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The regulatory subunits of CK2 complex mediate DNA damage response and virulence in Candida Glabrata

BMC Microbiology volume 23, Article number: 317 (2023) Cite this article

Abstract

Background

Candida glabrata which belongs to normal microbiota, has caused significant concern worldwide due to its high prevalence and drug resistance in recent years. C. glabrata has developed many strategies to evade the clearance of the host immune system, thereby causing persistent infection. Although coping with the induced DNA damage is widely acknowledged to be important, the underlying mechanisms remain unclear.

Results

The present study provides hitherto undocumented evidence of the importance of the regulatory subunits of CgCK2 (CgCkb1 and CgCkb2) in response to DNA damage. Deletion of CgCKB1 or CgCKB2 enhanced cellular apoptosis and DNA breaks and led to cell cycle delay. In addition, deficiencies in survival upon phagocytosis were observed in Δckb1 and Δckb2 strains. Consistently, disruption of CgCKB1 and CgCKB2 attenuated the virulence of C. glabrata in mouse models of invasive candidiasis. Furthermore, global transcriptional profiling analysis revealed that CgCkb1 and CgCkb2 participate in cell cycle resumption and genomic stability.

Conclusions

Overall, our findings suggest that the response to DNA damage stress is crucial for C. glabrata to survive in macrophages, leading to full virulence in vivo. The significance of this work lies in providing a better understanding of pathogenicity in C. glabrata-related candidiasis and expanding ideas for clinical therapies.

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Background

The incidence of candidiasis infections has increased progressively in recent decades. As the second most frequent pathogenic yeast associated with candidiasis, Candida glabrata has several unique biological features, including absence of hyphae, antifungal resistance, and virulence factors. Unlike Candida albicans, C. glabrata is a haploid budding yeast similar to the non-pathogenic yeast Saccharomyces cerevisiae evolutionarily [1]. C. glabrata now accounts for one-third or more of all candidemia isolates in USA and a trend of increasing C. glabrata rates is seen in Australia and in some European and Asia countries as well, which even causes a mortality rate of 30% [2]. Moreover, reduced susceptibility to azoles and acquired cross-resistance during clinical treatment have been observed in C. glabrata. C. glabrata has caused significant health concerns worldwide based on increasing evidence of its high prevalence and associated drug resistance [3, 4].

As part of the normal flora, C. glabrata often causes superficial skin and mucosal infection and even leads to life-threatening invasive infections, especially in immunocompromised patients. C. glabrata is an opportunistic pathogen with secretory hydrolytic and proteolytic enzymes and adhesion and biofilm formation abilities [5, 6]. In contrast with C. albicans, C. glabrata lacks many virulence factors but is highly evolved for interaction with the host. C. glabrata is a pathogen that induces a moderate immune response to prolong the disease course [7, 8]. Both innate immune and adaptive immune responses contribute to the host defenses of C. glabrata. Encountering phagocytic cells and further survival within them is crucial for keeping C. glabrata shielded from immune attack [9]. After being phagocytized, C. glabrata is confronted with several challenges. DNA damage under a series of phagocytic stress factors (such as reactive oxygen species and cytokines) has been documented within 2 h of phagocytosis. C. glabrata induces chromatin remodeling to manage the stress of DNA damage and even replicates inside macrophages after adapting to the hostile environment to cause stubborn infections [10]. Responses to DNA damage including cell cycle checkpoints and DNA repair pathways are crucial for maintaining genetic integrity and are highly diverse in fungi [11].

CK2 is a conserved ubiquitous serine/threonine kinase protein complex and is a heterotetramer composed of two catalytic subunits (Cka1 and Cka2, α and α’ subunit) which bind to the dimer of regulatory subunits (Ckb1 and Ckb2, β subunits) [12]. CK2 participates in various activities such as cell cycle regulation, cell stress response, and apoptosis [13]. In addition to the holoenzyme, the functions of individual subunits have been confirmed [14]. It has been reported that CK2 was recognized as a key director in DNA repair process of both single-strand break (SSB) and double-strand break (DSB) [15, 16]. Moreover, DNA damage repair pathways could be blocked by the inhibitors of CK2 [17]. Many recent studies have found that ScCK2 can directly phosphorylate downstream proteins and is involved in histone phosphorylation [18, 19]. The β subunit of ScCK2 in S. cerevisiae has a zinc finger structure, which may be related to the structural stability of the CK2 complex [20, 21]; the C-terminal of β subunits plays an important role in the interaction with catalytic subunits and other kinases, and may also be related to the recruitment of downstream target proteins [13]. The deletion of ScCKB1 and ScCKB2 results in the cells’ inability to recover or adapt to DSB [12]. In C. albicans, CaCka1 and CaCka2 have been observed to govern virulence and azole drug resistance, but the significance of the regulatory subunits of CK2 remains unknown [22]. Overall, the C. glabrata CK2 complex has been largely understudied over the past few years, warranting further research.

In the present study, the phenotypes of CgCKB1 and CgCKB2 null mutants in relation to stress survival and virulence were examined. In addition, global transcriptome profiling was performed by RNA-seq to investigate the molecular regulations in CgCkb1 and CgCkb2. We show for the first time that in C. glabrata, the β subunits of CK2 are crucial in DNA damage response possibly by maintaining a normal cell cycle. Moreover, our data suggest a link between genome integrity and survival upon phagocytosis, which is important for full virulence of C. glabrata in vivo.

