophages, and production of reactive oxygen species are essential for neutralization and killing of S. schenckii cells.135,136 In addition, differential ability of murine macrophages to interact with species of S. schenckii and relatives has been reported, with S. brasiliensis being significantly more phagocytosed than S. schenckii.3 However, not all the molecules of the oxidative burst during immune activation against Sporothrix have a positive contribution in the control of the fungus. Production of nitric oxide during experimental sporotrichosis has a detrimental role during infection, facilitating the establishment of the infection via apoptosis of immune cells, and stimulating an anti-inflammatory state.137 S. schenckii yeast cells can activate both classical and alternative complement pathways,116 which makes cells susceptible to the uptake by macrophages.138 However, conidial interaction with phagocytic cells does not depend on opsonization by complement activation,138 suggesting this cell morphology is recognized by receptors different from those involved in the yeast recognition. Knowledge on identity and molecular structure of PAMPs in Sporothrix species is still limited.139 Nevertheless, significant contributions have been made in the involvement of PAMPs in immune sensing. Toll-like receptor (TLR) 4 was found to interact with lipid preparations from S. schenckii yeast-like cells, stimulating the production of pro- and anti-inflammatory cytokines and oxidative mediators in a murine model of sporotrichosis.140–142 This immune receptor has also been involved in the production of both pro- and anti-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), in particular when stimulated with either conidia, yeast-like cells or germlings from S. brasiliensis.139 Interestingly, conidia and germlings from S. schenckii, but not yeast-like cells, are capable of stimulating cytokine production via TLR4.139 Cytokine production via TLR2 also occurs upon interaction with S. schenckii yeast-like cells, and macrophage functions against this organism are significantly impair in cells from TLR2-deficient mice.143 The interaction of the three morphologies of both S. schenckii and S. brasiliensis with human PBMCs is also dependent on TLR2, confirming this receptor is relevant in both the murine and human response against these pathogens.139 A mannose receptor (MR) has also been involved in sensing S. schenckii conidia by THP-1 macrophages.138 Using primary human PBMCs, it has demonstrated this receptor is relevant to cytokine production stimulated by S. schenckii conidia or S. brasiliensis yeast-like cells,139 underlining that cell morphology is accompanied by changes in the molecular structure of PAMPs on the surface of the fungal cells. One of the most important immune receptors involved in the establishment of an anti-fungal immune response is dectin-1. This receptor is key for cytokine stimulation when human PBMCs interact with the three morphologies of S. schenckii or S. brasiliensis conidia and yeast-like cells.139 However, it is dispensable for cytokine stimulation when human PBMCs interact with S. brasiliensis germlings.139 Accordingly, it has been reported that this receptor, along with IL-17 production, is not involved in the clearance of S. schenckii infection in a rat model of sporotrichosis.144 The adaptive branch of the immune response is essential for appropriate control of Sporothrix infections, involving the establishment of a Th1/Th17-based immune response.145–147 The successful establishment of this response, partially relies in the activation of the NLRP3 inflammasome,148 and activation of dendritic cells,149 which respond to exoantigens of S. schenckii yeast-like cells. However, a differential dendritic cell activation has been reported, depending on the origin of the etiologic agent, that is, a strain from a cutaneous lesion provides a better stimulus to establish a Th1 response than a strain of visceral origin.150 Despite this interesting evidence, the antigenic / molecular differences between these strains remain to be established. In addition, since this study was published before the separation of S. schenckii siblings4 it is tempting to speculate whether these two strains might belong to different species. Even though these evidences suggest that control of these pathogens is Th1/Th17 dependent, the activation of the Th2 response is also important during the generation of an anti-Sporothrix immune response, especially in advanced stages of the infection.151 After animal immunization with S. schenckii cells, antibodies raised against several fungal antigens can be detected.152 As mentioned before, the best characterized antigen thus far is Gp70, a secreted and cell wall glycoprotein of about 70 kDa, 3,9,47,125,153,154 which is a glycoprotein heterogeneous in molecular weight also known as Gp60.155 In mice, the passive immunization with anti-Gp70 antibodies has shown protection against sporotrichosis caused by S. schenckii and S. brasiliensis, promoting a significant reduction in fungal burden, even with deficiency in T-cells.127,128 It seems the mechanism to protect against the infection is based on opsonization, which facilitates the uptake by macrophages and enhances production of pro-inflammatory cytokines.156 Recently, humanized anti-Gp70 antibodies have been successfully used to opsonize both S. schenckii and S. brasiliensis cells with a positive influence on phagocytosis by human monocyte-derived macrophages,129 which bring us closer to the use of these antibodies as part of a treatment of human sporotrichosis. Future directions The next steps following the genomic studies are the development of an effective transformation system for Sporothrix spp. and the trascritpome analysis (RNA sequencing analysis; RNA-Seq). These studies are under development. A bank of mutants will be of undeniable value to unveil the role of several genes and/or gene products in the biology and pathogenicity of Sporothrix spp. Moreover, the new molecular and serological diagnostic tools already available, described in this review, will contribute to refine epidemiological studies. In addition, our perspective is to generate a consensus, in the near future, to recommend the application of more sensitive and rapid diagnostic tests in the clinical routine. The delay in the diagnosis of severe or unsusual clinical forms of sporotrichosis, especially related to the new emerging pathogenic species, is a critical aspect to initiate a correct and appropiate therapeutic regimen. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements L.M.L.B. is a research fellow of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 307169/2015-4) and of Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ; CNE 2015), Brazil. H.M.M.M. is supported by Consejo Nacional de Ciencia y Tecnología (ref. CB2011/166860; PDCPN2014-247109, and FC 2015-02-834), Universidad de Guanajuato (ref. 0087/13; ref. 1025/2016; Convocatoria Institucional para Fortalecer la Excelencia Académica 2015; CIFOREA 89/2016), Programa de Mejoramiento de Profesorado (ref. UGTO-PTC-261), and Red Temática Glicociencia en Salud (CONACYT-México). Y.Z. was supported by the Tianjin Municipal Natural Science Foundation (grant no.15JCYBJC49500). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Schenck B. 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Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis. Eur J Immunol . 2008; 38: 3080– 3089. Google Scholar CrossRef Search ADS PubMed  127. Almeida SR. Therapeutic monoclonal antibody for sporotrichosis. Front Microbiol . 2012; 3: 409. Google Scholar CrossRef Search ADS PubMed  128. Almeida JR, Kaihami GH, Jannuzzi GP, de Almeida SR. Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis. Med Mycol . 2015; 53: 42– 50. Google Scholar CrossRef Search ADS PubMed  129. Almeida JR, Santiago KL, Kaihami GH et al.   The efficacy of humanized antibody against the Sporothrix antigen, gp70, in promoting phagocytosis and reducing disease burden. Front Microbiol . 2017; 8: 345. Google Scholar PubMed  130. Ruiz-Baca E, Hernández-Mendoza G, Cuéllar-Cruz M, Toriello C, López-Romero E, Gutiérrez-Sánchez G. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa. Diagn Microbiol Infect Dis . 2014; 79: 328– 330. Google Scholar CrossRef Search ADS PubMed  131. Chen F, Jiang R, Wang Y et al.   Recombinant phage elicits protective immune response against systemic S. globosa infection in mouse model. Sci Rep . 2017; 7: 42024. Google Scholar CrossRef Search ADS PubMed  132. Portuondo DL, Batista-Duharte A, Ferreira LS et al.   A cell wall protein-based vaccine candidate induce protective immune response against Sporothrix schenckii infection. Immunobiology . 2016; 221: 300– 309. Google Scholar CrossRef Search ADS PubMed  133. Portuondo DL, Batista-Duharte A, Ferreira LS et al.   Comparative efficacy and toxicity of two vaccine candidates against Sporothrix schenckii using either Montanide™ Pet Gel A or aluminum hydroxide adjuvants in mice. Vaccine . 2017; 35: 4430– 4436. Google Scholar CrossRef Search ADS PubMed  134. Martinez-Alvarez JA, Perez-Garcia LA, Flores-Carreon A, Mora-Montes HM. The immune response against Candida spp. and Sporothrix schenckii. Rev Iberoam Micol.  2014; 31: 62– 66. Google Scholar CrossRef Search ADS PubMed  135. Shiraishi A, Nakagaki K, Arai T. Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice. Mycopathologia.  1992; 120: 15– 21. Google Scholar CrossRef Search ADS PubMed  136. Kajiwara H, Saito M, Ohga S, Uenotsuchi T, Yoshida S-I. Impaired host defense against Sporothrix schenckii in mice with chronic granulomatous disease. Infect Immun . 2004; 72: 5073– 5079. Google Scholar CrossRef Search ADS PubMed  137. Fernandes KSS, Neto EH, Brito MMS et al.   Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii. Immunology . 2008; 123: 469– 479. Google Scholar CrossRef Search ADS PubMed  138. 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Uenotsuchi T, Takeuchi S, Matsuda T et al.   Differential induction of Th1-prone immunity by human dendritic cells activated with Sporothrix schenckii of cutaneous and visceral origins to determine their different virulence. Int Immunol . 2006; 18: 1637– 1646. Google Scholar CrossRef Search ADS PubMed  151. Maia DC, Sassá MF, Placeres MC, Carlos IZ. Influence of Th1/Th2 cytokines and nitric oxide in murine systemic infection induced by Sporothrix schenckii. Mycopathologia . 2006; 161: 11– 19. Google Scholar CrossRef Search ADS PubMed  152. Ruiz-Baca E, Mora-Montes HM, Lopez-Romero E et al.   2D-immunoblotting analysis of Sporothrix schenckii cell wall. Mem Inst Oswaldo Cruz . 2011; 106: 248– 250. Google Scholar CrossRef Search ADS PubMed  153. Nascimento RC, Almeida SR. Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis. FEMS Immunol Med Microbiol . 2005; 43: 241– 247. Google Scholar CrossRef Search ADS PubMed  154. 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For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

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

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© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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10.1093/mmy/myx103
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Abstract

Abstract 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. Sporothrix schenckii, Sporothrix brasiliensis, Sporothrix globosa, genome, sporotrichosis Etiology of sporotrichosis: historical aspects The description of the first clinical case of cutaneous sporotrichosis was published in 1898 by Benjamin Schenck.1 The yeast-like pathogenic phase and the lymphocutaneous / extracutaneous forms of this disease were observed in humans and in rats in 1907 by Lutz and Splendore.2 In the century that followed, but particularly during the last decades, an enormous expansion took place in the knowledge of this mycosis on humans and animals. Now that genetic and molecular tools have become available,3 some classical viewpoints deserve renewed attention. Particularly, the description of cryptic species with pathogenic potential has changed the visions on this disease.4 The biology and genomic data of these fungal pathogens, cell wall antigens, the host immune response, as well as some epidemiological and diagnostic aspects associated with emerging etiological agents of sporotrichosis, will be the focus of this review. Today, nearly all research in fungal pathogens has become multidisciplinary. Since 2011 a Working Group on Sporotrichosis is active under the umbrella of ISHAM, where clinicians cooperate with basic scientists, bioinformaticians, and epidemiologists; the present review is one of the product of this broad cooperation. The recently described species S. brasiliensis and S. globosa, which are prevalent in South America and East Asia, respectively,5–7 together with S. schenckii (sensu stricto) and S. luriei make up the ‘pathogenic clade’ of the genus Sporothrix.8 It is important to notice that S. luriei has a low clinical-epidemiological impact within this genus. This revision will cover the clinically relevant species: S. schenckii, S. brasiliensis and S. globosa. Sporothrix species are dimorphic fungi that present a saprophytic mycelium phase at room temperature (25–28°C) and a yeast-like pathogenic phase at 36–37°C. Sporothrix propagules usually entry the warm-blooded host through minor cutaneous trauma from contaminated plant debris or through scratches or bites from animals (mostly felines) carrying the fungus. Often multiple infections arise from a single source, leading to outbreaks of the disease, potentially with thousands of patients.6 The classical species S. schenckii is related to a benign chronic subcutaneous mycosis, and in its restricted sense the species exhibits a moderate virulence profile in animal models.9,10 In contrast, S. brasiliensis is highly virulent in animal models and is associated with severe clinical forms of sporotrichosis.9–16 Interestingly, both S. brasiliensis and S. globosa are less susceptible to itraconazole, judging from clinical reports and animal model studies.17,18 Of the recently described siblings, S. globosa (formerly reported as S. schenckii) is a well-established agent of prevalently plant-transmitted infection,19 but S. brasiliensis is emerging only since the nineties of the previous century and shows preponderance of cat-transmission. Both routes of infection are common in the ancestral species S. schenckii. The differential biology of these newly emerging species may trigger different host recognition and immune response.3 Clinical-epidemiological profiles and the evolutionary processes of S. brasiliensis and S. globosa differ from each other and from S. schenckii in adaptation to mammal tissue.5,19–21 A recent evolutionary specialization seems to have taken place in the two siblings, which has led to these remarkably deviating sources of infection and transmission routes between these closely related species. There is a consensus that sporotrichosis basically is an implantation disease. However, some authors refer to inhalation as a possible route of infection,22 but not proved experimentally. Although the most common types of sporotrichosis are the lympho-cutaneous and fixed cutaneous forms, atypical, extracutaneous forms and mucosal sporotrichosis have also been reported.23 Cat-transmitted S. brasiliensis infection was observed for the first time around 2000, in Rio de Janeiro, Brazil,20,21 showing exponential expansion, even though a close interaction of stray cats and susceptible hosts existed already for hundreds of years. How does feline sporotrichosis arise? How Sporothrix did become adapted to animal infection? These are key questions to be answered that can help us to understand the change in sporotrichosis from an uncommon, benign disease to an important emerging mycosis with severe clinical forms in immunocompromised as well as in immunocompetent hosts. The work published by Lutz and Splendore in 1907,2 who described the yeast parasitic phase of S. schenckii, can bring some light to cat infection. They also studied Sporothrix infection in Rattus novergicus (brown rats) and part of their findings must be highlighted here: “One of us (Lutz), from many years had been aware of special lesions that manifest themselves in the common rat … we were able to collect a total of more than 40 cases of infected animals. The most common form of this mycosis, which is spontaneously observed in the rat, consists of lesions located at the extremities and the tail. It usually appears in the tarsal region of one or more extremities, or at any point of the tail, a local swelling, remembering the affections produced in the man by the articular tuberculosis …. There are also internal locations in the form of isolated and few numerous miliary tubers that are seen in the spleen, liver, lungs, kidneys, genital glands and internal serosa ….” - translated from the original manuscript of Lutz and Splendore, published in Portuguese. In summary, Lutz and Splendore had observed sporotrichosis in wild rats and experimental animals, describing osteo-articular and cutaneous lesions. They also hypothesized that sporotrichosis can be acquired by ingestion based on their experimental model.2 This can represent a host shift in S. brasiliensis infection (unknow species until 2007) from rats to cats in the last century. One of the major predators of rats are cats. Furthermore, Sporothrix infection by ingestion was proposed by other authors.23 Some aspects of the possible adaptation of S. brasiliensis to cat saliva will be discussed later. Another important aspect is the fact that the feline sporotrichosis epidemic has started in Rio de Janeiro State, Brazil, in areas of low socioeconomic status and, precarious sanitary and health system, with high demographic density of both the human and feline population.24,25 There is no information collected from rats in these endemic areas. The skin disease that had long been considered as a benign occupational disease of horticulturalists, acquired mainly by the manipulation of contaminated soil or plant material (such as Sphagnum moss), has in fact become an urban problem with a peri-domicile character. The high zoonotic potential of S. brasiliensis compared to S. schenckii21 has led to an increasing number of severe and /or systemic reported clinical cases.12–16,26,27 In parallel with the development of molecular tools, cell biological models are now available to understand pathogenesis and the balance between virulence factors of ancestral and evolved Sporothrix species, as well as the host's immune status. Global distribution patterns of S. brasiliensis and S. globosa Sporotrichosis occurs worldwide, but