Figure 5. Capsular enlargement in response to different extracts.

A) Activity of upper phase amoebae extracts (AE) on C. neoformans strain H99 after extract treatment with heat, Proteinase K (Ptn K), and glucanases. Neither heat nor glucanase treatments had a significant effect on capsule enlargement activity. In contrast, Proteinase K + heat treatment elicited cells with larger capsule volumes. This experiment was done two times on separate days and the results were reproducible. The numbers shown represent the average of a minimum of 50 C. neoformans cells. B) Thin liquid chromatography shows an increase in the mobility of the phospholipid bands upon treatment of amoebae extracts with Proteinase K, and fractionation of the free lipid compounds to the lower phase fraction. Lane 1: Phospholipid markers: (PE) phosphatidylethanolamine; (PS) phosphatidylserine; (PI) phosphatidylinositol; and (PC) phosphatidylcholine; Lane 2: Untreated extracts; Lane 3: Untreated extract + Proteinase K (before treatment); Lane 4: Proteinase K-treated extract; Lane 5: Lower phase fraction after treatment with Proteinase K; Lane 6: Upper phase fraction after treatment with Proteinase K. C) and D) Demonstration that the active substance in the amoeba extract (AE) shifts from the upper phase to the lower phase after digestion with Proteinase K (Ptn K). Panels C and D show results from overnight and 48 h, respectively. Chloroform extraction of the upper phase after Proteinase K digestion (Cn + AE upper phase) enhances the activity of the upper phase to promote cell body size increase, while the capsule inducing substance transfers to the lower phase (Cn + AE lower phase). (*) indicates P<0.05 for capsule enlargement for each condition when compared to control incubation in PBS. In Panel C, the (**) indicates P<0.05 for a decrease in capsule relative to incubation in PBS, while in Panel D (**), indicates P<0.05 for a decrease in cell body volume relative to incubation in PBS.