Results

C. glabrata Ckb1 and Ckb2 are phylogenetically closely related to S. cerevisiae Ckb1 and Ckb2

Although being the most common pathogens of candidiasis, C. albicans and C. glabrata are from different evolutionary clades. C. albicans belongs to the CTG clade, which includes pathogens like C. parapsilosis, C. dubliniensis and C. auris. However, C. glabrata belongs to the Nakaseomyces clade, which has a higher interspecific affinity for S. cerevisiae than C. albicans. Therefore, we used the Saccharomyces cerevisiae Ckb1/Ckb2 protein sequences to retrieve the Ckb1/Ckb2 protein sequences in BLASTP for various fungi, including C. glabrata. Phylogenetic trees of these protein sequences were constructed to show the sequence homology of the Ckb1/Ckb2 proteins across species (Fig. 1). For Ckb1 (CAGL0A00275g) and Ckb2 (CAGL0I00946g) of C. glabrata, the S. cerevisiae Ckb1 and Ckb2 were identified as closest orthologs respectively. The amino acid sequence of the Ckb1/Ckb2 protein from C. glabrata was highly similar to that of S. cerevisiae (Ckb1 76.3%, Ckb2 79.2%). The close relationship between C. glabrata Ckb1/ Ckb2 and ScCkb1/ScCkb2 suggests that the β subunit of CK2 of C. glabrata may have similar phenotypes as in S. cerevisiae.

Fig. 1
figure 1

Phylogenetic trees of fungal Ckb1and Ckb2. (A) and (B) A phylogenetic tree constructed based on fungal Ckb1 or Ckb2 protein sequences to show the sequence homology of Ckb1 or Ckb2 proteins across species. The human Ckb1 or Ckb2 protein sequence was used as the root, and the maximum likelihood method was used for calculation and comparison by MEGA (V11.0.13). (C) Overview of amino acid identity between the C. glabrata and S. cerevisiae Ckb1 and CKb2.

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CgCKB1 and CgCKB2 genes are essential in response to DNA damage

DNA damage reagents MMS (Methyl Methane Sulfonate) or 4-NQO (4-Nitroquinoline-1-oxide) were supplemented to YPD plates to examine the DNA repair ability of WT, Δckb1, and Δckb2 strains. The CgCKB1 or CgCKB2 null mutants exhibited increased susceptibilities to DNA damage reagents than WT strains, and these differences became more obvious as the concentration of reagents increased (Fig. 2A). In contrast, no differences were observed in WT strains lacking CgCKA1 or CgCKA2 genes encoding catalytic subunits of the CK2 complex (Fig. 2A).

In addition, growth curves of WT, Δckb1, and Δckb2 strains cultivated with different media revealed similar results with spot assays (Fig. 2B). Under the treatment of 0.01% MMS or 4 µM 4-NQO, the Δckb1 and Δckb2 strains grew slowly and reached significantly lower OD600 (Optical density) values in the plateau phase than WT. In contrast, Δcka1 and Δcka2 exhibited similar growth patterns with WT. Consistent results were observed in RPMI 1640 medium (Figure S1). These results suggest that the regulatory subunits of the CK2 complex might participate in the response to DNA damage.

Fig. 2
figure 2

CgCKB1 and CgCKB2 genes are essential in response to DNA damage. (A) Strains were diluted serially in 10-fold to being grown on YPD containing different concentrations of DNA damage reagents (MMS, methyl methane sulfonate and 4-NQO, 4-Nitroquinoline-1-oxide) at 30℃ for 2 days. WT, wildtype. (B) Growth curves of each strain after treatment with different concentrations of DNA damage reagents. The OD600 values were obtained by BioTek plate reader every 15 min at 30 °C for 48 h. Data were representative of three independent experiments. All the growth curves were repeated at least three times.

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Deletion of CgCKB1 or CgCKB2 increases cellular apoptosis and DNA breaks

Given that DNA damage induces certain changes in genome integrity, we detected the apoptosis and DNA fragmentation of CgCKB1 and CgCKB2 null mutants in the presence of DNA damage reagents. Annexin V-FITC was used to investigate the phosphatidylserine (PS) molecules exposed outside the cell membrane to identify apoptotic cells. PI (Propidium Iodide) was used to detect necrotic cells. After treatment with 0.01% MMS and 4µM 4-NQO for 2 h, the apoptosis and necrotic cells were detected by flow cytometry in each group. Larger amounts of apoptotic and necrotic cells were observed in CgCKB1 and CgCKB2 null mutants than in WT when cultured in the RPMI 1640 medium. Consistent results were observed in the presence of DNA damage stress (Fig. 3A and B). This finding suggests that CgCKB1 and CgCKB2 null mutants are more prone to apoptosis in response to DNA-damaging agents.

Furthermore, a TUNEL assay was conducted to detect DNA fragmentation by fluorescence microscope and flow cytometry. CgCKB1 or CgCKB2 null mutant cells displayed a higher abundance of TUNEL-positive nuclei than WT cells upon 0.01% MMS and 4µM 4-NQO treatment (Fig. 3C). Consistently, the proportions of TUNEL-positive cells significantly increased both in Δckb1 and Δckb2 strains when treated with DNA damage reagents (Fig. 3D). Additionally, the degree of chromosome agglutination by fluorescence intensity was observed by DAPI (Diamidino-phenylindole). In the presence of DNA-damaging reagents, blue fluorescence became brighter in CgCKB1 or CgCKB2 mutant cells than in WT cells, which indicated more agglutination of chromosomes (Fig. 3C).

