FIG 8.
Laccase expression of C. gattii suppresses phagocytosis and killing by neutrophils in vitro. Freshly isolated murine neutrophils were cultured with live C. gattii R265, LAC1 deletion (lac1Δ), and LAC1 complementary (lac1Δ::LAC1 [::LAC1]) strains. For neutrophil phagocytosis, cryptococci were opsonized with fresh mouse serum and labeled with calcofluor white (CFW; 1:100), followed by incubation with neutrophils for 3 h. The percent phagocytosis was evaluated by flow cytometry. (A) Representative dot plot showing strategy for characterization of neutrophils associated with Cryptococcus (CD11b+ Gr.1hi CFW+ neutrophils). (B) Graph showing percent phagocytosis of Cryptococcus by neutrophils. For neutrophil killing, cryptococci were opsonized with fresh mouse serum and incubated with neutrophils for 4 h. After neutrophil lysis, the suspension was plated on an SDA plate to measure the number of viable fungi (CFU/ml) and the percent killing was calculated by comparison with a Cryptococcus culture without neutrophils. (C) Graph showing percent killing of Cryptococcus by neutrophils. Graphs depict means ± SD and are representative of results of three independent experiments. Significance was determined using one-way ANOVA with Tukey’s post hoc analysis; **, P < 0.01; ***, P < 0.0001.