the infectious agents are not evenly distributed since the main pathogenic species of Sporothrix have high degrees of endemicity. Sporothrix is unique in the fungal kingdom by its prevalent occurrence in the form of outbreaks and in that these outbreaks differ fundamentally from each other between closely related species. In S. brasiliensis a huge zoonosis is taking place today, while the contemporary outbreak of similar dimensions in China is a sapronosis.19S. globosa exhibits a global distribution pattern and is involved in an expanding sapronosis with preponderant plant transmission.5,28 The contemporary zoonotic outbreak with similar dimensions in Brazil is caused by S. brasiliensis and geographically limited to Brazil.20,21 In China, sporotrichosis exists nationwide but is mainly observed in the Northeastern provinces. S. globosa is the overwhelming pathogen, with several thousands of cases having been reported from China,19,28–30 while there are only four known isolates of S. schenckii from Jiangxi province and Hunan province, respectively.19 With lower endemicity outside Asia, S. globosa exhibits a global distribution. It mainly causes benign, fixed or lymphocutaneous infections and is never vectored by animals.19,31 The species is hypothesised to grow exponentially in rotten, self-heated plant debris like corn or reed that serves as a potential inoculum for plant harvesters in northeast China, where the prevalent clinical type is facial.32 Upon removal of the infectious material, the human sapronosis will die out with some delay, matching with the observation that most cases in China's Jilin Province become apparent during winter following harvest. Several studies showed a relationship between geographical distribution and genotypes among species of Sporothrix.4,19,33 With the exception of Africa and Australia, S. globosa has been reported worldwide, similar to its hypothesized ancestral species S. schenckii, but at different prevalence: the latter species is preponderant in eastern South America, Africa, and Australia.19 Epidemiological studies proved that strains of S. globosa from Brazil, China, Japan, Spain, and the United Kingdom had nearly identical genotypes.19,30 An as yet unknown mechanism seems to be involved in the rapid dispersal of S. globosa. Given the large geographical distances between the localities harboring genetically indistinguishable isolates, airborne distribution seems to be the most plausible explanation. The absence of reported S. globosa infections and environmental isolates from Africa and Australia may perhaps be explained by sampling effects. Notably, S. globosa infections are derived from plant debris and this infection is classically known as ‘reed toxin’.30 Sporothrix globosa strains analysed thus far were nearly identical. Isolates studied by Yu et al.30 were divided into two highly supported subclades (S. globosa I and S. globosa II). Group I comprised the majority of Chinese clinical isolates, three Chinese environmental isolates from reed, corn stalks, and soil, the type strain of S. globosa, and some isolates from the United States, India, Japan, Brazil, and the United Kingdom. Sporothrix globosa II included some Chinese clinical isolates and a single isolate from Italy. More material is needed to ascertain whether S. globosa is preponderantly clonal. Interestingly, cats have never been observed as sources of infection in endemic areas of S. globosa, in contrast to the large-scale, expanding outbreak of S. brasiliensis among humans and cats in South America. Sporothrix brasiliensis also shows low degrees of variability and has been suggested to be clonal.7 The low variability of this taxon is supported by low chromosomal polymorphisms and homogeneous susceptibility profiles to antifungal agents. In Southeast Brazil, transmission nearly always occurs by cats.20 Cat saliva is a stable environment, and despite the presence of antibodies—which generally have a low impact on fungal infections—repeated colonization by Sporothrix once it has adapted to these conditions may be expected. The large outbreak in this area suggests that the number of cases increases relative to the number of patients and cat vectors. Several peculiarities of cats may facilitate the dispersal of the fungus in the environment within limited endemic areas. First, they are the most common pet animals with close contact to humans. Second, given the hypothesized origin of Sporothrix in cat saliva and its transmission to claws during licking, cat mobility and clawing enable them to take up and transmit the fungus, either to each other during play or fight with house cats or stray cats, or to human hosts via scratches or bites.20 Thus, despite the preponderance of cat vectors, the animal host species may vary, just as the plant host species did. This leads us to a hypothesis of wild animals occasionally providing conditions similar to those in fermented plant material. Cats take up propagules from the soil and easily transmit them to their mouth by licking. Conditions in animal saliva at the feline body temperature (normal range 37.7−39.1°C) might be a stimulating factor for the production of the Sporothrix yeast phase.20 Cat saliva has a pH of 7.5−8.0, which is similar to that of self-heating bulk corn debris (around 8.0) and optimal for the mould-to-yeast conversion.20 We evaluated the impact of the feline host on the epidemiology, spatial distribution, prevalence and genetic diversity of human sporotrichosis. Nuclear and mitochondrial markers revealed large genetic differences between S. brasiliensis and ancestral S. schenckii populations, suggesting that the interplay of host, pathogen and environment has a structuring effect on the diversity, frequency, and distribution of Sporothrix species. Phylogenetic data support a recent habitat shift within S. brasiliensis from plant to cat. According to the above it should be plant to rat and then rat to cat that seems to have occurred in Southeastern Brazil and is responsible for its emergence. A clonal structure was found in the early expanding phase of the cat–human epidemic.34 However, the preponderant recombination structure in the prevalently plant-associated pathogen S. schenckii generates a diversity of genotypes that did not show any significant increase in frequency as pathogens of human infection over time.7,21 These characters suggest that closely related causative agents can follow different strategies in epidemics. Thus, species-specific types of transmission may require public health strategies to consider these distinctions. Sapronoses, providing very special conditions promoting fungal growth, basically can be controlled by removal of the plant biomass allowing this contamination. In contrast, the zoonoses of cats compose a much more diffuse source of infection, which is difficult to control. With a hypothesis of conditional similarities between fermenting plant material and animal digestive saliva, the unique host shift of Sporothrix from plant to animal becomes understandable. Therefore, we hypothesise that Sporothrix species are not plant pathogens but require particular conditions in decaying plant material, which are reached only occasionally during advanced decomposition. We postulate that a particular state of decay and fermentation of the plant material promotes excessive growth of Sporothrix. High temperature and humidity, associated with metabolic changes (induction of respiratory system) and oxidative stress during decay and fermentation may shift the morphology, favoring the invasive yeast growth form. What does the genome tell us? To date, the genomes of S. schenckii, S. brasiliensis, and S. globosa, for the clinically important species, as well as the nonvirulent environmental species, Sporothrix pallida have been sequenced, although only annotated for S. schenckii and S. brasiliensis.35,36,37 The genome is similar in size for S. schenckii, S. brasiliensis, and S. globosa, although the number of predicted protein encoding genes are notably variable for the three species, with a larger number of putative genes in S. schenckii and the lowest number present in S. globosa (Table 1). However, when compared with the environmental, nonvirulent species S. pallida, some outstanding differences are apparent. S. pallida genome is almost 5 Mb larger than the medically important species and shows higher number of predicted protein encoding genes. It has been proposed that pathogenic fungi with an environmental phase in their life cycle, have evolve to survive in the mammalian host through processes of genetic expansion and contraction of gene families, which ultimately allows the fungus to colonize and survive the environmental hazards of the mammalian immune system.35,38,39 Since the genomes of S. globosa and S. pallida are only present as draft genomes in the public databases, it is not possible to produce a comparative analysis of the four genomes above mentioned, to address the question of adaptation to the mammalian host. However, recently Teixeira et al.35 published a thorough comparative analysis of the S. schenckii and S. brasiliensis genomes with 14 fungi, either dimorphic or plant-associated, addressing that same question. In Coccidioides spp., a reduction of plant cell wall degrading enzymes and an expansion of peptidase genes has been related to adaptation to the mammal host and lead to the suggestion that Coccidioides spp. are not soil saprophytes but have evolved to remain associated to their dead host in soil.38 On the other hand, dimorphic pathogens with a saprophyte stage in their life cycle, such as Paracoccidioides spp. present expansion of peptidase gene families, related to their adaptation to the mammal host, but keep the plant cell wall degrading enzymes genes, related to their saprophyte stage.39 In their work, Teixeira et al.35 did not find an expansion of peptidase genes in the S. schenckii and S. brasiliensis genomes; however, they found lack of plant decay-associated polysaccharide lyase genes, present in other soil related Sordariomycetes, which lead the authors to suggest evolutionary adaptation of S. schenckii and S. brasiliensis from plant pathogens or saprobes to an animal pathogenic life style.35 Table 1. Genomes size and number of predicted coding genes for Sporothrix spp.