Fig. 3
figure 3

Deletion of CgCKB1 or CgCKB2 increases cellular apoptosis and DNA breaks. (A) Cells were treated with 0.01% MMS or 4 µM 4-NQO or left untreated for 2 h and stained with Annexin V-FITC and PI. The Fluorescence intensity was detected by BD Fortessa Flow cytometer. Q1 (Annexin V-/PI+): dead cells; Q2 (Annexin V+/PI+): necrotic cells; Q3 (Annexin V+/PI-): apoptotic cells; Q4 (Annexin V-/PI-): live cells. (B) The percentages of each strain that are apoptotic (black bars) and death (gray bars) were obtained. The relative cell ratio was compared with WT in RPMI 1640 medium (set at 1.0) (C) Representative fluorescence micrographs showing DNA fragmentation and unusual chromosome agglutination. Cells were incubated in RPMI 1640, 0.01% MMS or 4 µM 4-NQO for 12 h at 30 ℃ and then stained with DAPI and FITC. The DNA fragmentation was visualized with FITC (green fluorescence) and unusual chromosome agglutination was visualized with DAPI (blue fluorescence). Data were representative of three independent experiments. (D) The TUNEL-positive cells were quantified. DNase I treatment was used as the positive control. The percentages of TUNEL-positive cells were presented as mean ± SD and assessed for statistical analysis by one-way ANOVA followed by Tukey test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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Cell cycle delay occurs in CgCKB1 and CgCKB2 null mutants

Since deletion of CgCKB1 or CgCKB2 increases the sensitivity to DNA damage agents, apoptosis and DNA fragmentation in C. glabrata, we wondered whether the cell cycle of CgCKB1 and CgCKB2 null mutants was altered. A carbon starvation strategy was used to synchronize C. glabrata cells at the G1 phase. After being released in the 2% glucose YPD medium with or without 0.01% MMS or 4µM 4-NQO, we used flow cytometry to analyze the distribution of cell cycles once an hour. In the YPD medium, similar cell cycle patterns were observed in WT, Δckb1 and Δckb2 strains. DNA replicated from 1 to 2 C gradually within 3 h. S phase cells slightly accumulated in Δckb1 and Δckb2 at 4 h. In the MMS or 4-NQO-containing YPD medium, both WT and null mutants slowed down DNA replication obviously. In contrast with the YPD culture, a larger proportion of the population remained at the S phase at 2 h under DNA damage stress in both null mutant cells. After 3 h, parts of WT cells completed DNA replication gradually; however, Δckb1 and Δckb2 cells remained at the S phase and failed to replicate DNA within 4 h (Fig. 4 and S2). Taken together, these data demonstrated that regulation of the cell cycle is disrupted in Δckb1 and Δckb2, and CgCkb1 and CgCkb2 are crucial in maintaining a normal cell cycle upon DNA damage stress.

Fig. 4
figure 4

The cell cycle is delayed in CgCKB1 and CgCKB2 null mutants. Yeast cells were synchronized in YP (carbon starvation condition) and released to YPD either in the absence or presence of 0.01% MMS or 4 µM 4-NQO and collected once an hour. Flow cytometry was used to measure DNA content.

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CgCKB1 and CgCKB2 null mutants have defects in replication in macrophages

Dysregulation of CgCKB1 or CgCKB2 genes leads to defects in response to DNA damage and cell cycle delay in C. glabrata. We assessed cell survival upon phagocytosis to further explore whether CgCKB1 and CgCKB2 play essential roles in interacting with the host immune system. PMA (Phorbol 12-myristate 13-acetate) -induced human monocytic cell line THP-1 and mouse leukemia macrophage cell line RAW 264.7 were used in this study. Similar numbers of WT, Δckb1 and Δckb2 cells were engulfed after co-incubated with macrophages for 2 h. Nevertheless, WT cells yielded 4.90 -fold and 6.66-fold higher replication rates after cocultured with THP-1 and RAW 264.7 cells for 24 h, respectively (Fig. 5A). In contrast, CgCKB1 or CgCKB2 null mutants exhibited only a 1.53- or 1.60-fold increase in replication in THP-1 cells (p < 0.0001) and 1.34-fold or 1.25-fold (p < 0.0001) increase in RAW264.7 cells (Fig. 5B). These results substantiated that Δckb1 and Δckb2 strains induced dysregulated macrophage replication, and CgCKB1 and CgCKB2 are required for intracellular survival in macrophage.

In addition, we evaluated the adhesion ability to epithelial cells Caco-2 and biofilm formation in WT, Δckb1, and Δckb2 strains. The adhesion rates of Δckb1 and Δckb2 strains were slightly reduced, although the difference was not statistically significant (p > 0.05) (Fig. 5C). Moreover, Δckb1 and Δckb2 strains displayed similar biofilm formation abilities to WT (Fig. 5D).

Fig. 5
figure 5

Effect of CgCKB1 and CgCKB2 on interaction with host cells. (A) After co-incubation of fungi with RAW264.7 macrophages or PMA-treated THP-1 cells for 2 h, the cells were lysed with 0.05% Triton X-100 to release the fungi, and the phagocytosis rate was defined as the number of fungi phagocytosed by macrophages after 2 h to the number of fungi before co-incubation. (B) After cocultured with macrophages for 24 h, survival upon phagocytosis was measured as described above. The ratio of the intracellular yeast cells after 24 h of coculture to that after 2 h coculture was defined as fold replication. (C) Epithelial Caco-2 cells were cocultured with CgCKB1 or CgCKB2 null mutants, respectively. Δepa1 which exhibited deficient adhesion ability was used as a control. (D) Log-phased yeast cells were seeded in a 24-well plate for biofilm formation assay. Crystal violet [0.4% (w/v)] was used for staining mature biofilms. The destaining solution was measured for absorbance at 595 nm. Wells without yeast cells were used as background. CBS138 was the used as the WT and the knockout strains were generated using CBS138 as the parent. Experiments were performed three times. Error bars show standard deviation. Comparisons were performed using one-way ANOVA followed by Tukey test, and asterisks indicate statistically significant differences (*** p < 0.001, **** p < 0.0001).

