The C. albicans sur7Δ mutant has an abnormal response to induction of filamentation and hyphal cells are markedly defective in plasma membrane structure An important virulence attribute in
C. albicans is the ability to switch between yeast, pseudohyphal, and filamentous forms [25–27]. When spotted onto M199 agar, hyphal structures were formed from each colony (Fig. 4A). However, the extent of filamentation was reduced in the sur7Δ null mutant compared to DAY185 and the SUR7 complemented strain. Similar results were observed when grown on GSK872 Spider agar medium at 37°C (Fig. 4A). When BSA agar plates were incubated for an extended period of time, filamentous structures emerged from the edge of each colony except in the sur7Δ null mutant (Fig. 4A). This reduced filamentation in response to inducing conditions was also seen on solid media containing selleck kinase inhibitor fetal calf serum (Fig. 4A). In GDC-0941 clinical trial liquid media (YPD supplemented with 10% FCS, high glucose D-MEM with 10% FCS, or RPMI-1640), time of germination and the extent of filament elongation of the C. albicans sur7Δ mutant were grossly similar to the wild-type and SUR7 complemented strains (data not shown). However, when grown in weak hyphal-inducing liquid Spider medium, a population of yeast cells and hyphae with aberrant morphology and branching was observed (Fig. 4B). Figure 4 Filamentation assays on various media.
(A) Overnight cultures were spotted onto weak-inducing media such as M199 agar plates, Spider agar, and BSA plates, and monitored daily. Overnight cultures were also spotted onto YPD containing 10% (v/v) fetal calf serum (FCS), a strong inducer of filamentation. Representative figures at the indicated times and incubation temperatures are shown. (B) Filamentation was also assayed in liquid media. Inoculums of 5 × 106 cells ml-1 were incubated at 37°C with constant shaking at 200 rpm. The time of germination, extent of elongation,
and overall Inositol oxygenase hyphal morphology were observed and compared between each strain at given time points using standard light microscopy. Results from growth in weak-inducing medium (Spider medium) are shown here at 2 and 4 hours where aberrant branching is evident at the latter timepoint. Standard light microscopy was performed using a 60× and 40× objective for the 2 and 4 hour timepoint, respectively. Next, structures of the filamentous form were compared using light microscopy. After 24 hours of growth, the wild-type (DAY185; Table 1) and SUR7 complemented strains produced mature, elongated hyphal cells with clear septa, whereas the sur7Δ null mutant produced irregularly shaped hyphae with obvious intracellular invaginations (Fig. 5A). Thin-section electron microscopy demonstrated subcellular structures in the filaments formed by the sur7Δ null mutant strain (Fig.