FIG 4.

Coinfection enhances C. albicans pathogenicity. Tg(Mpx:EGFP) zebrafish larvae infected and screened as previously described and 3 to 4 random fish per group were subsequently homogenized for quantifying CFU of C. albicans by using selective media, with the remaining fish monitored out to 48 hpi. Random fish were imaged at 24 and 48 hpi via confocal microscopy, and another 3 to 4 fish per group at 48 hpi were homogenized to quantify CFU as described above. Data are representative of 6 to 8 pooled, independent experiments. (A) A total of 22 individual fish were homogenized per time point for CFU quantification and plotted on a log₁₀ scale. There was no significant difference in C. albicans CFU between single infection and coinfection according to Student's t test. (B) Median percent coverage of the swimbladder by C. albicans was quantified via ImageJ analysis of confocal images acquired of representative fish at 24 hpi, with n = 55 and 63 for C. albicans and C. albicans plus P. aeruginosa infections, respectively. An unpaired Mann-Whitney test revealed a significant difference between C. albicans single infection and coinfection, as indicated. (C) Confocal images acquired at 24 hpi were stratified based on the survival of individual fish at 48 hpi relative to the median percent swimbladder coverage by C. albicans, with n = 18 dead and n = 44 alive. Scale bar, 100 μm. According to an unpaired Mann-Whitney test, fish that died by 48 hpi had significantly higher swimbladder coverage by C. albicans than those that survived, as indicated. (D) Cumulative frequency distribution plots representing percent swimbladder coverage by C. albicans at 24 hpi. Fish that died at 48 hpi are indicated by red points; for C. albicans, n = 26; for C. albicans plus P. aeruginosa, n = 36. (E) Percentage of fish with epithelial invasion at 24 hpi; for C. albicans, n = 52; for C. albicans plus P. aeruginosa, n = 63. According to a Fisher's exact test, there was a significant difference between C. albicans and C. albicans plus P. aeruginosa infection, as indicated. (F and G) Confocal images acquired at 24 hpi were quantified by ImageJ and stratified based on the survival of individual fish at 48 hpi relative to epithelial invasion by C. albicans, with n = 15 with invasion and n = 45 without invasion. According to a Fisher's exact test, fish that died by 48 hpi had significantly higher incidences of C. albicans invasion than those that survived, as indicated. Statistical significance was assigned based on GraphPad Prism conventions (n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). The swimbladder is outlined in a dotted magenta line for clarity.