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CgCKB1 and CgCKB2 are important in the virulence of systemic candidiasis mouse model

We used a systemic candidiasis mouse model through tail vein injection to investigate the importance of CgCKB1 and CgCKB2 for virulence. In the survival assay, immunocompromised ICR mice were injected with equal doses of C. glabrata and observed for up to 14 days. Mice infected with WT strains exhibited 50% mortality within 3 days, and only 25% percent of mice survived at the end of the experiment on day 14 (Fig. 6A). In contrast, CgCKB1 and CgCKB2 null mutants exhibited significant differences in survival curves with the wild-type strain (p < 0.05). Infection with CgCKB1 and CgCKB2 knock-out strains only caused 25% and 41.7% mortalities, respectively, and most mice recovered from infection (Fig. 6A).

Fungal burden was also measured 3 days post-infection in immunocompromised ICR mice. The mean number of wildtype yeast cells colonies (1.9 × 105 CFUs/g) in the spleen (Fig. 6B) was significantly higher than in CgCKB1 null mutants (5.6 × 103 CFUs/g, p < 0.001) and CgCKB2 null mutants (3.4 × 103 CFUs/g, p < 0.001). Moreover, the fungal burden in the liver (Fig. 6C) was significantly higher in wildtype (9.9 × 104 CFUs/g) than in Δckb1 (1.1 × 104 CFUs/g, p < 0.0001) and Δckb2 (5.5 × 103 CFUs/g, p < 0.0001). In addition, fungal burden in the kidney (Fig. 6D) was significantly higher in wildtype (3.1 × 104 CFUs/g) than in Δckb1 (2.3 × 103 CFUs/g, p < 0.01) and Δckb2 (1.6 × 103 CFUs/g, p < 0.01).

Moreover, histological analysis confirmed these findings (Fig. 6E). Yeast colonies were observed in PAS (Periodic Acid-Schiff) stained kidney sections of WT-infected mice but were barely found in Δckb1 and Δckb2-infected mice. In summary, the fungal burden is significantly decreased in Δckb1 and Δckb2 strains and CgCKB1 and CgCKB2 are required for organ colonization in the systemic candidiasis mouse model.

Fig. 6
figure 6

CgCKB1 and CgCKB2 are essential to virulence in vivo. (A) 200 mg/kg cyclophosphamide was intraperitoneally injected per ICR mouse (5–6 weeks old, 24–26 g) on day − 3 and every fourth day later. Then mice were infected with 1 × 108 WT, Δckb1 or Δckb2 cells in 200 µL in 0.9% (w/v) saline (n = 12). Mice in the agonal stage were humanely euthanized by cervical dislocation. Experiments were terminated on day 14. Analysis by the Gehan-Breslow-Wilcoxon test indicated that in contrast to WT, the virulence of CgCKB1 and CgCKB2 null mutants was significantly attenuated (p < 0.05). (B)(C)(D) Fungal burden assays were performed using ICR mice (n = 6). ICR mice were immunosuppressed with 200 mg/kg cyclophosphamide on day − 3 and were further injected with 5 × 107 yeast cells through the tail veil on day 0. Organs were harvested, weighed and mechanically homogenized 3 days post-infection. Tissue homogenates were diluted, plated onto YPD agar, and incubated at 30℃ for 2 days. CFUs were calculated and analyzed. Error bars show standard deviation. Comparisons were performed using one-way ANOVA followed by Tukey test, and asterisks indicate statistically significant differences (**: p < 0.01; ***: p < 0.001, ****: p < 0.0001). CBS138 was the used as the WT and the knockout strains were generated using CBS138 as the parent; (E) Representative HE (Hematoxylin-Eosin) and PAS (Periodic Acid-Schiff) stained sections of kidneys from immunosuppressed ICR mice on day 3 post-infection. Magnification was ×20. PAS-positive yeast cells were indicated by red arrowhead. Data are representative of at least three replicates.

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Global transcriptional profiling analysis of Δckb1 and Δckb2 strains

To obtain a better understanding of molecular mechanisms underlying the DNA damage response in Δckb1 and Δckb2 mutants, transcriptome analyses were performed between WT, Δckb1, and Δckb2 strains. Strains treated with 0.01% MMS for 1 h or untreated in YPD were analyzed (Fig. 7A). The transcriptome data revealed that Δckb1 and Δckb2 strains showed similar transcriptional profiles for YPD and YPD supplemented with 0.01% MMS (Fig. 7B C). The data also indicated that compared with WT, about 200 genes were up or downregulated in Δckb1 and Δckb2 strains with or without treatment with 0.01% MMS (Fig. 7D and E; Supplementary Table S2 and S3). This finding suggests a certain degree of overlap in the functions of ckb1 and ckb2 in C. glabrata.

To functionally characterize the transcriptional phenotypes, the differential genes found both in Δckb1 and Δckb2 (compared with WT under the same treatments) were analyzed by GO (Gene ontology) enrichment to identify key molecular processes. GO analysis highlighted that the differentially expressed genes in Δckb1 and Δckb2 were marked enriched in the processes related to the programmed formation of DNA double-strand breaks (DSBs), recombination, and DNA repair coordination of the meiotic cell cycle. In contrast to WT, the processes mentioned above were altered in Δckb1 and Δckb2 strains (Fig. 7F; Supplementary Table S4 and S5). It should be noted that after the deletion of CgCKB1 and CgCKB2, the expression of many genes related to the meiotic cell cycle was upregulated, and these differences were magnified in the presence of 0.01% MMS (Fig. 7G). These genes included early meiotic genes SPO11, meiotic recombination-related gene MSH4, reciprocal meiotic recombination-related gene REC104 and REC114, which have been associated with the repair of DSB of meiosis both in S. cerevisiae and C. albicans [23,24,25,26]. These findings suggested that CgCKB1 and CgCKB2 may maintain genome integrity, affecting the DNA damage response through certain pathways, such as cell cycle regulation.

