Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition
(A) The mar1Δ strain has a capsule defect. Cells were incubated in capsule-inducing conditions (CO2-independent tissue culture (TC) medium, 37°C) with shaking for 72 hours. Capsule was assessed by India ink counterstaining, followed by imaging. (B) The mar1Δ strain sheds capsule comparable to WT. Shed capsule polysaccharide was measured by blotting of culture supernatant, using an anti-GXM antibody to probe for capsule as described previously . (C) The mar1Δ mutant displays increased staining for exposed chitin and chitooligomers in tissue culture medium. Cells were incubated for 16–18 hours at 30°C in rich medium (YPD) or 37°C in tissue culture medium (TC), followed by staining with FITC-conjugated wheat germ agglutinin (WGA) for exposed chitin/chitooligomers, or calcofluor white (CFW) for total chitin. Stained cells were imaged by fluorescent microscopy with the appropriate filters. (D) Average fluorescence of at least 100 individual cells was measured using ImageJ/Fiji software. ****, p < 0.0001 as determined by two-way ANOVA with Tukey’s multiple comparisons test. (E) The mar1Δ mutant displays increased chitosan staining. Cells were incubated for 16–18 hours at 30°C (YPD) or 37°C (TC), followed by staining with eosin Y (EY) for chitosan. Stained cells were imaged by fluorescent microscopy. (F) The mar1Δ cell wall does not have increased total chitin or chitosan. Cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. Chitin and chitosan levels were quantified using a modified 3-methyl-2-benzothiazolinone hydrazine hydrochloride (MBTH) colorimetric assay as described previously . Data represent means of 3 independent cell wall isolations (n = 3 for each strain). Ns, not significant as determined by two-way ANOVA with Sidak’s multiple comparisons test.