Figure 5 Data

Figure 5. Using timelapse microscopy to gain insight into C. albicans yeast and hyphae interactions with the melanization immune response. (A) Kinetics of the melanization reaction within hemolymph incubated with C. albicans yeast, with a distinct tri-phastic response in which the yeast begin non-encapsulated with melanin (1), followed by an initial peak of the melanization reaction (2), an extended plateau (3), and a greater second peak of the melanization reaction (4). (B) Using microscopy, we saw that the yeast start off non-melanized (B1), and most reached their peak melanin-encapsulation by 120 minutes (B2). After which, the yeast began to filament, and hyphae grew between 120 min and 750 minutes with minimal melanin encapsulation occurring. At 750 minutes (B3), blastoconidium (yeast) began to form on the hyphae, which then corresponded to a rapid increase in melanization (B4). Scale bars in (B) indicate 100 µm. (C) The time until this melanin bloom was approximately 14 hours (n = 15, mean = 874 minutes, 95% Confidence Interval 762 to 985 minutes). (D) Through these movies, we also saw that some melanin-encapsulated yeast were able to survive the immune response and grew from underneath the layer of pigment. The melanin-encapsulated C. albicans in panel (D) began to grow through the melanin by 250 minutes. Scale bars in (D) indicate 10 µm. (E) Overall, approximately 23% of melanin-encapsulated yeast were able to grow following melanin-encapsulation. Pairs of melanin-encapsulated yeast had the highest percentage of growth following melanin encpasulation, with a 33% frequency. (F) In the yeast that grew following melanin-encapsulation, the time until germ tube formation was delayed 230 minutes on average (n = 26), while in non-encapsulated yeast, the average time was 90 minutes (n = 111) (Two-tailed unpaired t-test, p