Fig. 7
figure 7

Transcriptional analysis in WT, Δckb1, and Δckb2 strains. Log-phase yeast cells were inoculated into fresh YPD medium either with 0.01% MMS added or left untreated, total RNA was extracted and further sequenced. (A) Volcano plot of RNA seq transcriptome data displaying the gene expression pattern. The values of gene expression in each group were compared with those of WT strain under the same conditions. Significantly differentially expressed genes (FDR, p ≤ 0.05) are highlighted in red (upregulated) or blue (downregulated). (B) Heatmap plot of DEGs (Differential expression genes) displaying the pattern of gene expression. The gene expression value is normalized and converted to Z score. (C) Scatterplot where each gene is represented by a dot and its “no MMS/MMS” log2 ratio for Δckb1 is plotted on the x axis and the ratio for Δckb2 is plotted on the y axis. (D and E) Venn plot comparing the upregulated DEGs (URGs) and downregulated DEGs (DRGs) of Δckb1 and Δckb2 under all culture conditions. The differential genes of each group were compared with WT strain under the same conditions. (F) Gene ontology (GO) terms in the biological processes describing the upregulated and downregulated genes in each condition. The differential genes both in Δckb1 and Δckb2 (compared with WT under the same conditions) were clustered to GO analysis. (G) Heatmap of RNA-seq data for meiotic genes in Δckb1 and Δckb2 strains compared with WT under all culture conditions, including programmed formation of DNA double‐strand breaks (DSBs), recombination, and DNA repair coordination of the meiotic cell cycle.

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The role of CgCkb1 and CgCkb2 in C. glabrata intracellular survival

As the first defense line of the host against microbial invasion, macrophages recognize and engulf C. glabrata and then produce ROS (reactive oxygen species) or other active substances to induce DNA damage of C. glabrata. The role of CgCkb1 and CgCkb2 in intracellular survival of C. glabrata in macrophages and their involvement in DNA damage stress, led us to compare the genes regulated by CgCkb1 and CgCkb2 under control conditions or MMS treated with the activated genes of C. glabrata upon phagocytosis [10].

A total of 55 genes overlapping were found, including MSH4, IME1, MAM1, MEK1, RED1, which involved in DNA DSB formation, DNA recombination and DNA repair processes (Fig. 8A; Supplementary Table S6 and S7). Based on the report of Rai.et al [10], 38 of 55 genes were down-regulated and 17 were up-regulated in macrophage-engulfed C. glabrata compared with control conditions. Interestingly, we noticed that a major part of these genes (34/38 and 9/17) exhibited the opposite expression patterns in ΔCkb1 and ΔCkb2. These observations indicated the importance of CgCkb1 and CgCkb2 for C. glabrata in maintaining survival in macrophages. What is more, GO analysis indicated that these genes are involved in biological processes such as meiotic cell cycle, homologous recombination, and reactive oxygen species metabolism, which is consistent with our previous analysis (Fig. 8B; Supplementary Table S8). Therefore, these results suggesting that the response to DNA damage stress and the maintenance of genomic stability are crucial for intracellular survival of C. glabrata.

Fig. 8
figure 8

Transcriptome data reveal the link between Ckb1/2 and C. glabrata survival in macrophages. (A) Comparison of differential genes (compared with WT) regulated by CgCKB1 and CgCKB2 under control conditions and MMS conditions with the activated genes (compared with RPMI 1640 medium condition) of C. glabrata upon macrophage engulfment. (B) GO analysis demonstrates the biological processes involved in overlapping genes. A schematic diagram of a bar chart for the top enriched GO terms ranked according to the values of − log10 (adjusted p value). BP: Biological Process, CC: Cell Component.

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Discussion

Antifungal resistance associated with C. glabrata has become a research hotspot in recent years; however, the pathogenicity of this life-threatening fungus remains unclear. Casein kinase 2 (CK2), a conserved ubiquitous serine/threonine kinase protein complex, has been extensively studied in many species and is advocated as a potential drug target for cancer. Nonetheless, no study has hitherto reported CK2 in C. glabrata. Herein, we identified roles for CgCkb1 and CgCkb2 as the regulatory subunits of CK2 and revealed their functions in response to DNA damage and maintaining a normal cell cycle. Moreover, we highlighted an association between genome integrity and virulence.

DNA damage is universal in life. DNA damage response is composed of DNA breaks repair and DNA damage checkpoint. In eukaryotic cells, broken DNA ends are detected and initiate DNA repair process. Meanwhile, a checkpoint is activated to delay cell cycle progression. In S. cerevisiae, receptors receive the signal of DNA damage and transfer it to central effectors through transduction factors [27]. The sensor kinase Mec1 recruits and phosphorylates the effector kinase Rad9 finally leading to the recruitment and phosphorylation of Rad53 [28]. The activated Rad53 can target multiple biological processes and is triggered upon DNA damage to promote cell cycle arrest and the transcription of DNA repair related genes [29]. Our RNA seq data indicated that the expression levels of MEC1 in Δckb1 and Δckb2 significantly increased compared to WT either in YPD or under 0.01% MMS treatment. The evidence allows us to assume that Ckb1 and Ckb2 may be involved in the DNA damage response mediated by Mec1 in C. glabrata.