* Organism  Genome size (Mb)  Predicted protein coding genes  G + C content  tRNA  S. schenckii  32.4  10 293  62%  139  S. brasiliensis  33.2  9091  62%  140  S. globosa  33.5  7719–7760  54.37%  126  S. pallida  37.8  11 356  52.8%  151  Organism  Genome size (Mb)  Predicted protein coding genes  G + C content  tRNA  S. schenckii  32.4  10 293  62%  139  S. brasiliensis  33.2  9091  62%  140  S. globosa  33.5  7719–7760  54.37%  126  S. pallida  37.8  11 356  52.8%  151  *Adapted from Teixeira et al.35; D’Alessandro et al.36; and Huang et al.37 View Large Fungal dimorphism The dimorphism exhibited by S. schenckii sensu lato is also found in other human pathogenic fungi, such as Coccidioides immitis, C. posadasi, Blastomyces dermatitidis, B. gilchristii, Talaromyces marneffei, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, and Emmonsia spp., and is essential for the establishment of the infection. Interestingly, Sporothrix belongs to a different taxonomic order than the above listed organisms, indicating that this phenotypical trait is a result of convergent evolution.35 Even though there is genetic distance between Onygenales and Ophiostomatales, it is likely that dimorphic members of both orders share similar molecular mechanisms controlling the dimorphic process. The histidine kinase drk1 participates controlling the mycelium to yeast transition in both B. dermatitidis and H. capsulatum.40,41 The putative ortholog to this gene has been identified in S. schenckii and is likely to be involved in regulating the dimorphic process.42 Another important player in regulating the dimorphic process in H. capsulatum is the gene required for yeast phase growth (RYP1), which is a transcriptional regulator of yeast-specific genes and a functional ortholog of Candida albicans WOR1, the master regulator not only of the yeast-to-hypha transition but the mating and the phenotypical switch.40 Bioinformatics analyses found the putative RYP1 ortholog as part of both S. schenckii and S. brasiliensis genomes.35 In addition, RYP2, RYP3, and VEA1 have been involved in the formation of H. capsulatum yeast-like cells,43 and the putative orthologs are found within the Sporothrix genome (GenBank accession codes: XP_016590923.1, ERS98419.1, and XP_016588232.1, respectively). In T. marneffei, the p21-activated kinase pakB is required to avoid yeast-like cell formation at 25°C, temperature where the fungus grows like hyphae.44 The putative orthologs are found in the genome of both S. schenckii sensu stricto and S. brasiliensis (GenBank accession codes: XP_016583587.1 and KIH88219.1, respectively). The transcription factor hgrA has a hypha-specific expression responsible of the hyphal growth program in T. marneffei, and its expression must be repressed to undergo dimorphism.45 Interestingly, the putative ortholog is found within the S. brasiliensis genome (GenBank accession code KIH95137.1) but not as part of S. schenckii. Cell wall: a glycobiology overview The fungal cell wall is an essential structure for cell viability and interaction with the environment, and contains macromolecules involved in the virulence of pathogenic fungi.46 Since it is composed of compounds that are not synthesized by the human and animal hosts, the study of the biosynthetic pathways involved could potentially lead to the discovery of additional molecules with antifungal properties. The sequencing and gene annotation of the S. schenckii and S. brasiliensis genomes have provided a closer view of the synthesis of glycoproteins, β-glucans, and chitin in these organisms.35 Protein glycosylation Glycoproteins are key components of the Sporothrix cell wall and among the first aspects studied in this organism.47 In the fungal cell, as in other kind of eukaryotic cells, there are three main types of protein glycosylation: addition of oligosaccharides to residues of Asn (N-linked glycosylation), to Ser/Thr (O-linked glycosylation) or incorporation of the glycolipid glycosylphosphatidylinositol (GPI) at the C-terminal end of the polypeptide. The first steps in the elaboration of N-linked oligosaccharides are extremely conserved in eukaryotic cells, and as expected, the bioinformatic analysis of both S. schenckii and S. brasiliensis genomes revealed the presence of all the elements required for synthesis and processing of the N-linked glycan core (see supplementary Table 1S). The first step during elaboration of the N-linked oligosaccharides is the synthesis of a precursor attached to dolichol phosphate (Dol-P), which is within the endoplasmic reticulum (ER) membrane. This stepwise process is highly conserved in all eukaryotes and is performed in two stages: first at the ER cytosolic face, where the glycolipid Dol-P-Man5GlcNAc2 is synthesized, followed by a luminal stage where the glycolipid is further elaborated to generate Dol-P-Glc3Man9GlcNAc2, also named N-linked glycan precursor.48,49 The N-linked glycan precursor is then transferred in bloc to an Asn residue within the N-linked glycosylation sequon Asn-X-Ser/Thr (where X can be any amino acid except Pro) by the oligosaccharyl transferase complex.50 The N-linked glycan on the glycoprotein surface then undergoes processing by ER α-glucosidases I and II that remove the outermost α-1,2-glucose unit and the two remaining glucose residues, respectively,51–53 and the ER α-1,2-mannosidase that trims one α-1,2-mannose residue generating Man8GlcNAc2 isomer B.52–54 The N-linked glycan biosynthesis has been thoroughly studied in Saccharomyces cerevisiae and in a lesser extent in Aspergillus fumigatus. Table 2 shows a comparative analysis of these organisms with S. schenckii and S. brasiliensis, indicating that Sporothrix spp. contain all the genes involved in the synthesis of Glc3Man9GlcNAc2 and transfer to nascent proteins and processing by glycosidases. Table 2. Genes involved in O-linked glycosylation in S. cerevisiae, A. fumigatus, S. schenckii, and S. brasiliensis. Protein function  S. cerevisiae1  A. fumigatus2  S. schenckii3  S. brasiliensis3  ER mannosyltransferases          Dol-P-Man:protein-O-D-mannosyltransferase  PMT1  pmt1  SS05892  SB04624  Dol-P-Man:protein-O-D-mannosyltransferase  PMT2  pmt2  SS08548  SB01344  Dol-P-Man:protein-O-D-mannosyltransferase  PMT4  pmt4  SS08628  SB08186  Golgi mannosyltransferases          α1,2-Mannosyltransferase  KTR1  mnt1  SS09069  SB08384  Protein function  S. cerevisiae1  A. fumigatus2  S. schenckii3  S. brasiliensis3  ER mannosyltransferases          Dol-P-Man:protein-O-D-mannosyltransferase  PMT1  pmt1  SS05892  SB04624  Dol-P-Man:protein-O-D-mannosyltransferase  PMT2  pmt2  SS08548  SB01344  Dol-P-Man:protein-O-D-mannosyltransferase  PMT4  pmt4  SS08628  SB08186  Golgi mannosyltransferases          α1,2-Mannosyltransferase  KTR1  mnt1  SS09069  SB08384  1Standard names were retrieved from http://www.yeastgenome.org/. 2Systematic names were retrieved from http://www.aspergillusgenome.org/. 3Systematic names were retrieved from Teixeira et al.35 View Large Subsequently, the glycoproteins are transported to the Golgi complex where they are further modified by glycosyltransferases. In filamentous fungi and higher eukaryotes there are Golgi mannosidases IA, IB and IC that process Man8GlcNAc2 to Man5GlcNAc2, just before action of transferases.55–57 This processing step is critical to establish the final structure of N-linked oligosaccharides, which in fungal cells can be either high mannose or hybrid N-linked glycans. Organisms lacking Golgi mannosidases, such as S. cerevisiae, exclusively elaborate high mannose N-linked glycans.48 The presence of Golgi-resident mannosidases IA, IB, and IC in S. schenckii cells,53 strongly suggest that this organism modifies the N-linked glycan core to generate hybrid N-linked glycans. This is in line with the previously described N-linked rhamnomannan structure.47 Interestingly, Sporothrix does not have any obvious ortholog for Golgi mannosidase II, a key enzyme in the elaboration of complex N-linked glycans in higher eukaryotes.58 In S. cerevisiae, there are exclusively Golgi mannosyltransferases and they are responsible of the elaboration of the N-linked glycan outer chain. The first reaction is catalyzed by the α1, 6-mannosyltransferase Och1 followed by the α1, 6-mannosyltransferase complexes MolP-I and MolP-II, generating an α-1, 6-polymannose backbone.59 Both S. schenckii and S. brasiliensis genomes have the genes encoding these enzyme activities and the