Galactofuranose antigens, a target for diagnosis of fungal infections in humans

The use of biomarkers for the detection of fungal infections is of interest to complement histopathological and culture methods. Since the production of antibodies in immunocompromised patients is scarce, detection of a specific antigen could be effective for early diagnosis. D-Galactofuranose (Galf) is the antigenic epitope in glycoconjugates of several pathogenic fungi. Since Galf is not biosynthesized by mammals, it is an attractive candidate for diagnosis of infection. A monoclonal antibody that recognizes Galf is commercialized for detection of aspergillosis. The linkage of Galf in the natural glycans and the chemical structures of the synthesized Galf-containing oligosaccharides are described in this paper. The oligosaccharides could be used for the synthesis of artificial carbohydrate-based antigens, not enough exploited for diagnosis.

involved in the biosynthesis of antigenic O-glycans was identified [28]. The glycans that contain Galf are involved in immunological reactions [29,30,31], therefore they are envisioned as targets for diagnosis. This article aims to explore the perspective of methods based on synthetic sugar antigens for the production of monoclonal antibodies for serological detection of fungus-specific antigens.

β-galactofuranosyl structures in human infective fungi
Galf is mainly present in the β-configuration in fungi, many of them involved in mammal infections. It is also present in plant pathogenic fungi, but these will not be discussed in the present review.
Fungi that infect mammals and contain glycans with Galf are listed in Table 1. Galf is usually present as terminal sugar, linked to each other by β(1→6) or β(1→5) linkages or branching a mannan core at the O-2 or O-3 position of mannose.
The cell walls of A. fumigatus have at least four different types of molecules containing Galf, which are important for cell wall integrity (Table 1) [29,32,33,34,35,46,47]. Galf units were identified in O-and N-linked chains of glycoproteins and also in glycoinositolphosphorylceramides [48]. The glycoinositol phosphorylceramides with Galf(β1→6) linked to a mannose core were highly immunogenic.
Fusarium is a pathogen of plants, but some species are pathogenic for humans, particularly Fusarium verticillioides and F. proliferatum [51]. Fusarium species cause a broad range of opportunistic infections in humans. In healthy individuals, the most common clinical manifestations are onychomycosis, skin infections and keratitis, whereas in immunocompromised patients disseminated infections with multiple necrotic lesions may occur [52].
It is important to discriminate Aspergillus from Fusarium infections since both differ in their response to common antifungals. Using a combination of two antibodies, both species could be differentiated by immunohistology (see below) [31]. Galactofuranose antigens, a target for diagnosis of fungal infections in humans Special Report future science group Trichophyton spp., in particular T. mentagrophytes and T. rubrum cause chronic dermatophyte infections, often associated with infection of the nails (onycho mycosis) [53]. Although they first infected animals, they adapted to infect humans and are now considered a major health problem [54]. Cell wall antigens secreted by the fungus may diffuse into the dermis and establish the infection, due to immunosuppressive effects. Impairment of lymphocyte proliferation was shown [55]. One of the major cell wall components secreted to the medium is a GM. Structure determinations showed that Galf terminal units are β(1→3) linked to a mannan core. Polygalacto furanosyl chains, similar to those produced by A. fumigatus, have not been found in Trichophyton [39]. Accordingly, a monoclonal antibody against the GM of A. fumigatus showed very low cross-reactivity with exoantigens from cultures obtained from clinical specimens [56]. Glycosylphosphatidy linositols labeled with [ 3 H]-galactose and [ 3 H]-mannose were biosynthesized by membrane prepar ations of T. rubrum. The lability to acid of the galactose suggested its furanosic configuration [57].