An increasing body of evidence suggests that cell cycle checkpoint and DNA repair pathways contribute to coping with DNA damage, and appropriate response to DNA damage is related to genome integrity and is vital for adaptation to surroundings [30, 31]. In S. cerevisiae, Spo11 forms a subcomplex with Ski8, Rec102, and Rec104, which promotes the formation of DSB together with the subcomplex formed by Rec114, Rec107, and Mei4, and then triggers the cells to enter the meiotic recombination process [24]. Studies in mammals, S. cerevisiae, and plants have found that Zip2 and Msh4 can promote the formation of synaptonemal complexes in meiosis [25]. Our study observed a delayed cell cycle in CgCKB1 or CgCKB2 null mutants. Meanwhile, the expression levels of SPO11, REC102, REC104, REC114, MEI4, MSH4 and ZIP2 were altered significantly in these strains. It has been established that these genes are functionally conserved and essential to cell cycle resumption and genomic stability. Accordingly, it is reasonable to infer that CgCkb1 and CgCkb2 might regulate the cell cycle through the above components. In the presence of DNA damage stress, eukaryotic cells trigger intricate DNA damage checkpoint programs to slow down the S-phase and prevent cell division [32, 33]. In S. cerevisiae, the cell cycle is governed by the expression level of transcription factor PHO4 [34]. Our RNA seq data showed that the expression level of CgPHO4 does not decrease in Δckb1 and Δckb2 strains either in YPD or under DNA damage stress. Based on these findings, we hypothesize that the CK2 regulatory subunits do not regulate the cell cycle by inhibiting the transcription level of PHO4 in C. glabrata. Nonetheless, more research is required to elucidate the specific mechanism of Ckb1/2 in cell cycle regulation in C. glabrata.

Although C. albicans has been studied comprehensively, there is still a knowledge gap on the virulence and infection processes of other non-C. albicans yeast, such as C. glabrata. In C. albicans, the catalytic subunit Cka2 of CK2 has been documented to play important roles in interactions with endothelial and oral epithelial cells in vitro and virulence in the murine model of oropharyngeal candidiasis [22]. There is no relevant report on the role of regulatory subunits (Ckb1/2) in virulence in Candida species currently. It is widely acknowledged that macrophages represent the first line of defense against infections. It has been stated that C. glabrata is a successful pathogen without promoting a robust immune response. It can replicate within macrophages to cause recalcitrant infections [7, 9]. Hence, protecting against DNA damage stress induced by macrophages might be essential for C. glabrata to adapt to the microenvironment. Rai et al. found that C. glabrata mutants defective in DNA repair (Δrtt107, Δsgs1) exhibit attenuated virulence and C. glabrata remodeled its chromatin when interacting with macrophages, including chromatin structure modified, altered epigenetic signature, decreased protein acetylation, and so on [10]. In the present study, we examined the survival of CK2 mutant strains upon phagocytosis in THP-1 and RAW 264.7 cells. Contrary to WT, Δckb1 and Δckb2 exhibited significant deficiencies in replication inside macrophages, which indicated weakened adaption to the host and easier elimination of yeasts. The virulence of Δckb1 and Δckb2 was also attenuated in the candidiasis model. In addition, comparison the differential genes regulated by CgCKB1 and CgCKB2 with the activated genes of C. glabrata upon macrophage engulfment revealed that response to DNA damage is crucial for intracellular survival of C. glabrata. Overall, these observations provide compelling evidence that the response to DNA damage is essential for interaction with host immune and pathogenicity.

Some studies have reported the abnormal expression of CK2 in tumor cells, given that inhibitors of CK2 are widely thought of as potential drugs against cancer [35, 36]. There is evidence that CK2 inhibitors can effectively inhibit growth, hypha formation, and biofilm formation in Candida species [37, 38]. Based on the poor viability and attenuated virulence of Δckb1 and Δckb2, inhibition of β subunits of CK2 in C. glabrata provides a novel approach for antifungal treatment.

Conclusions

This is the first study to corroborate that CgCkb1 and CgCkb2 are required to respond to DNA damage and maintain a normal cell cycle. Moreover, CgCkb1 and CgCkb2 are crucial for the survival of engulfed C. glabrata and yield full virulence in the mouse model of invasive candidiasis. Our findings suggest that CgCkb1 and CgCkb2 might promote cellular recovery and adaptation of DSB and genomic stability, thereby maintaining the pathogenicity of C. glabrata. This study contributes to a better understanding of the interaction between C. glabrata and the host and paves the way for more efficient antifungal therapies.

Materials and methods

Strains and growth media

All strains used in this study were constructed from C. glabrata CBS138 and are listed in Table 1. CgCKB1, CgCKB2, CgCKA1 and CgCKA2 genes were disrupted using a recyclable, nourseothricin resistance marker through a homologous recombination strategy as described before [39]. The CEN/ARS episomal plasmid PCU-PDC1-GFP was used to construct CgCKB1, CgCKB2, CgCKA1 and CgCKA2 complemented strains. Briefly, genes were amplified form WT strains including the promoter and terminator regions and digested by SacI/XhoI. pCU-PDC1-GFP was digested by SacI/XhoI to remove GFP expression cassette. The residual plasmid backbone was ligated to digested gene fragments respectively to generate complemented plasmids. Plasmids were further transformed into CgCKB1, CgCKB2, CgCKA1 and CgCKA2 null mutants respectively using the lithium acetate method. Positive transformants grew on SD Medium-URA plates (synthetic medium containing 2% glucose and 2% agar without uracil, Solarbio, China) were picked and further verified. To exclude the potential effects of auxotrophic strain in cell and animal experiments, Ura + phenotype CBS138 was used as wildtype, and the mutant strains were generated from CBS138 through a homologous recombination strategy. Primers used are listed in supplementary Table S1.

