α mannosidase

Both laccase variants were deglycosylated using 1000 U mg⁻¹ endoglycosidase Hf (New England BioLabs, USA) and 0.1 U mg⁻¹ α-mannosidase (Sigma, USA) in 50 mM sodium citrate buffer pH 5.5 containing 10 mM ZnCl₂ for 3 h at room temperature. The deglycosylation mixture was loaded onto a Superdex 75 gel-filtration column (GE Healthcare, USA) equilibrated with 50 mM sodium citrate buffer pH 5.5 to remove the deglycosylating enzymes. Pure fractions were concentrated and stored at 4°C. The deglycosylated forms of BaL and L499M BaL were only used for crystallization experiments.

Glycans were treated, prior to re-analysis by MALDI-TOF–MS, with α-fucosidase (bovine kidney from Sigma-Aldrich), α-mannosidase (jack bean from Sigma), β-glucuronidases (E. coli from Megazyme or Helix pomatia from Sigma; desalted and concentrated ten-fold with a centrifugal device with a 10 kDa molecular weight cut-off before use) or β-N-acetylhexosaminidases (jack bean from Sigma-Aldrich, Xanthomonas manihotis from New England Biolabs, Streptomyces plicatus chitinase from New England Biolabs or in-house-produced recombinant forms of Caenorhabditis elegans HEX-4 specific for β1,4-GalNAc-linked residues or Apis mellifera FDL specific for the β1,2-linked product of GlcNAc-transferase I^{19 (link)}) in 50 mM ammonium acetate, pH 5, at 37 °C overnight (except for pH 6.5 in the case of HEX-4, or pH 7 in the case of E. coli β-glucuronidase or an incubation time of only 3 h in the case of FDL or <2 h for H. pomatia β-glucuronidase). Hydrofluoric acid was used for removal of core or antennal α1,3-fucose or of phosphorylcholine^{17 (link)}. As appropriate, treated glycans were re-chromatographed by RP-amide HPLC to ascertain retention time shifts prior to MALDI-TOF–MS. See also Supplementary Note 2 for discussion of glycosidase specificities and Supplementary Figure 23 for the HEX-4 and chitinase sensitivity of defined disaccharide conjugates.

Proteinase-K, non-specific esterase, phospholipase, phosphatase, urease, or α-mannosidase, (each at 0.1 IU, Sigma-Aldrich, Saint Louis, MO) was added to 150 μl of LAM-spiked urine, and lactase and caseinase (both at 0.1 IU, Sigma-Aldrich) were added to 150 μl of LAM-spiked milk at the concentrations indicated in the figures and figure legends, hand mixed and further incubated for 15 minutes at room temperature. Enzymatically treated LAM-spiked urine or milk was then directly used to perform LAM-tests following manufacturer’s instructions.

Biofilm quantification and susceptibility assays were conducted on biofilms grown in 96-well polystyrene plates. C. auris (100 μl at 10⁸ cells/ml) was added to each well and incubated statically for 24 h at 37°C. For experiments examining susceptibility, fresh media and the antifungal and/or enzymes (1000 μg/ml) were added after the 24 h of incubation and incubated for an additional 24 h. Reagents included α-mannosidase (0.78 U/ml, jack bean; Sigma), zymolyase (0.63 U/ml; MP Biomedicals), and fluconazole (1,000 μg/ml). Biofilms were quantified using a tetrazolium salt XTT reduction assay. Briefly, media and nonadherent cells were removed, and biofilms were washed with sterile PBS. XTT (80 μl; 0.75 mg/ml), phenazine methosulfate (PMS) (10 μl; 320 μg/ml), and 10 μl of 20% glucose were added, and plates were incubated for 60 min at 37°C in the dark. Absorbance at 492 nm was measured using an automated plate reader. Biofilm reduction was calculated by comparing untreated biofilms with those treated. Assays were performed in triplicate, and differences were assessed by analysis of variance (ANOVA) with pairwise comparisons using the Holm-Sidak method.

Glycans were treated, prior to re-analysis by MALDI-TOF–MS, with α-fucosidase (bovine kidney from Sigma-Aldrich or almond α1,3/4-specific from Prozyme), α-mannosidase (jack bean from Sigma), or β-N-acetylhexosaminidases (jack bean from Sigma-Aldrich, Streptomyces plicatus chitinase from New England Biolabs or in-house-produced recombinant Caenorhabditis elegans HEX-4 specific for β1,4-GalNAc-linked residues) in 50 mM ammonium acetate, pH 5, at 37 °C overnight (except for pH 6.5 in the case of HEX-4). Hydrofluoric acid was used for removal of core or antennal α1,3-fucose, phosphorylcholine or phosphate. As appropriate, treated glycans were re-chromatographed by RP-amide HPLC to ascertain retention time shifts prior to MALDI-TOF-MS.

The Ts4 mAb (mouse IgM) and 6035 (a mAb for TEX101 peptide portion (mouse IgG2a)) were established and purified as previously described [2 (link)]. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG polyclonal antibody (pAb) and control mouse IgM were obtained from DAKO (Glostrup, Denmark). HRP-conjugated goat anti-mouse IgM pAb were purchased from Chemicon (Temecula, CA). Various biotin-labeled lectins, such as Dolichos biflorus agglutinin (DBA), Datura stramonium agglutinin (DSA), Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Pisum sativum agglutinin (PSA), Sophora japonica agglutinin (SJA), Triticum vulgaris (wheat germ) agglutinin (WGA) were purchased from Vector Laboratories (Burlingame, CA). HRP-conjugated streptavidin was obtained from Invitrogen (Carlsbad, CA). Endoglycosidases (N-glycanase, endoglycosidase-F1, endoglycosidase-F2, endoglycosidase-F3, and endoglycosidase-H) and exoglycosidases (α-mannosidase, β-mannosidase, β-N-acetylglucosaminidase (from Canavalia ensiformis), neuraminidase, and α-L-fucosidase) were from Sigma-Aldrich (St. Louis, MO). β-N-acetylglucosaminidase (from Xanthomonas manihotis) was purchased from New England Biolabs (Ipswich, MA). Other chemicals used in this study were obtained commercially and were of the highest purity available.