future science group future science group Special Report Marino, Rinflerch & de Lederkremer Sporothrix schenckii is the agent of sporotrichosis in humans and animals, producing skin and subcutaneous lesions. It is present in all continents, especially in tropical and subtropical areas [59,60]. In an early work, Galf-containing polysaccharides were isolated from the supernatants of S. schenckii cultures [41]. It was found that a mannan core is substituted by β-Galf chains which are responsible for cross-reactions with other fungal antigens. Apparently, no further studies on this GM were described. In turn, peptidorhamnomannans with both N-and O-linked carbohydrate chains as the immunodominant structures were obtained from extracts of the cell walls [61]. Later studies characterized a peptidorhamnomannan (Gp70) isolated from the yeast phase of S. schenckii as an adhesin involved in the host-pathogen interaction, but Galf was not reported as a constituent of this glycoprotein [62].
Cladosporium spp. are mainly plant pathogens but some may trigger allergic reactions in sensitive individuals. Prolonged exposure to a high concentration of spores may produce chronic asthma. A glyco protein named Ag-54, which contains 80% carbohydrate, was an allergen purified from C. herbarum. Structural studies showed chains of β1→6 linked Galf bound to O-2 of 1→6 linked mannoses (Table 1) [42].
Paracoccidioides brasiliensis is endemic to regions of Latin America, causing a mycosis which spreads from the lungs to many organs and if not treated could be fatal [63]. Paracoccidioidomycosis (PCM) is commonly diagnosed by identifying budding yeast cells in biological fluids or histologically [64]. Glycolipids extracted from yeast and mycelium forms of P. brasiliensis reacted with sera from patients. The antibodies were directed mainly to the galactofuranosyl units in the glycolipids [65]. A review on glycolipids in fungi and trypanosomatids discusses the role of Galf in the infect ivity [66]. However, the main diagnostic antigen of P. brasiliensis is the exocellularly secreted glycoprotein gp43, which contains a terminal Galf unit attached (β1→6) to a mannose in an N-linked oligosaccharide chain [30].
Neotestudina rosatii is one of the etiologic agents of subcutaneous infections in humans. This species occurs with other fungi causing eumycetoma, and its taxonomic classification is uncertain [67].
A glycoprotein with N-and O-glycans was extracted from Fonsecaea pedrosoi, the etiologic agent of chromoblastomycosis. The hexasaccharide containing internal Galf (Table 1) was the main O-linked chain [44,68]. Specific antibodies against F. pedrosoi were strongly inhibited by the hexose, but the contribution of the internal Galf to the antigenicity was not evaluated. Probably, the main epitope in F. pedrosoi is the external α-Galp, a recognized antibody in humans [69].
In the previously mentioned examples, Galf is present in the β-anomeric configuration. However, the less common α-Galf has also been found in some organisms, such as, P. brasiliensis. Interestingly, the configuration of the sugar depends on the phase, whereas β-Galf is present in the yeast form infecting the mammal, α-Galf is a constituent of the mycelial GM (Table 2) [38,70]. Galf was also found in glycoinositol phospholipids obtained from the yeast phase of Histo plasma capsulatum, the causative agent of histoplasmosis, an endemic mycosis [71]. Histoplasma capsulatum is a dimorphic fungus which grows in the soil as a filamentous mycelium, but it converts to a yeast-like form in the tissues of infected animals. The purified glycoinositolphospholipids were shown to react with sera from histoplasmosis patients [71]. The authors tentatively assigned the α-configuration for the Galf from the yeast form, in contrast with the configuration found for the Galf in the same phase in P. brasiliensis. The configuration of galactofuranosides is unequiv ocally assigned by NMR spectroscopy [12], which was not used by Barr et al. in 1984 [71]. The glycosphingolipids from the mycelial phase were not described.
Galactofuranose antigens, a target for diagnosis of fungal infections in humans Special Report future science group  [89,90,91] future science group future science group Special Report Marino, Rinflerch & de Lederkremer The antigenic properties of β-galactofuranosides are well known [29,30,31] but the immunological role of α-Galf was apparently not described. The differences in the Galf configuration in the galactomannans of the infective P. brasiliensis and A. fumigatus could explain the low cross-reactivity in serological tests using a specific antibody [73].