Strains were cultured in YPD medium (Becton, Dickinson and Company, USA) composed of 1% yeast extract, 2% peptone, and 2% glucose, and gene-disrupted yeast transformants were selected on YPD plus 100 µg/mL nourseothricin (Sigma Aldrich, USA) as appropriate. Uracil auxotrophs were selected on YNB plates (Becton, Dickinson and Company, USA) composed of 0.67% yeast nitrogen base, 2% glucose, and 2% agar with amino acids and 0.1% 5-fluoroorotic acid (5-FOA, Sigma Aldrich, USA).

Spot assays

Exponential phase WT, Δckb1, and Δckb2 cells were washed with phosphate-buffered saline (PBS, Gibco, USA) and diluted to a series of concentrates from 107 to 103 CFU/mL [40]. 5µL dilutions were spotted on YPD plates containing DNA damage reagents methyl methanesulfonate (MMS, Sigma Aldrich, USA) or 4-Nitroquinoline-1-oxide (4-NQO, Sigma Aldrich, USA) then cultured at 30℃. Photos were taken 48 h later.

Growth curve assay

Overnight inocula of WT, Δckb1 and Δckb2 in YPD medium were washed in PBS and diluted to OD600 of 0.02 in 200 µL YPD medium with 0.01% MMS or 4µM 4-NQO added, respectively. Dilutions were inoculated in a flat-bottomed 96-well plate, and the OD600 values were obtained by BioTek plate reader every 15 min at 30 °C [41]. Experiments were repeated at least three times and analyzed by GraphPad Prism Software.

Table 1 strains used in this study
Full size table

Cell apoptosis assay and TUNEL assay

WT, Δckb1 and Δckb2 were cultured to exponential phase in RPMI 1640 medium and diluted to an OD600 of 0.2 and were either treated with 0.01% MMS or 4 µM 4-NQO for 2 h or left untreated. Then cells were collected and washed with PBS three times. Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology, China) was used. Cells were double stained with Annexin V-FITC and PI (Propidium Iodide, Thermo Fisher Scientific, USA). Fluorescence intensity was detected by BD Fortessa Flow cytometer and data were analyzed by FlowJo software [42].Briefly, forward scatter area (FSC-A) vs. side scatter area (SSC-A) gate was used to gate out obvious debris. Then single cells were identified by side scatter area (SSC-A) vs. side scatter height (SSC-H) gate. The quadrant gates of Annexin V FITC-A vs. PI-A showed the non-apoptotic cells (Annexin V-FITC-/PI-), apoptotic cells (Annexin V-FITC+/PI-), necrotic cells (Annexin V-FITC+/PI+) and dead cells (Annexin V-FITC-/PI+), respectively.

The one-step TUNEL detection kit (Beyotime Biotechnology, China) was used for the TUNEL assay [43]. Briefly, log phase cells were treated with 0.01% MMS or 4 µM 4-NQO for 12 h at 30℃. Cells were digested with 10U Zymolyase 20T (Seikagaku Biobusiness, Japan) at 35℃ for 30 min and then fixed with 4% paraformaldehyde. Cells were incubated in a permeabilization solution for 5 min. For positive control, cells were treated with 2000 U DNase I for 30 min at 37℃. The cells were further incubated with a TUNEL reaction mixture and were observed under fluorescence microscopy or analyzed by flow cytometry. As for negative control, the TdT enzyme in TUNEL reaction mixture was replaced by ddH2O. The same strategies were applied to gate out obvious debris and identify single cells as described above. The proportions of TUNEL + cells were determined by the quantification of fluorescence intensity.

Cell cycle analysis

2 × 108 CFU/mL of WT, Δckb1, and Δckb2 yeast cells were synchronized in YP (1% yeast extract and 2% peptone) for 24 h, respectively. Then yeast cells were released to YPD with or without 0.01% MMS at 30℃, 200 rpm, and collected once an hour. After being washed with PBS, aliquots were fixed with 70% pre-cold ethanol and stored at -20℃ overnight. Samples were further resuspended in 1 mL 0.2 M Tris-HCl, sonicated, and treated with RNase (Thermo Fisher Scientific, USA) at 37℃ overnight. After being washed with 0.2 M Tris-HCl, samples were stained with 0.05 mg/mL PI at 0℃ for 15 min, finally pelleted and resuspended in 0.2 M Tris-HCl with 0.01 mg/mL PI added. At least 50,000 events were analyzed by flow cytometry using BD Fortessa, and data were analyzed by FlowJo software [44]. The same strategies as in cell apoptosis assay were used to discard cell debris and identify the single cells. DNA content was measured by the PI histogram plot. The DNA content of G2 phase cells (2 C) is twice that of G1 phase cells (1 C). G1 and G2 phases cells were distinguished by different fluorescence intensity. G2 phase cells displayed approximately twice brighter fluorescence than G1 phase cells.

Survival upon phagocytosis and adhesion assays

Human monocyte THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific, USA ) at 37℃, 5% CO2. First, THP-1 cells were seeded into 24-well plates at a density of 5 × 105 cells per well with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, USA) added. After 18 h, a fresh medium without PMA was replaced, and cells were cultured for another 18 h. Mouse leukemia macrophages RAW264.7 (ATCC TIB-71) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-Glutamine, 100 U/ml penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific, USA) at 37℃ under a 5% CO2 atmosphere. Afterward, C. glabrata cells were cocultured with THP-1 or RAW264.7 cells at MOI 1:10. 2 h later, supernatants were removed, and cells were washed with PBS three times. 300 µL 0.05% Triton X-100 (w/v) was added into per well to lyse macrophages, and the lysates were diluted and plated on YPD to calculate the number of yeast cells phagocyted by macrophages [45]. As for C. glabrata survival upon phagocytosis, THP-1 or RAW264.7 cells were cultured for 24 h after being washed with PBS and then lysed with the same method. Survival rates were calculated by the ratio of 24 h to 2 h yeast cell numbers. Experiments were performed three times.

