Fig 2.

Replication of C. glabrata inside BMDMs following phagocytosis. (A) Microscopy images of three representative examples acquired following 5 h and 24 h of coincubation of C. glabrata DSY565 with BMDMs. Log-phase cultures of RFP-labeled yeasts (VSY58) were stained with FITC, as described in Materials and Methods, and added to preconfluent BMDM monolayers (established on top of round cover slides in 24-well plates) at an MOI of 1. Cocultures were incubated in IMDM at 37°C in 5% CO₂ and mounted onto microscopy slides at selected time points for visualization. Since the FITC stain does not propagate to daughter cells during budding, the absence of FITC staining denotes yeast cells resulting from replication inside BMDMs. Bar, 10 μm. DIC, differential interference contrast. (B) Two-hour and 24-h inoculum CFU count ratios. Log-phase C. glabrata (VSY59 and VSY60) cultures containing either a single strain (single inoculum) or a 1:1 mix of two strains (mixed inoculum) were added to preconfluent BMDM monolayers at an MOI of 1. After 2 h of coincubation, the cultures were washed to remove noninternalized yeast cells. Immediately following the washes, as well as after 24 h of coincubation, BMDMs were lysed and dilutions of the resulting yeast suspensions were plated onto solid medium. (C) Count ratios (24-h CFU/2-h CFU) of the data displayed in panel B. Results on panels B and C are means + standard deviations (SD) of a minimum of three independent experiments. P values of <0.05 were considered significant, and differences between strains DSY562 and DSY565 are not statistically significant (P > 0.05, unpaired Student's t test).