Fig. 4: Candidalysin induces double-strand DNA breaks and suppresses DNA damage repair by binding directly to CCNH.

a Viability and death rate of FaDu cells in control, 10 μM and 20 μM candidalysin-treated group (4 h) (n = 3 samples), detected via Calcein/PI viability/cytotoxicity assay. Data was shown as mean ± SD. The P value was determined by one-way ANOVA with Tukey’s post-hoc tests. ****P < 0.0001. b Cell-cycle analysis of CHO-K1 cells after 10 μM or 20 μM candidalysin treatment for 4 h. G2/G1 indicates the ratio of DNA fluorescence intensity in G2/M phase cells to DNA fluorescence intensity in G1 phase cells. Example of the illustration: the proportion of G1 phase cells in the Control group was 64.14%, the average fluorescence intensity was 140721, and the CV was 6.86; the ratio of G2 phase cells to G1 phase cells was 1.95. The P value was determined by two-sided Pearson’s Chi-Square test, P < 0.001. c The representative image of comets enriched in FaDu cells. The cells were stimulated by 10 μM of candidalysin for 4 h. Percentage of tail DNA (%) in control and candidalysin-treated group (n = 90 cells). Ctrl, Control; Clys, candidalysin. Data was shown as the median with interquartile range. The P value was determined by two-sided Mann–Whitney U test. P < 0.0001. d The micronucleus (MN) ratio induced by candidalysin in FaDu cells. e The γ-H2AX formation and CCNH expression in FaDu cells. The representative image (from 3 biological repeats) indicates the γ-H2AX formation (red) and CCNH expression (green) in the nuclei. The expression of CCNH and γ-H2AX was evaluated by the immunofluorescence assay. Scale bar, 10 μm, 5 μm, respectively. f The transcriptional expression level of DNA damage repair and nucleotide excision repair-related genes in FaDu cells stimulated by candidalysin. Cells were stimulated by 10 µM of candidalysin for 4 h and 24 h. The mRNA expression levels were measured by qRT-PCR (n = 3 samples). Data was shown as mean ± SD. The P value was determined using two-way ANOVA followed by Šídák’s multiple comparisons test. *P = 0.0112; **P = 0.001; ****, P < 0.0001. g The DNA damage repair efficiency in FaDu cells. The FaDu cells were stimulated with 10 µM of candidalysin for 4 h or 24 h, respectively. The cyclin H (CCNH) and γ-H2AX protein expression levels were measured by western blot (from 3 biological repeats). h Protein expression levels of CCNH and γ-H2AX in A549 stimulated by 10 μM THZ1 or 10 μM candidalysin, and in CCNH-KD A549 stimulated by 10 μM candidalysin for 4 h (from 3 biological repeats). i Co-immunoprecipitation assay demonstrated the interaction between CCNH and candidalysin in HEK-293T cells (from 3 biological repeats). j The bimolecular fluorescence complementation (BiFC) assay by transfecting N-terminal EGFP-tagged Clys and C-terminal EGFP-tagged CCNH into HEK-293T cells. The green fluorescence signal confirmed the interaction between Clys and CCNH proteins within the host cells (from 3 biological repeats). k Upper panel: the binding efficiency of candidalysin to CCNH protein was evaluated by BIAcore. By curve fitting, we measured the binding rate constant K_a = 9.916 × 10⁴/Ms, dissociation rate constant K_d = 0.001706/s, and equilibrium dissociation constant K_D = 1.720 × 10⁻⁸M for candidalysin. Lower panel: comparison of equilibrium dissociation constant (K_D) of CCNH-Clys binding and CCNH-Amp binding. Amp, human cathelicidin (FKRIVQRIKDFLRNLVPRTES). K_D fold change = K_D(CCNH-Clys)/ K_D(CCNH-Amp). l Docking patterns performed by HPepDock. The structure of candidalysin was predicted by AlphaFold and was colored as orange. m The kinase activity of CAK complex induced by candidalysin in vitro. n The protein expression levels of CCNH and γ-H2AX and phosphorylation level of CDK1/2 (substrate of CAK complex) were measured by western blot after co-culturing FaDu cells with the C. albicans WT, ece1Δ/Δ, ece1Δ/Δ+ECE1 and ece1Δ/Δ+ECE1^Δ184-279 (from 3 biological repeats). WT, wild type; ece1Δ/Δ, null mutant; ece1Δ/Δ+ECE1, ECE1 re-integrant; ece1Δ/Δ+ECE1^Δ184-279, candidalysin re-integrant.