OCH1-like gene family, including OCH2, OCH3 and OCH4.60 Again, this data support elaboration of the N-linked glycan outer chain reported previously.47 The S. schenckii and S. brasiliensis genomes also contain genes encoding members of the KRE2/MNT1 gene family, and at least one member is likely to participate in the modification of the outer chain backbone, with branches of α1, 2-mannose residues.61 The presence of a gene with significant similarity to those encoding the N-acetylglucosaminidase III (see supplementary Table 2S) that adds the bisecting GlcNAc residue found in both hybrid and complex N-linked glycans62 supports our hypothesis on the N-linked glycan structure in S. schenckii sensu lato. Presence of galactose-containing glycans has been reported63 and some putative galactosyltransferases have been identified during the genome analysis (see supplementary Table 1S), although it remains to be addressed whether these enzymes participate in elaboration of glycoproteins and/or glycolipids. Furthermore, S. schenckii and S. brasiliensis contain an ortholog of Aspergillus nidulans ugmA, whose products generates UDP-galactofuranose from UDP-galactopyranose, the galactomannan-building sugar donor in Aspergillus.64 Sialic acid has been reported as component of the S. schenckii cell wall glycolipids.65 So it is likely that the putative Golgi CMP-sialic acid transporter (see supplementary Table 2S) is involved in modification of such lipids. Figure 1A shows the current working model on the N-linked glycan structure based on bioinformatics. The processing of the N-linked oligosaccharide in the ER does not only have a significant role during N-linked glycan elaboration, but also in the glycoprotein ER-associated degradation, responsible of labeling misfolded glycoproteins for degradation by the cytosolic proteasome.66 The glycoproteins carrying the monoglucosylated GlcMan9GlcNAc2N-linked oligosaccharide generated by ER glucosidase II52 are recognized by the complex calnexin-Pdi1 to promote protein refolding.66 The glycoprotein-chaperone complex interaction is then broken by glucosidase II that trims the last glucose residue. The UDP-glucose:glycoprotein α-glucosyltransferase senses the surfaces of the released glycoproteins and those that are still misfolded undergo glucosylation of the Man9GlcNAc2 oligosaccharide, allowing interaction again with the chaperon complex.66 This glucosylation / deglucosylation cycle will continue until the protein is properly folded or alternatively, can be disrupted by ER α-1,2-mannosidase and EDEM proteins that demannosylate the Man9GlcNAc2 oligosaccharide53 generating glycans unable to be recognized by the UDP-glucose:glycoprotein α-glucosyltransferase. If the glycoprotein is still misfolded upon the cycle has been disrupted by the ER α-1,2-mannosidase and EDEM proteins, it suffers retrograde transport to the cytosolic compartment for degradation.67 This glycoprotein quality control system is likely to occur in both S. schenckii and S. brasiliensis, as all basic components are present in their genomes.53 Figure 1. View largeDownload slide Hypothetical structures of N-linked (panel A) and O-linked glycans (panel B) generated from the bioinformatics analysis of S. schenckii and S. brasiliensis genomes Figure 1. View largeDownload slide Hypothetical structures of N-linked (panel A) and O-linked glycans (panel B) generated from the bioinformatics analysis of S. schenckii and S. brasiliensis genomes Similar to N-linked glycosylation, elaboration of O-linked glycans starts in the ER, where a mannose residue is transferred from the Dol-P-Man donor to a Ser- or Thr-residue at the surface of a nascent protein by protein mannosyltransferases, encoded by members of the PMT family,68 which is composed of three enzymes in both S. schenckii and S. brasiliensis (Table 2). Then, the O-linked glycans are elongated by members of the KRE2/MNT1 gene family, which generate linear α1,2-mannose polymers69 that in S. schenckii may contain two mannose units.70 Since some members of the KRE2/MNT1 gene family can add mannose units to both N-linked and O-linked glycans,71–73 it is possible that the three members of this gene family found in Sporothrix can work in both pathways. Experimental evidence suggests that at least one of them can participate in both O-linked and N-linked glycosylation pathways.61 The best characterized O-linked glycans were isolated from the peptido-rhamnomannan (PRM) fraction and contains an α1, 2-mannobiose core, one α1,2-glucuronic acid unit substituted by one or two rhamnose residues.70 The S. schenckii and S. brasiliensis genomes contain three putative glucuronosyltransferases that might participate in the elaboration of this O-linked glycan (Table 2). Our bioinformatics analysis could not find any obvious ortholog for rhamnosyltransferases, but both Sporothrix species contain all the required genes for synthesis of UDP-L-rhamnose (Table 2) and the sugar donor in the enzyme reaction catalyzed by rhamnosyltransferases.73–75 Figure 1B shows the current working model of the O-linked glycan structure based on these bioinformatic analyses. Finally, synthesis of GPI is extremely conserved in eukaryotic cells and the genomes of both Sporothrix species contain all genes to elaborate this glycolipid (see supplementary Table 2S). Interestingly, the S. schenckii genome lacks a putative ortholog of S. cerevisiae GPI11 (see supplementary table 2S) that encodes for a small subunit of the luminal phosphoethanolamine transferase complex.76 Since S. brasiliensis has a putative GPI11 ortholog, it remains to be established whether this difference significantly affects the structure of GPI anchors in S. schenckii and S. brasiliensis. Cell wall polysaccharides The cell wall provides protection to the fungus, acting as an initial barrier against hostile environments, while preserving the cell's integrity against internal turgor pressure. It is a dynamic structure, presenting continuous changes in composition and structural organization as the cell grows or presents morphological changes.77 These changes are strongly regulated during the cell cycle and in response to changing environmental conditions, stress, and mutations in cell wall biosynthetic processes.78,79 Over 90% of its components are polysaccharides, which are present as alkali-insoluble and alkali-soluble polysaccharides. The alkali-insoluble polysaccharides are arranged into a fibrillar skeleton, and are mainly composed of branched β1,3-glucan cross-linked to microfibers of chitin by β-1,4-linkages.77 Their proportions change according to the fungal species, and in some cases, to the cell type in dimorphic or polymorphic fungi.80 The alkali-soluble matrix is composed of amorphous polysaccharides, whose chemical composition also changes according to the fungal species.80,81 In S. schenckii, cell wall components of the yeast (Y), mycelial (M), and conidial (C) forms have been determined.82 Chitin is a minor component (7 to 8%) of the cell wall's fibrillar skeleton in all three cell types, while β-Glucan is a major component. β-glucan is present as a linear glucan, mainly composed of β-1,3-linkages (up to 66%), with β-1,6-(26 to 29%) and β-1,4-linkages (5 to 10%) in all three cell forms.82 The alkali-soluble fraction of S. schenckii cell wall is mainly composed of β-glucans and rhamnomannans, the later also complexed as a peptido-rhamnomannan after extraction by milder methods.83 The structure of the alkali-soluble β-glucan is like the alkali-insoluble β-glucan, although with differences in the proportions of linkages present in the polysaccharide (β-1,3: 44–45%; β-1,6: 28–31%; β-1,4: 24–28%).82 No variations in β-glucan-chitin composition can be related to the morphology of the fungus, since their relative composition is similar in the yeast, mycelial and conidial forms.82 In fungi, the synthesis of cell wall β-1,3-glucan is carried out through a complex formed by the catalytic unit (β-1,3-glucan synthase; Fks1), activated by Rho1, the GTP-dependent regulatory subunit in the β-1,3-glucan complex.84,85 The genomes of S. schenckii and S. brasiliensis contain single FKS orthologues35 (Table 3), which are different from those of C. albicans and S. cerevisiae, both of which present three β-1,3-glucan synthase genes with different functions in vegetative and conidial growth.84,86 However, several dimorphic and filamentous fungi present single FKS orthologues, as in Sporothrix species.35 No genes related to the synthesis of either β-1,6- or β-1,4-glucans could be identified, although hydrolase orthologs for the three types of β-glucan linkages present in the S. schenckii cell wall are present in both genomes (Table 3).35 Table 3. Genes identified as potentially involved in glucan synthesis and hydrolysis in S. schenckii and S. brasiliensis.