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Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

ABSTRACT: A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide ?-D-Galf-(1?5)-[?-D-Galf-(1?5)]3-?-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts. Immunofluorescence studies showed that both 7B8 and 8G4 could stain A. fumigatus cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that Candida albicans, Bifidobacterium longum, Lactobacillus plantarum, and numerous gram-positive and gram-negative bacteria were not labeled by mAbs 7B8 and 8G4. The generated mAbs can be considered promising for the development of a new specific enzyme-linked assay for detection of A. fumigatus, which is highly demanded for medical and environmental controls.

SUBMITTER: Matveev AL

PROVIDER: S-EPMC5843280 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

Matveev Andrey L AL Krylov Vadim B VB Emelyanova Ljudmila A LA Solovev Arsenii S AS Khlusevich Yana A YA Baykov Ivan K IK Fontaine Thierry T Latgé Jean-Paul JP Tikunova Nina V NV Nifantiev Nikolay E NE

PloS one 20180308 3

A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Galf-(1→5)-[β-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor ass ...[more]

PMID: 29518144

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Project description:Fungal-type galactomannan (FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-D-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.

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Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

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Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

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