P4-ATPase subunit Cdc50 plays a role in yeast budding and cell wall integrity in Candida glabrata

Abstract

Background: As highly-conserved types of lipid flippases among fungi, P4-ATPases play a significant role in various cellular processes. Cdc50 acts as the regulatory subunit of flippases, forming heterodimers with Drs2 to translocate aminophospholipids. Cdc50 homologs have been reported to be implicated in protein trafficking, drug susceptibility, and virulence in Saccharomyces cerevisiae, Candida albicans and Cryptococcus neoformans. It is likely that Cdc50 has an extensive influence on fungal cellular processes. The present study aimed to determine the function of Cdc50 in Candida glabrata by constructing a Δcdc50 null mutant and its complemented strain.

Results: In Candida glabrata, the loss of Cdc50 led to difficulty in yeast budding, probably caused by actin depolarization. The Δcdc50 mutant also showed hypersensitivity to azoles, caspofungin, and cell wall stressors. Further experiments indicated hyperactivation of the cell wall integrity pathway in the Δcdc50 mutant, which elevated the major cell wall contents. An increase in exposure of β-(1,3)-glucan and chitin on the cell surface was also observed through flow cytometry. Interestingly, we observed a decrease in the phagocytosis rate when the Δcdc50 mutant was co-incubated with THP-1 macrophages. The Δcdc50 mutant also exhibited weakened virulence in nematode survival tests.

Conclusion: The results suggested that the lipid flippase subunit Cdc50 is implicated in yeast budding and cell wall integrity in C. glabrata, and thus have a broad influence on drug susceptibility and virulence. This work highlights the importance of lipid flippase, and offers potential targets for new drug research.

Keywords: Candida glabrata; Cdc50; Cell wall integrity; Drug susceptibility; Lipid flippase; Virulence.

Fig. 1

The Δcdc50 mutant showed defects in cell growth, yeast budding, and actin polarization. A Wild-type (WT), Δcdc50, and Δcdc50 + CDC50 strains were adjusted to the same cell density and incubated in fresh YPD medium at 30 ℃ for 24 h. Their OD at 600 nm was recorded to construct the cell growth curve. In the exponential phase, the Δcdc50 mutant exhibited significantly slower growth compared with the other two strains. B The cell cycle distribution of different strains as determined using microscopy. Mid-log phase yeast was collected and stained to be classified as having no, small, or large buds. More than 300 cells were counted in each group. The Δcdc50 mutant showed increased numbers of small buds and decreased numbers of large buds compared with those of the wild-type. Data represent the mean ± SD of three independent experiments. C Typical views of wild-type and Δcdc50 actin fluorescent imaging during yeast budding. After staining with FITC-phalloidin, the WT and Δcdc50 yeast cells were observed using fluorescence microscopy. Most WT budding yeasts exhibited normal actin patch polarization (enriched in buds and bud necks), while a large amount of Δcdc50 budding yeasts showed actin mislocalization: The actin patches were depolarized and scattered. Bar, 10 μm. *, P < 0.05. Data represent the mean ± SD of three independent experiments

Fig. 2

The Δcdc50 mutant showed hypersensitivity to antifungal drugs and cell wall stressors. Mid-log phase yeast was adjusted to 0.1 OD at 600 nm and diluted tenfold serially to be spotted on YPD plates containing different drugs. A The Δcdc50 mutant exhibited weakened resistance to the clinically used antifungal drugs fluconazole, itraconazole and caspofungin. FLC, fluconazole; ICZ, itraconazole; CSF, caspofungin. B The Δcdc50 mutant was more sensitive to cell wall stressors CR, CFW and SDS, suggesting cell wall remodeling. CR, Congo Red; CFW, Calcofluor White; SDS, sodium dodecyl sulfate

