10A). Average OD630 nm measurements of the crystal violet extracts, which are https://www.selleckchem.com/Proteasome.html directly related to biofilm mass, were 0.204 ± 0.003, 0.137 ± 0.006, and 0.194 ± 0.003 for the wild-type, sur7Δ null mutant, and SUR7 complemented strains, respectively (p < 0.0001). Examination of the biofilm by scanning electron microscopy demonstrated a sparse biofilm architecture compared to control strain DAY185 (Fig. 10B). Figure 10 Analysis of C. albicans sur7 Δ biofilm formation. (A) Biofilm mass was assayed by staining the biofilm formed with Crystal Violet [45]. Data analyzed consisted of 14 replicates and statistical significance was determined by ANOVA (p-value < 0.0001), indicated on the figure with
an asterisk (*). (B) The structure and morphology of the biofilm formed by the sur7Δ null mutant strain and wild-type strain DAY185 was examined by scanning electron microscopy. Size bars indicate 20 μm. Next, in order to determine if the reduced biofilm mass of the
sur7Δ mutant is related to decreased attachment of the biofilm, we quantified the amount of planktonic cells in the biofilm GSK458 wash of each strain. Compared to the control strains, there were significantly fewer planktonic cells (colony forming units) present in the biofilm formed by the sur7Δ null mutant (p < 0.0001; data not shown). These results are therefore consistent with the previous adhesion studies. Thus, reduced attachment of cells does not account for the lesser biofilm mass of the sur7Δ null mutant. Furthermore, as there is only a minor delay or impairment in filamentation in this growth Astemizole medium, it appears that the defect in biofilm formation is most likely due to a defect in cell wall or plasma membrane structure related to the absence of SUR7. The C. albicans sur7Δ mutant is defective in macrophage killing Lastly, we sought to determine the effect of the loss-of-function of SUR7 on the ability of C. albicans to kill macrophage cells. At early time points (1 and 5 hours co-incubation), the number of live macrophage cells co-incubated with the sur7Δ null mutant was similar
to the numbers found when co-incubated with either DAY185 or the SUR7 complemented strain (>1,000 macrophages per field; data not shown). After 24 hours of co-incubation, significantly more macrophages per field remained when co-incubated with the sur7Δ null mutant (841 ± 87) than either of the control strains (5 ± 2 and 3 ± 1 for wild-type and SUR7 complemented strains, respectively) (Fig. 11A and 11B p < 0.0001). These results indicate that C. albicans SUR7 is required for in vitro macrophage killing. Figure 11 In vitro test of virulence using the macrophage killing assay. Macrophages were seeded onto a glass slide and subsequently co-incubated with C. albicans strains at a multiplicity of infection of 2. (A) Live macrophage cells from four fields per strain tested were counted and the averages were compared using ANOVA.