Structural analysis of Galf-containing polysaccharides or glycoconjugates
In earlier works, the identification of Galf units in glyco conjugates was performed using chemical methods. Taking advantage of the greater lability of furanosic linkages with respect to the pyranosic bonds, terminal Galf units may be selectively released from the molecule by mild acid hydrolysis, separated by chromatographic techniques or by dialysis, depending on the MW of the remaining degraded molecule, and then analyzed by chromatographic methods or even GC-MS. Also, the exocyclic chain of terminal Galf could be selectively oxidized by periodate under mild conditions, and upon reduction with NaBH 4 converted to arabinofuranose, which may be identified after acid hydrolysis by GC-MS. Application of these methods is exemplified in the report on the determination of the structure of glycolipids of P. brasiliensis [65]. Inform ation on the substitution position of the galacto furanosyl linkages is provided by methylation analysis [74]. NMR has also been useful for the identification of Galf units in oligosaccharides. 1 H NMR spectra usually show deshielded anomeric signals for Galf units, compared with those of pyranosic units. Particularly useful is the 13 C NMR spectroscopy because the furanosyl ring has a specific signal pattern, easily distinguishable from the pyranosyl analog. For β-Galf units, signals corresponding to the anomeric carbon, and C-2 and C-4 resonate significantly deshielded in comparison with pyranosic signals. In addition, α-Galf has also a particular pattern. Advances in NMR spectroscopy have facilitated the characterization of oligo-saccharides. Thus, bidimensional techniques allow not only the identification of the monosaccharides and their anomeric configuration but also to stablish the position of substitution and sequences [35,39,75].

Chemically synthesized oligosaccharides of antigenic glycans
The use of chemically synthesized oligosaccharides of defined structure avoids the disadvantages of glycans obtained from natural sources, such as, the difficulty of isolating and purificating sufficient quantities, and problems associated with their heterogeneity. Also, more specificity may be achieved by using a neoantigen. Following this line, a biotinylated tetrasaccharide with Galf(β1→5)Galf linkages was synthesized (Table 3A; n = 2). This synthetic antigen was recognized by the monoclonal antibody EB-A2 against Aspergillus [76].
The synthesis of Galf-containing molecules requires the efficient preparation of derivatives of  3 Fu et al. [92]  Most sugars lead by conventional methods to pyranosic derivatives, which are thermodynamically more stable. However, in conditions under which other monosaccharides lead mainly to pyranosic products, large proportions of galactofuranosic derivatives can be obtained from galactose. This is the case of the traditional Fisher glycosylation, which yields a mixture of furanosic and pyranosic glycosides that must be separated by chromatography [27]. An example may be found in the preparation of the pentenyl galactofuranosides which were used as precursors in glycosylation reactions [97].
One of the most popular glycosylation procedures is the trichloroacetimidate method [99], which is compatible with acid-sensitive acceptors and was used first for the synthesis of Galf (β1→3)GlcNAc [100]. As this is an area of active research, several more methods have been designed and extens ively reviewed [26,95,96]. The synthetic oligosaccharides could be precursors for artificial antigens to be used for diagnosis [101] or for the construction of synthetic vaccines [102,103].