Fig. 3

Increased cell wall mannan, chitin and β-1,3-glucan contents, along with exposed chitin and β-1,3-glucan were found in Δcdc50. A Yeast cells in mid-log phase were collected and incubated in FITC-ConA, CFW and Aniline Blue to assess the total cell wall mannan, chitin, and β-1,3-glucan contents, respectively, then analyzed using flow cytometry and spectrofluorometry. In the Δcdc50 mutant, the cell wall mannan content increased by 14.5%, total chitin contents increased significantly by 39%, and total β-1,3-glucan contents increased by 44.3%. B Exposure of chitin and β-1,3-glucan were tested using FITC-WGA and anti-β-1,3-glucan antibodies. The exposure of inner chitin has increased by 14.9%, while the β-1,3-glucan exposure has increased by 25.5% in the Δcdc50 mutant. Data represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 4

Loss of Cdc50 led to decreased phagocytosis rate in macrophages and increased pro-inflammatory cytokine secretion. A Aliquots of THP-1 cells were co-incubated with wild-type (WT), Δcdc50, and Δcdc50 + CDC50 yeast for 2 h, then washed and lysed to release intracellular yeast for CFU counting. Other THP-1 cells were cultured for another 4 h after the 2-h co-incubation and washing, and lysed for CFU counting. After being exposed to macrophages for 2 h, the Δcdc50 mutant exhibited a much lower phagocytosis rate compared with the WT and Δcdc50 + CDC50 strains. Accordingly, the number of surviving intracellular Δcdc50 yeast were reduced by almost 50% compared with that of the surviving WT yeasts after 6 h. B After a 2-h co-incubation with yeast cells, THP-1 cells were brushed and Gram stained to count the intracellular yeast. The Δcdc50 cells were about 58.6% of WT cells, exhibiting less uptake by macrophages. C Typical field views of THP-1 after co-incubation with WT and Δcdc50 cells, respectively. D Different C. glabrata strains were co-incubated with THP-1 cells for 4 h, and the culture supernatants were collected and centrifugated to measure the secreted TNF-α and IL-1β levels. THP-1 cells without exposure to yeast were used for baseline secretion level estimation as a negative control. Exposure to WT and Δcdc50 + CDC50 hardly triggered enhanced secretion of pro-inflammatory cytokines in macrophages, whereas the loss of Cdc50 activity in C. glabrata caused increased secretion of TNF-α and IL-1β. Data represent the mean ± SD of three independent experiments. NC Negative control. *, P < 0.05; **, P < 0.01

Fig. 5

The Δcdc50 mutant showed defective virulence in Caenorhabditis elegans compared with other strains. C. elegans were raised and fed on different strains of C. glabrata, and the number of deaths in each group was counted daily to construct a survival curve and assess in vivo virulence. E. coli OP50 strain was used as a quality control. Nematodes in the Δcdc50 group exhibited a longer lifespan compared with the wild-type (WT) and Δcdc50 + CDC50 group, suggesting weakened virulence in the Δcdc50 mutant. ***, P < 0.001

Fig. 6

Stress response pathways associated with cell wall biogenesis and remodeling were constitutively upregulated in the Δcdc50 mutant. A Mid-log phase yeast was collected, washed, and frozen to isolate total RNA, followed by qRT-PCR. The relative expression of genes in different strains were obtained by comparison with that in the wild-type (WT). In the Δcdc50 mutant, the genes that encode the constituents of the cell integrity pathway, including Slt2, Rlm1, Swi4, Swi6, Fks1, Fks2 and Chs3, all showed increased expression to varying degrees. B Mid-log phase WT and Δcdc50 yeast were collected for complex transcriptome sequencing, and the expression level of every gene was quantified using the TPM method. Differentially expressed genes were enriched via GO functional enrichment analysis to identify significantly different GO pathways between the WT and Δcdc50. The top 12 most significant pathways were mainly implicated in cell wall organization and biogenesis. C The TPM of genes enriched in the GO pathways in B were gathered and taken the logarithm to generate a heatmap for visualized comparison. TPM, transcripts per million reads; GO, Gene Ontology. *, P < 0.05; **, P < 0.01; ***, P < 0.001