As described before, adhesion assays were performed using human colorectal adenocarcinoma cells (Caco-2 ATCC HTB-37) and were repeated three times [39]. Δepa1 was verified as deficient in adherence and was used for control.

Biofilm formation

5 × 107 Log-phased yeast cells in YPD were collected and seeded in a 24-well plate, followed by a 37℃ incubation for 90 min. RPMI 1640 medium with 10% FBS contained was added to each well after being washed with PBS twice. Then the plate was incubated at 37℃ for 24 h and replaced with the fresh medium to incubate for another 24 h. Unattached yeast cells were washed with PBS three times. After 30 min of staining with crystal violet [0.4% (w/v)], 95% ethanol was used for destaining. The destaining solution was measured for absorbance at 595 nm. Wells without yeast cells were used as background [46]. Three biological replicates were performed.

Animal candidiasis models

The virulence of WT, Δckb1 and Δckb2 strains were evaluated by mouse models of invasive candidiasis [47, 48]. Both survival curves of mouse and fungal burdens were assessed. Female outbred ICR mice (5–6 weeks old, 24–26 g) were obtained from Charles River Company, China. As for survival assays, 200 mg/kg cyclophosphamide (Sigma Aldrich, USA) was intraperitoneally injected per mouse on day − 3 and every fourth day later. Mice were randomly designated into groups according to the random number table method, with a total of 12 mice in each group. Then 1 × 108 yeast cells in 200 µL in 0.9% (w/v) saline were injected through the tail veil on day 0. The survival conditions were monitored and recorded. Mice in the agonal stage were humanely euthanized by cervical dislocation. Experiments were terminated on day 14. As for fungal burden assays, groups of 6 mice were immunosuppressed with 200 mg/kg cyclophosphamide on day − 3 and were further injected with 5 × 107 yeast cells through the tail veil on day 0. Mice were sacrificed on day 3 post-infection, and organs (spleen, liver, and kidney) were weighed and transferred to ice-cold PBS. Tissue homogenates were diluted, plated onto YPD agar, and incubated at 30℃ for 2 days. CFUs were calculated and analyzed. Evaluation of histology was performed by kidney tissue sections stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) (Servicebio Technology company, China).

RNA sequencing

Log phase WT, Δckb1, and Δckb2 cells were inoculated into fresh YPD medium either with 0.01% MMS added or left untreated. After 1 h of incubation, yeast cells were harvested, and total RNA was extracted by the acid phenol method [49]. Three biological replicates were performed per condition. RNA was frozen at -80℃ and sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China) for further sequencing by Illumina novaseq6000 PE150. Quality-filtered RNA-seq reads were aligned to the C. glabrata genome (http://www.candidagenome.org) by STAR software (version 2.7.4). Gene expressions were obtained and normalized by StringTie (V2.1.2) and DESeq2 (V1.20). Significantly expressed genes were screened based on a 2-fold cut-off and p-value < 0.05.

Data Availability

The dataset generated from this study has been deposited in the NCBI database under the GEO accession number GSE232439 (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/geo/query/acc.cgi?acc=GSE232439). All primary data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

4-NQO:

4-Nitroquinoline-1-oxide

CFU:

Colony-forming unit

DAPI:

Diamidino-phenylindole

DEG:

Differential expression gene

DMEM:

Dulbecco’s modified Eagle’s medium

DSB:

DNA double-strand break

GO:

Gene ontology

HE:

Hematoxylin-Eosin

MMS:

Methyl methane sulfonate

OD:

Optical density

PAS:

Periodic acid-Schiff

PBS:

Phosphate-buffered saline

PI:

Propidium Iodide

PMA:

Phorbol 12-myristate 13-acetate

PS:

Phosphatidylserine

ROS:

Reactive oxygen species

RNS:

Reactive nitrogen species

SSB:

Single-strand breaks

WT:

Wild-type

YPD:

Yeast extract peptone dextrose medium

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Acknowledgements

We thank Dr. Ningning Liu for kindly providing the BioTek plate reader and Dr. Xiaoqing Chen for advice on statistical analysis.

Funding

This work was supported by the National Natural Science Foundation of China (82002200, 81971993, 82172324, 81902117 and 82202581), the Shanghai Sailing Program (grant no. 19YF1431300).

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  1. Qi Ni and Xianwei Wu contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No.197 Ruijin ER Road, Shanghai, 200025, China

    Qi Ni, Tongxuan Su, Cen Jiang, Danfeng Dong, Daosheng Wang, Wei Chen, Yingchao Cui & Yibing Peng

  2. Institute of Respiratory Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, No.725 South Wanping Road, Shanghai, 200032, China

    Xianwei Wu

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Contributions

QN and XW contributed to the conceptualization and wrote the original draft. QN, XW, TS, DW, CJ, DD, WC, and YC performed the experiments and provided essential reagents. QN, XW, TS, and YC analyzed the data. QN, DD, YC, and YP contributed to the funding acquisition. YP performed the project administration. All authors read and approved the final manuscript.

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Correspondence to Yibing Peng.

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All animal experiments were reviewed and approved by The Ruijing Hospital Ethics Committee, and were performed in compliance with the Regulations for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China, which enforces the ethical use of animals. This study was carried out in compliance with the ARRIVE guidelines.

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Ni, Q., Wu, X., Su, T. et al. The regulatory subunits of CK2 complex mediate DNA damage response and virulence in Candida Glabrata. BMC Microbiol 23, 317 (2023). https://0-doi-org.brum.beds.ac.uk/10.1186/s12866-023-03069-4

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