Immunologic detection of Galf-containing molecules
A monoclonal antibody, which recognized Galf (β1→5)Galf epitopes was produced by immunizing rats with an extract of A. fumigatus mycelia. The antibody called EB-A2 also detected the GM of other Aspergillus species, using an ELISA assay [104,105]. It was commercialized as Platelia™ Aspergillus ELISA (Bio-Rad Laboratories and Sanofi Diagnostics, CA, USA). The specificity and sensitivity of the assay were reviewed [106]. Multivalent gold nanoparticles carrying Galf were recognized by this antibody [107]. The presence of GM in circulation depends on the manifestation of the disease. For instance, negative results are commonly obtained for the detection of GM in sera of allergic bronchopulmonary aspergillosis. Reviews on diagnosis of aspergillosis have been published [108,109,110]. Apparently, this is the only commercialized test based on detection of Galf. It was shown that EB-A2 detects GM in supernatants of several Fusarium species [111,112]. However, Wiedemann et al. described a novel monoclonal antibody, AB 135-8, which recognizes Galf in Fusarium but as part of a different antigen [31]. In fact, the structural unit Galfβ(1→6)Galf, was characterized in a polysaccharide extracted from Fusarium [75] but it was not reported in Aspergillus (Table 1). The same antigenic disaccharide was found in Malassezia galactomannans, which did not react with the EB-A2 antibody against A. fumigatus, but apparently was not tested with AB 135-8 against Fusarium. Antibodies against M. furfur did not react with galactomannans of A. fumigatus, T. rubrum or F. pedrosoi, confirming the presence of a different linkage for the Galf units, but apparently these were not tested with AB 135-8 against Fusarium [40].
The Platelia test was negative for yeast and mold forms of S. schenckii [113]. Controversial results were Galactofuranose antigens, a target for diagnosis of fungal infections in humans Special Report future science group reported for Cryptococcus neoformans. While a positive test was reported for a patient with C. meningitis [114], a later report revealed no cross-reaction with culture extracts, purified polysaccharides, clinical specimens and specimens from animals following experimental infection [115]. These results are explained by the composition of the cell wall of C. neoformans. Although galactoxylomannan is a constituent of the cell wall, and D-Gal is its major component, this sugar is mainly in the pyranosic configuration, with only 2% tentatively assigned to Galf, considering a small peak in the 13 C NMR spectrum [116], which is as explained above, is not enough to guarantee the configuration. More recently, it was demonstrated that Galf is not required for growth or virulence of C. neoformans [117].

D-Gal
In P. brasiliensis, Galf is the immunodominant unit in a glycosylinositolphosphoryl ceramide [43], however, the main diagnostic antigen is the glycoprotein gp43. Studies of N-deglycosylation gave rise to a protein of 38 kDa, which strongly reacted with sera from patients with PCM [118]. Also, recombinant gp43 isoforms as N-mannosylated proteins were expressed in the yeast Pichia pastoris and showed good specificity for detection of PCM in sera [119]. These results indicate the presence of other epitopes in gp43, although the cause of cross-reactivity with sera from patients with other mycoses would be the terminal Galf [118120]. Immunodiagnosis of gp43 in PCM using a latex test was recently described [73].
In the previous examples, Galf is present in the galactomannans in the β-anomeric configuration. A few examples of α-Galf may be seen in Table 2. The antigenic properties of α-Galf were apparently not described. As expected, isolates from H. capsulatum in yeast and mold phases were not recognized by the monoclonal antibody against the Aspergillus GM [113]. However, it was reported that some patients with dis-seminated histoplasmosis gave positive results with this antibody [121].

Conclusion & future perspective
Galf is an antigenic structural unit in many infectious fungi, which is not biosynthesized by humans. The preparation of synthetic neoglycoconjugates containing β-Galf for the diagnosis of some extended mycoses as aspergillosis is a field worth to explore. The synthetic antigens would help define the structure of the corresponding epitopes and in a more ambitious project they could be the starting line for the synthesis of carbohydrate-based vaccines [102]. In reviewing the natural structures, it is challenging to understand why in some dimorphic fungi the commonly found β-Galf changes the anomeric configuration to α-Galf, in the transition between phases. The α-Galf transferases necessary for the construction of the linkages in some galactomannans have not yet been described. No writing assistance was utilized in the production of this manuscript.

Biomarkers
• Diagnosis of fungal infections and identification of the causative agent is important for the selection of an accurate therapy. The use of biomarkers may provide faster results and complement culture and histological methods.

Carbohydrate-based markers
• Galactofuranose (Galf) is an attractive candidate as a biomarker for diagnosis of infections, since it is the antigenic epitope in glycans of several pathogenic fungi and is not biosynthesized by mammals. An ELISA assay based on the EB-A2 antibody that detects the Galf in the GM of Aspergillus is commercialized. The different linkages of the Galf to other sugars in the fungal carbohydrates may provide specificity, a line of research that needs further studies.

Chemically synthesized oligosaccharides of antigenic glycans
• The impressive advance achieved in the chemical synthesis of the natural oligosaccharides encourages their use as specific antigens. • Describes different anomeric configurations for the D-Galf in the yeast and mycelial phases of Paracoccidioides brasiliensis.