* Protein function  S. schenckii  S. brasiliensis  Endo-β-1,3-glucanase (Cazy 81)  SS05995  SB04729  β-1,3-glucanase (Cazy 64)  SS03158  SB00339  β-1,3(4)-glucanase (Cazy 16)  SS02566  SB03506  β-1,6-glucanase (Cazy 2)  SS06336  SB05043  β-1,3-glucan synthase  SS01365  SB04029  Protein function  S. schenckii  S. brasiliensis  Endo-β-1,3-glucanase (Cazy 81)  SS05995  SB04729  β-1,3-glucanase (Cazy 64)  SS03158  SB00339  β-1,3(4)-glucanase (Cazy 16)  SS02566  SB03506  β-1,6-glucanase (Cazy 2)  SS06336  SB05043  β-1,3-glucan synthase  SS01365  SB04029  *Modified from Teixeira et al.35 View Large Chitin synthesis in fungi is regulated by multigene families, encoding chitin synthase isoenzymes.87–89 Their activities are spatially regulated to fulfil their roles.87 Seven chitin synthase (CHS) genes are present in both S. schenckii and S. brasiliensis genomes. Each one of the translation products of the seven CHS Sporothrix genes identified can be classified into each one of the seven chitin synthase classes known (I to VII, Table 4).35 The large number of chitin synthase genes identified in S. schenckii and S. brasiliensis contrasts with the low chitin content of the Sporothrix cell wall (7 to 8% of total cell wall content). In S. cerevisiae, where only three CHS genes have been identified, chitin synthesis is regulated both temporarily and spatially in relation to the cell cycle.90 However, in this fungus, none of the CHS genes is essential, although the triple mutant is impaired. In contrast, C. albicans has four CHS genes, of which CHS1 (whose translated product, Chs1 belongs to class II) is essential for cell viability.90 The significance of each of the seven chitin synthase classes is not yet understood, as mutations affecting members of the same enzyme class, do not always result in a similar phenotype.91 This suggests that the different fungal Chs enzymes perform distinct and specific functions in every fungus, even though they have homologous sequences. Table 4. Genes identified as potentially involved in chitin synthesis in S. schenckii and S. brasiliensis. Protein function  S. schenckii  S. brasiliensis  CHS class  Proposed gene name  Chitin synthase  SPSK08492  SPBR08106  I  CHS1    SPSK06891  SPBR02298  II  CHS2    SPSK06989  SPBR02297  III  CHS3    SPSK07523  SPBR06424  IV  CHS4    SPSK06859  SPBR02173  V  CHS5    SPSK00405  SPBR08833  VI  CHS6    SPSK6887  SPBR02195  VII  CHS7  Protein function  S. schenckii  S. brasiliensis  CHS class  Proposed gene name  Chitin synthase  SPSK08492  SPBR08106  I  CHS1    SPSK06891  SPBR02298  II  CHS2    SPSK06989  SPBR02297  III  CHS3    SPSK07523  SPBR06424  IV  CHS4    SPSK06859  SPBR02173  V  CHS5    SPSK00405  SPBR08833  VI  CHS6    SPSK6887  SPBR02195  VII  CHS7  *Modified from Teixeira et al.35 View Large Large numbers of chitinases in fungi have been proposed as being directly related to the chitin content in the cell wall.92 However, this clearly does not apply to Sporothrix spp., where 10 chitinase genes have been found in the S. schenckii genome while nine are present in that of the S. brasiliensis genome.35 The functions of such large numbers of chitinases is yet to be elucidated. The polysaccharide synthesis and hydrolysis-related genes identified in S. schenckii and S. brasiliensis genomes35 correlate with the polysaccharide composition of the cell wall previously reported for S. schenckii.82 As in many fungi, there is still a long way to go in determining whether individual cell wall polysaccharides synthase and/or hydrolase-related genes here discussed, might be involved in shaping the yeast, mycelial and/or conidial cell forms of Sporothrix spp., how they could be related in host-fungal interactions, or even if any of them might have a role in the fungal survival, and therefore being targets for the development of specific antifungal drugs. Diagnosis and identification of clinically relevant Sporothrix species Direct mycological examination using potassium hydroxide (KOH) or differential staining is low sensitive for the diagnosis of human sporotrichosis due to the scarcity of fungal elements in the lesions, particularly in lymphocutaneous and fixed cutaneous forms. However, Gram, Giemsa, Periodic-Schiff (PAS), and Grocott-Gomori's staining (silver staining) can be successfully used in disseminated manifestations. On the other hand, in feline sporotrichosis there is a high fungal load in the lesions favoring direct examination for the rapid diagnosis of the disease.93 In general, round, oval, often elongated yeasts are observed, resembling a cigar shape. Although direct examination does not allow differentiation of Sporothrix species, it is important to rule out other cutaneous sporotrichoid infections.94 The gold standard for diagnosing Sporothrix is based on conventional culture of clinical specimens obtained from active lesions, pus, secretions or biopsy.95 Samples are cultured on Sabouraud agar and may be followed by antifungal susceptibility testing and additional phenotypic characterization on a case-by-case basis. Positive cultures appear in the first 2 weeks of incubation, however, in some cases it will be necessary to observe for up to 30 days before discarding them as negative. Cultures held at 25°C develop thin hyphae with erect conidiophores bearing several hyaline single-celled conidia, disposed in a flower-like arrangement. Numerous sessile, brown, (sub)globose, or triangular conidia are visible along undifferentiated hyphae. Note that this phenotypic character is shared by several environmental Sporothrix species.96 Demonstration of dimorphism is important to confirm the supposed agent.97 Phenotypic identification often requires 7 to 14 days for culture, and 10 to 21 additional days for physiological assays.96 Moreover, morphological characteristics are insufficiently diagnostic for the clinically relevant species.98 Molecular tools are required for the recognition of cryptic entities. Accurate molecular diagnostic tests have a key role in patient management guiding therapy99,100 and from an epidemiological point of view it will help to recognize and control nascent outbreaks due to distinct Sporothrix species.21 Sequencing reduces identification time to about 12 h, with an improved diagnosis. Phylogenetic analyses based on the rRNA operon supports monophyletic of human-pathogenic Sporothrix group.8,21,101 Global sampling of clinical Sporothrix does not affect the success of species recognition by ITS sequencing confirming the robustness of this marker.102 A striking phylogenetic bipartition is observed between the pathogenic species S. brasiliensis, S. schenckii, S. globosa, and S. luriei and remaining environmental Sporothrix species living in association with soil and plant debris.8 It is noteworthy that the taxonomic resolution of the ITS region (ITS1/5.8 s/ITS2) is low among agents embedded in the S. pallida and S. stenoceras complexes. In these cases, it is necessary to use protein-coding loci for the recognition of cryptic entities.33 Frequently used protein-coding loci include beta-tubulin (BT2), calmodulin (CAL), and elongation factor 1α (EF-1α). Multilocus analysis significantly increases the taxonomic resolution among species in the clinical clade and is helpful to recognize rare agents such as S. pallida, S. mexicana, and S. chilensis.33 Among the loci above, the region spanning exons 3 to 5 of the calmodulin gene is the main marker for the recognition of Sporothrix (Fig. 2).4,103,104 DNA polymorphisms can also be used to study genetic diversity, population structure, recombination analysis, and molecular epidemiology of Sporothrix species.34,105,106 Figure 2. View largeDownload slide Phylogenetic relationships among members of the clinical clade and environmental clade in Sporothrix, based on sequences of calmodulin encoding gene (exon 3–5). Method: Maximum Likelihood and Neighbor-joining. The numbers near the branches (ML / NJ) refer to re-sampling percentages (1000 bootstraps). Genbank accession number can be found close to species name. All sequences have been previously published [8, 34, 130, 138] and are available from GenBank. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Phylogenetic relationships among members of the clinical clade and environmental clade in Sporothrix, based on sequences of calmodulin encoding gene (exon 3–5). Method: Maximum Likelihood and Neighbor-joining. The numbers near the branches (ML / NJ) refer to re-sampling percentages (1000 bootstraps). Genbank accession number can be found close to species name. All sequences have been previously published [8, 34, 130, 138] and are available from GenBank. This Figure is reproduced in color in the online version of Medical Mycology. Amplification of a target sequence in the fungal genome through polymerase chain reaction (PCR) followed by digestion of the amplicon with one or a combination of restriction enzymes (RFLP) has been successfully employed for the identification of Sporothrix species of clinical interest. An 800 bp fragment of the calmodulin gene is amplified using primers CL1 and CL2A and digested with the enzyme HhaI (5΄-GCGC-3΄) to produce species-specific profiles assigned to S. brasiliensis, S. schenckii, S. globosa, and S. luriei.106 As the primers (CL1 and CL2A) are not specific for Sporothrix, it is necessary to isolate the fungus in culture, not allowing direct detection from clinical samples (Fig. 3). Figure 3. View largeDownload slide Recognition of Sporothrix species using classical and molecular approaches. Classical methods present a great phenotypic overlap among closely related species such as S. brasiliensis, S. schenckii and S. globosa, being therefore their use discouraged as the only identification method. Molecular methods based on DNA sequencing followed by phylogenetic analysis allows the identification of all 51 Sporothrix described to date. Methods such as species-specific PCR, PCR-RFLP and RCA are important tools for