Effects of the Tobacco Defensin NaD1 Against Susceptible and Resistant Strains of Candida albicans

Abstract

Today, Candida albicans is still the most common cause of both local and life-threatening systemic candidiasis. The spread of resistant fungal strains has resulted in an urgent need to search for new promising antimycotics. Here, we investigated the antifungal action of the tobacco defensin NaD1 against susceptible and resistant to azoles and echinocandins strains of C. albicans. We demonstrated that NaD1 was equally effective and fungicidal against all tested strains. The MIC and MFC values were 6.25 and 12.5 µM, respectively. We showed for the first time that NaD1 could act synergistically not only with caspofungin but also with human host defense antimicrobial peptides cathelicidin LL-37 and β-defensin-2 (HBD2) against susceptible and resistant fungal strains. Using flow cytometry, we demonstrated that NaD1 in combinations with LL-37 or HBD2 can reinforce each other by enhancing membrane disruption. Using the Caco-2 cell monolayer model, we demonstrated that NaD1 impaired the adhesion of C. albicans cells to the human epithelium. Moreover, NaD1 inhibited the formation of fungal biofilms in Sabouraud broth and less markedly in nutrient-rich RPMI-1640 medium, and enhanced the antibiofilm activity of caspofungin. Thus, we hypothesized that NaD1 might affect the development of candidiasis in vivo, including that caused by resistant fungal strains.

Keywords: Candida albicans; adherence; biofilms; candidiasis; caspofungin; clinical isolates; human cathelicidin LL-37; human β-defensin 2 (HBD2); membrane-disrupting activity; plant defensins; resistance; synergism; tobacco NaD1.

Figure 1

Assessment of the ability of clinical isolates of C. albicans to produce secretory proteinases. The diameters of the unstained amido black zones (clear zones) where BSA hydrolysis occurred around the fungal colonies were measured. Error bars represent a standard deviation (±SD) between nine technical replications.

Figure 2

Adhesion of C. albicans cells to human Caco-2 epithelial monolayer. (A) Comparison of the ability of resistant strains ATCC 10231, v47a3, 9.1 and 8.2 to adhere to the Caco-2 monolayer and the influence of the tobacco defensin NaD1 at different concentrations on this process. (B) Inhibition of C. albicans 9.1 adhesion to epithelium by NaD1, caspofungin and their combination at various concentrations. Error bars represent a standard deviation (±SD) between three technical replications. Significance levels are * p ≤ 0.05, ** p < 0.01, *** p < 0.005. The numbers of adhered cells in untreated controls and samples treated by NaD1, caspofungin or their combinations were compared by unpaired two-sample t-test.

Figure 3

Effects of the tobacco defensin NaD1 at various concentrations on the growth of susceptible and resistant strains of C. albicans (400× magnification).

Figure 4

Flow cytometry analysis of viability of C. albicans ATCC 18804 cells after incubation for 2 h with NaD1 and its combinations with caspofungin (Casp), LL-37 or HBD2, measured by PI uptake. Live (A) and heat-killed (B) yeast-like cells were taken as negative and positive controls, respectively. Effects of NaD1 (C,D), caspofungin (E,F), LL-37 (H,I) and HBD2 (L,M) at the MIC (C,E,H,L) and 0.25× the MIC (D,F,I,M) are shown. Effects of combinations of NaD1 with caspofungin, LL-37 or HBD2 (G,J,K,N) on C. albicans viability are also demonstrated. Events on PI vs. count and FSC vs. PI plots are gated from the FSC vs. SSC diagram.

Figure 5

Effects of NaD1 (B,D) and caspofungin (A,C) on formation of biofilms by resistant strains ATCC 10231 (A,B) and 8.2 (C,D) of C. albicans in Sabouraud broth and RPMI-1640. Error bars represent a standard deviation (±SD) between three technical replications. Significance levels are * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The untreated controls and samples treated by NaD1 or caspofungin were compared by unpaired two-sample t-test.

Figure 6

Effects of combinations of NaD1 with caspofungin on biofilm formation by resistant strains ATCC 10231 (A,C), 8.2 (B) and 9.1 (D) of C. albicans in Sabouraud broth (A,B) or RPMI-1640 (C,D). Error bars represent a standard deviation (±SD) between two technical replications. Significance levels are * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The samples treated by caspofungin in different concentrations were compared with combinations of caspofungin in the same concentrations with NaD1 by unpaired two-sample t-test.