Takuji OKA Faculty of Biotechnology and Life Science, Sojo University
Research aims
Aspergillus fumigatus is the major causative fungus of invasive pulmonary aspergillosis. The structure of galactomannan (GM) produced by A. fumigatus was described in the 1930s and GM presence has been used clinically as an index for the early diagnosis of invasive aspergillosis. GM consists of α-(1→2)-/α-(1→6)-mannosyl and β-(1→5)-/β-(1→6)-galactofuranosyl residues, and it is located in the outermost layer of the A. fumigatus cell wall 1 ). There are two types of GM in A. fumigatus , namely fungal-type (FTGM) and O -mannose-type (OMGM) 2). FTGM contains a linear α-mannan structure called core mannan in which 9 or 10 α-(1→2)-mannotetraose units are concatenated by α-(1→6) linkages. Moreover, FTGM contains galactofuran side chains consisting of β-(1→5)-galactofuranotetraose units concatenated by β-(1→6) linkages that are bound to core mannan by β-(1→2), β-(1→3), and β-(1→6) linkages. OMGM consists of β-(1→5)-galactofuranosyl chains bound to the nonreducing terminal side of an O -mannose-type glycan, in which mannosyl chains are attached to a serine or threonine hydroxyl group in the proteins. Similar to FTGM, the galactofuranosyl residues of OMGM are elongated by β-(1→6)-galactofuranose 2 ) . Because plants and higher animals, including humans, do not produce GM, inhibition of the GM biosynthetic pathway represents a potential mode of action for medicines and agricultural chemicals without the risk of side effects. However, information on the glycosyltransferase involved in GM biosynthesis is limited. We identified and characterized the galactofuranosyltransferases (GfsAs) responsible for OMGM and FTGM biosynthesis in the model filamentous fungi A. nidulans and A. fumigatus in 2013 ). Therefore, in this research, we will proceed with the detailed enzymatic functional analysis of A. fumigatus GfsA (AfGfsA), AfGfsC, a paralogue of AfGfsA, and AfCmsA, a putative mannosyltransferase. We hope these findings will facilitate the development of novel antifungal agents by elucidating the functions of glycosyltransferases responsible for FTGM synthesis in filamentous fungi.
Methods
We obtained recombinant AfGfsA, AfGfsC, and AfCmsA proteins using an Escherichia coli expression system. The genes encoding the soluble forms of AfGfsA, AfGfsC, and AfCmsA, lacking the N-terminal region, which harbors the putative signal sequence and a transmembrane domain, were introduced into an E. coli expression vector fused in frame with a 6× histidine (6×His) tag at the N-terminus. Recombinant 6×His-tagged proteins were purified via Ni + affinity chromatography. GM was extracted from A. fumigatus mycelia via autoclaving at 121°C for 120 min. The β-elimination reaction was performed by exposing the fractionated GM to generate FTGM. Glycosidic linkage investigation of the polysaccharides and oligosaccharides was performed using methylation analysis. The Nuclear Magnetic Resonance (NMR) spectra were recorded using a JNM-LA600 spectrometer (JEOL).
Results
The galactofuranosyltransferase activity of the purified AfGfsA and AfGfsC proteins was assayed using uridine-5'-diphosphate (UDP)-galactose, UDP-galactose mutase, 10 mM  sodium  dithionite,  1  mM  Mn 2+,  and  1  mM  para-nitrophenyl (pNP)-β-D-galactofuranose. The reacted mixture was analyzed using HPLC with an column. As  a  result, AfGfsA and  AfGfsC could  synthesize amino pNP-β-(1→5)-galactofuranotetraose (unpublished data). GMs were prepared using WT and AgfsAC strains and characterized via 13 C-NMR spectroscopy and methylation analysis. Interestingly, the signal for β-(1→5)-galactofuranose disappeared in the 13C-NMR spectrum for ∆AfgfsAC-GM (unpublished data). In addition, no 5-galactofuranose produced by ΔAfgfsAC-GM was detectable via GC-MS of the O-methyl alditol acetate derivatives from the methylation analysis (unpublished data). These observations indicate that all β-(1→5)-galactofuranosyl residues of FTGM and OMGM in A. fumigatus are synthesized by AfGfsA and AfGfsC 1) . The mannosyltransferase activity of the purified AfCmsA protein was assayed using guanosine 5'-diphosphate-D-mannose, 1.5 mM Mn 2+ , and 1.5 mM pNP-α-D-Man-. The reacted mixture was analyzed using HPLC with an amino column. As a result, AfCmsA could synthesize pNP-α-(1→2)-mannobiose, indicating that AfCmsA is an α-1,2-mannosyltransferase (Fig. 2) 4) . Next, FTGMs were extracted from WT, ΔcmsA, and ΔcmsA::cmsA strains and subjected to acid hydrolysis to remove galactofuranosyl residues from FTGM, and the products (designated as FTGM-HCls) were analyzed via ¹H-NMR spectroscopy. The H-1 signals for the chemical shifts of the α-(1→2)-mannan backbone appeared from 5.0 to 5.2 ppm in the ¹H-NMR spectra of WT-FTGM-HCl. Conversely, these core mannan signals were absent or substantially suppressed in the ¹H-NMR spectra for ∆cmsA-FTGM-HCl, indicating that core mannan structures were altered and/or lost in the absence of cmsA (Fig. 3). Next, these FTGM-HCls were analyzed via gel filtration chromatography and detected using the phenol-sulfuric acid method. The peak FTGM-HCl content in the WT strain was detected in fractions 35 to 50. In the ΔcmsA strain, the molecular weights of FTGM-HCls were shifted downward, whereas the gene-complemented ∆cmsA::cmsA strain displayed restoration of the molecular masses of FTGM-HCls similar to those of the WT strain (Fig. 4). These results indicate that the average molecular weights of FTGM-HCl are reduced in the absence of cmsA. Taken together, these findings identify CmsA as the α-1,2-mannosyltransferase responsible for FTGM core mannan biosynthesis.
Conclusion
All β-(1→5)-galactofuranosyl residues of FTGM and OMGM in A. fumigatus are biosynthesized by AfGfsA and AfGfsC. CmsA is an α-1,2-mannosyltransferase that is responsible for FTGM core mannan biosynthesis in A. fumigatus . These enzymes represent promising targets for novel antifungal therapies.