identifying species of clinical interest. Direct detection and simultaneous identification of Sporothrix from complex samples can be performed with species-specific PCR or RCA. Characterizing Sporothrix in molecular epidemiology studies requires the development of new markers to access genetic diversity, aiming to answer questions related to population structure, transmission routes, intra- and interspecific variability, recombination and reproduction modes, speciation, among many other biological questions. Sybr, SYBR Green dye; PCR, polymerase chain reaction; PCR-RFLP, PCR-restriction fragment length polymorphism; RCA, rolling circle amplification; MLST, multilocus sequence typing; WGS, whole genome sequencing; SNPs, single-nucleotide polymorphisms; AFLP, amplified fragment length polymorphism; RAPD, random amplification of polymorphic DNA. This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. View largeDownload slide Recognition of Sporothrix species using classical and molecular approaches. Classical methods present a great phenotypic overlap among closely related species such as S. brasiliensis, S. schenckii and S. globosa, being therefore their use discouraged as the only identification method. Molecular methods based on DNA sequencing followed by phylogenetic analysis allows the identification of all 51 Sporothrix described to date. Methods such as species-specific PCR, PCR-RFLP and RCA are important tools for identifying species of clinical interest. Direct detection and simultaneous identification of Sporothrix from complex samples can be performed with species-specific PCR or RCA. Characterizing Sporothrix in molecular epidemiology studies requires the development of new markers to access genetic diversity, aiming to answer questions related to population structure, transmission routes, intra- and interspecific variability, recombination and reproduction modes, speciation, among many other biological questions. Sybr, SYBR Green dye; PCR, polymerase chain reaction; PCR-RFLP, PCR-restriction fragment length polymorphism; RCA, rolling circle amplification; MLST, multilocus sequence typing; WGS, whole genome sequencing; SNPs, single-nucleotide polymorphisms; AFLP, amplified fragment length polymorphism; RAPD, random amplification of polymorphic DNA. This Figure is reproduced in color in the online version of Medical Mycology. Molecular assays based on DNA amplification can easily detect Sporothrix from clinical samples or small amounts of cells in culture without the need for sequencing, thereby improving sensitivity and considerably shortening the time required for identification to a few hours.107 Species-specific primers are available that selectively amplify S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and S. stenoceras from minimal amounts of target DNA (10–100 fg DNA) (Fig. 3). Rolling circle amplification (RCA) was first introduced in the mid-1990s as a simple and powerful technique capable of synthesizing large amounts of DNA from very low initial concentrations.108 The method is particularly useful for padlock probe amplification, that is, linear DNA probes become circular (via Pfu DNA ligase enzyme) when specific recognition of a given target sequence occurs. The combination of probe-padlock circularization and amplification through RCA under isothermal conditions has been shown to be useful for the sensitive detection of Sporothrix sequences (up to 3 × 106 copies of the target), although the technique is not used in routine diagnostics. RCA can be applied from pure cultures to complex environmental samples such as soil and vegetables allowing ecological studies that are still scarce in Sporothrix (Fig. 3).109 Methods independent of DNA sequencing (e.g., PCR-RFLP, species-specific PCR and RCA) are important because they provide fast, accurate results in addition to lower costs for identification. This is particularly important during epizootics or zoonotic outbreaks where hundreds to thousands of cases emerge in a short time33 and a precise diagnosis is required to assist in choosing the best therapeutic regimen for the patient.34 Future perspectives might concern the development of multiplex real-time PCR assays using fluorescent probes for simultaneous detection and identification of clinically relevant Sporothrix species. Despite the technical difficulty in standardizing such reactions (annealing temperature, specificity, interactions between multiple primers and probes, etc.), multiplexing significantly reduces the cost of the reaction, the volume of samples, increases sensitivity, and allows the detection of mixed infections. Characterizing Sporothrix in molecular epidemiology studies requires the development of new powerful markers such as those based on whole genome sequencing (WGS) or single-nucleotide polymorphisms (SNPs)-arrays panels (Fig. 3) to access genetic diversity, aiming to answer questions related to population structure, transmission routes, intra- and interspecific variability, recombination and reproduction modes, speciation, among many other biological questions. Sporothrix antigens and vaccine development Although the classical mycological test is still useful in a routine basis, the development of immunochemical tests together with molecular tools can improve the diagnostic time and help to follow-up treatment. For example, resistance to azoles is already reported in S. brasiliensis and S. globosa.17,110–113 Moreover, due to the emergence of S. brasiliensis and S. globosa and the growing incidence of sporotrichosis in humans and cats, new strategies are necessary for controlling sporotrichosis in endemic areas. Proteomics and glycosciences can help to map and identify new antigens for these purposes.47,114 Furthermore, a peptido-rhamnomannan or PRM is one of the main components of Sporothrix spp. cell wall.47 Distinct cell wall components of pathogenic fungi can be of key importance in host recognition and immune response.115 The peptido-rhamnomannan was first isolated by Lloyd and Bitoon.83 These authors showed that this peptido-polysaccharide could be recognized by human serum antibodies of patients with sporotrichosis. Travassos and coworkers,116,117 studying the sugar moieties of PRM, showed that the epitopes present on the rhamnomannans differed between the yeast parasitic phase (α-L-Rhap 1→3 α-D-Manp) and the mycelium phase (α-L-Rhap 1→2 α-L-Rhap 1→3 α-D-Manp) of S. schenckii. For a long time these N-linked epitopes were considered as the main antigenic structures present on the cell wall PRM.116 Interestingly, PRM is recognized by the lectin Concanavalin A (ConA), but the rhamnomannans isolated by hot alkali extraction (N-linked glycan chains) loose ConA reactivity.116 Years later, Lopes-Alves et al.70 determined the fine structure of the O-linked glycan chains of PRM and reported that they bear the ConA binding sites, a mannobiose core of α-D-Manp 1→2 α-D-Manp. Among the PRM O-linked glycan chains, two new epitopes were described (α-L-Rhap 1→ 2 α-D-GlcAp and α-L-Rhap 1→ 2 [α-L-Rhap 1→ 4] α-D-GlcAp) at the non-reducing ends of a tetra- and pentasaccharides.47,118 These O-glycan epitopes are not described in any other fungal pathogen being exclusive of Sporothrix spp.47 Moreover, the PRM O-linked glycan chains were the main epitopes recognized by IgG antibodies present in patient's sera.119 Based on these biochemical findings, a Con-A binding cell wall antigen was further isolated and purified from the PRM fraction.120 This antigenic fraction denominated as SsCBF (Sporothrix schenckiiConA binding fraction) was used to develop an ELISA test for the serodiagnosis of human and feline sporotrichosis.121–123 The ELISA test was clinically validated for human sporotrichosis with 90% global efficacy (sensibility and specificity)122 and is used as an “in house” test in two University Hospitals in Rio de Janeiro, Brazil. The SsCBF ELISA test can give a serodiagnostic result in few hours and is very useful for treatment monitoring.24,26,27,124 A cell wall antigenic molecule described in S. schenckii and S. brasiliensis clinical isolates is a glycoprotein of 60–70 kDa (Gp70).9,125 The 70 kDa antigen was first described as a component of the cell wall PRM fraction of S. schenckii.120 A decade after its first description, the first evidence that this cell wall antigen has protective properties came out.126 A IgG1 monoclonal antibody raised against the extracellular Gp70 (MAb P6E7) was used successfully to induce a passive immunization to mice infected with Sporothrix schenckii126 and several subsequent studies showed its therapeutic efficacy towards Sporothrix spp. infection.126–129 A proteomic study was performed and the Gp70 antigen was sequenced by MS/MS.9 In a subsequent proteomic study, a 2-D fluorescence difference gel electrophoresis (DIGE) approach was used to compare the expression of this antigen isolated from S. schenckii and S. brasiliensis yeast cell extracts.125 The authors showed that this antigen has proteoforms in both species. A 60 kDa glycoform is predominant in S. brasiliensis.125 In conclusion, both antigenic bands of 60 and 70 kDa (Gp60 and Gp70, respectively), previously described in the literature as distinct antigens,130 are actually related to the same gene sequence.125 A recent study showed that a recombinant phage displaying a Gp70 peptide (kpvqhalltplgldr) on the major coat protein (pIII) was efficiency as a vaccine for preventing S. globosa infection.131 The data described above allow us to conclude that the Gp70 antigen identified so far has a potential for the development of a vaccine to control Sporothrix infection.125–129,131 A cell wall protein extract previously described by Castro et al.9 in their work showing the cell wall expression and peptide sequence of the Gp70 antigen, was used in recent vaccination studies.132,133 This DTT protein extract, denominated ssCWP, was associated with aluminum hydroxide (AH).132 Two AH-adsorbed ssCWP based vaccine formulations were tested in mice systemically infected with S. schenckii. In this study, Portuondo et al.132 characterized in the ssCWP extract a new antigen of 47 kDa identified as enolase and a 44 kDa non-antigenic band identified as a peptide hydrolase. These authors also corroborated the presence, in the DTT extract, of an antigenic band localized at the 70 kDa gel region (71 kDa).132 The passive immunization with sera of mice treated with an AH-adsorbed ssCWP formulation (AH+CWP10) showed a reduction in the number of colony forming units (cfu) in liver and spleen. In a recent study, these authors evaluated a Montanide™ Pet Gel A (PGA) formulated with ssCWP and compared with the AH-SsCWP formulation.133 Immune response against Sporothrix The cell wall of members of the pathogenic Sporothrix clade is the main source of pathogen-associated molecular patterns (PAMPs). Thus far the best characterized component is the peptido-rhamnomannan.47,116,117 It is now clear that the cell-mediated immune response plays a key role in controlling Sporothrix dissemination, as demonstrated by protection against infection upon transference of normal immune spleen cells to athymic nude mice.134,135 The innate immunity is key during the establishment of a protective anti-Sporothrix response. Phagocytosis by neutrophils and macrophages, and production of reactive oxygen species are essential for neutralization and killing of S. schenckii cells.135,136 In addition, differential ability of murine macrophages to interact with species of S. schenckii and relatives has been reported, with S. brasiliensis being significantly more phagocytosed than S. schenckii.3 However, not all the molecules of the oxidative burst during immune activation against Sporothrix have a positive contribution in the control of the fungus. Production of nitric oxide during experimental sporotrichosis has a detrimental role during infection, facilitating the establishment of the infection via apoptosis of immune cells, and stimulating an anti-inflammatory state.137 S. schenckii yeast cells can activate both classical and alternative complement pathways,116 which makes cells susceptible to the uptake by macrophages.138 However, conidial interaction with phagocytic cells does not depend on opsonization by complement activation,138 suggesting this cell morphology is recognized by receptors different from those involved in the yeast recognition. Knowledge on identity and molecular structure of PAMPs in Sporothrix species is still limited.139 Nevertheless, significant contributions have been made in the involvement of PAMPs in immune sensing. Toll-like receptor (TLR) 4 was found to interact with lipid preparations from S. schenckii yeast-like cells, stimulating the production of pro- and anti-inflammatory cytokines and oxidative mediators in a murine model of sporotrichosis.140–142 This immune receptor has also been involved in the production of both pro- and anti-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), in particular when stimulated with either conidia, yeast-like cells or germlings from S. brasiliensis.139 Interestingly, conidia and germlings from S. schenckii, but not yeast-like cells, are capable of stimulating cytokine production via TLR4.139 Cytokine production via TLR2 also occurs upon interaction with S. schenckii yeast-like cells, and macrophage functions against this organism are significantly impair in cells from TLR2-deficient mice.143 The interaction of the three morphologies of both S. schenckii and S. brasiliensis with human PBMCs is also dependent on TLR2, confirming this receptor is relevant in both the murine and human response against these pathogens.139 A mannose receptor (MR) has also been involved in sensing S. schenckii conidia by THP-1 macrophages.138 Using primary human PBMCs, it has demonstrated this receptor is relevant to cytokine production stimulated by S. schenckii conidia or S. brasiliensis yeast-like cells,139 underlining that cell morphology is accompanied by changes in the molecular structure of PAMPs on the surface of the fungal cells. One of the most important immune receptors involved in the establishment of an anti-fungal immune response is dectin-1. This receptor is key for cytokine stimulation when human PBMCs interact with the three morphologies of S. schenckii or S. brasiliensis conidia and yeast-like cells.139 However, it is dispensable for cytokine stimulation when human PBMCs interact with S. brasiliensis germlings.139 Accordingly, it has been reported that this receptor, along with IL-17 production, is not involved in the clearance of S. schenckii infection in a rat model of sporotrichosis.144 The adaptive branch of the immune response is essential for appropriate control of Sporothrix infections, involving the establishment of a Th1/Th17-based immune response.145–147 The successful establishment of this response, partially relies in the activation of the NLRP3 inflammasome,148 and activation of dendritic cells,149 which respond to exoantigens of S. schenckii yeast-like cells. However, a differential dendritic cell activation has been reported, depending on the origin of the etiologic agent, that is, a strain from a cutaneous lesion provides a better stimulus to establish a Th1 response than a strain of visceral origin.150 Despite this interesting evidence, the antigenic / molecular differences between these strains remain to be established. In addition, since this study was published before the separation of S. schenckii siblings4 it is tempting to speculate whether these two strains might belong to different species. Even though these evidences suggest that control of these pathogens is Th1/Th17 dependent, the activation of the Th2 response is also important during the generation of an anti-Sporothrix immune response, especially in advanced stages of the infection.151 After animal immunization with S. schenckii cells, antibodies raised against several fungal antigens can be detected.152 As mentioned before, the best characterized antigen thus far is Gp70, a secreted and cell wall glycoprotein of about 70 kDa, 3,9,47,125,153,154 which is a glycoprotein heterogeneous in molecular weight also known as Gp60.155 In mice, the passive immunization with anti-Gp70 antibodies has shown protection against sporotrichosis caused by S. schenckii and S. brasiliensis, promoting a significant reduction in fungal burden, even with deficiency in T-cells.127,128 It seems the mechanism to protect against the infection is based on opsonization, which facilitates the uptake by macrophages and enhances production of pro-inflammatory cytokines.156 Recently, humanized anti-Gp70 antibodies have been successfully used to opsonize both S. schenckii and S. brasiliensis cells with a positive influence on phagocytosis by human monocyte-derived macrophages,129 which bring us closer to the use of these antibodies as part of a treatment of human sporotrichosis. Future directions The next steps following the genomic studies are the development of an effective transformation system for Sporothrix spp. and the trascritpome analysis (RNA sequencing analysis; RNA-Seq). These studies are under development. A bank of mutants will be of undeniable value to unveil the role of several genes and/or gene products in the biology and pathogenicity of Sporothrix spp. Moreover, the new molecular and serological diagnostic tools already available, described in this review, will contribute to refine epidemiological studies. In addition, our perspective is to generate a consensus, in the near future, to recommend the application of more sensitive and rapid diagnostic tests in the clinical routine. The delay in the diagnosis of severe or unsusual clinical forms of sporotrichosis, especially related to the new emerging pathogenic species, is a critical aspect to initiate a correct and appropiate therapeutic regimen. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements L.M.L.B. is a research fellow of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 307169/2015-4) and of Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ; CNE 2015), Brazil. H.M.M.M. is supported by Consejo Nacional de Ciencia y Tecnología (ref. CB2011/166860; PDCPN2014-247109, and FC 2015-02-834), Universidad de Guanajuato (ref. 0087/13; ref. 1025/2016; Convocatoria Institucional para Fortalecer la Excelencia Académica 2015; CIFOREA 89/2016), Programa de Mejoramiento de Profesorado (ref. UGTO-PTC-261), and Red Temática Glicociencia en Salud (CONACYT-México). Y.Z. was supported by the Tianjin Municipal Natural Science Foundation (grant no.15JCYBJC49500). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Schenck B. 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Medical MycologyOxford University Press

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