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. 2023 Feb 16:14:1129155.
doi: 10.3389/fmicb.2023.1129155. eCollection 2023.

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans

Kedric L Milholland  1 , Ahmed AbdelKhalek  2 , Kortany M Baker  1 , Smriti Hoda  1 , Andrew G DeMarco  1 , Noelle H Naughton  1 , Angela N Koeberlein  1 , Gabrielle R Lorenz  1 , Kartikan Anandasothy  1 , Antonio Esperilla-Muñoz  3 , Sanjeev K Narayanan  2 , Jaime Correa-Bordes  3 , Scott D Briggs  1   4 , Mark C Hall  1   4
Affiliations

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans

Kedric L Milholland et al. Front Microbiol. .

Abstract

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

Keywords: Candida albicans; Cdc14; cell wall integrity; echinocandins; hyphal differentiation; pathogenesis; phosphatase; septation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
A conserved motif in Cdc14’s disordered C-terminal tail is required for full catalytic activity. (A) Domain map of Cdc14. DSPn and DSPc are the N-and C-terminal dual-specificity phosphatase domains, respectively. CX5R is the invariant PTP family catalytic motif. Sites of truncations and the invariant QPRK motif are indicated in the disordered C-terminus following DSPc. (B) Steady-state kinetic analyses with full length ScCdc14 (1–551) and the indicated truncation variants toward pNPP yielded kcat values of 0.91 (1–551), 0.80 (1–449), and 0.04 (1–374) sec−1. (C) Same as (B) with phosphopeptide substrate HT[pS]PIKSIG, yielding kcat values of 1.21 (1–449) and 0.01 (1–374) sec−1. (D) Multiple sequence alignment of fungal Cdc14 orthologs from the indicated species was generated by Clustal Omega using default settings. Only a portion of the C-terminal region showing the invariant QPRK motif is shown. (E) Steady-state kinetic analyses comparing wild-type ScCdc14 and QPRK motif mutants towards pNPP; kcat values were 0.02 (1–449QARA) and 0.03 (1–449APRK) sec−1. (F) Same as (E) with the phosphopeptide substrate; kcat values were 0.02 (1–449QARA) and 0.03 (1–449APRK) sec−1. The ScCdc14 (1–449) data in panels (E,F) are identical to panel (C). All kinetic data in panels (B,C,E,F) are averages of at least 3 independent trials with standard deviation error bars. Fit lines and kcat values were generated in GraphPad Prism using the Michaelis–Menten function.
Figure 2
Figure 2
QPRK mutation causes sensitivity to cell wall stress in S. cerevisiae. (A–E) Cultures of the indicated S. cerevisiae strains were serially diluted and spotted on YPAD agar plates containing indicated concentrations of (A) methyl methanesulfonate (MMS), (B) calcofluor white (CFW), (C) micafungin (MF), (D) caspofungin (CF), and (E) MF or MF + sorbitol. “#1” and “#2” refer to independent isolates of cdc14hm. yen1Δ mus81Δ is a positive control for MMS sensitivity (Ho et al., 2010). All plates were incubated at 30°C for 72 h. Images are representative of three biological replicates. “Wild-type” is W303. (F) Immunoblot detecting phosphorylated, active form of Slt2 (pSlt2) from cell lysates of mid-log phase cells with anti-p44/42 MAP kinase antibody. pSlt2 signals were corrected based on the G6PDH (glucose-6-phosphate dehydrogenase) load control and then normalized to the untreated wild-type sample to generate the Relative pSlt2 value, which is an average of two independent trials.
Figure 3
Figure 3
Cdc14-dependent cell wall stress sensitivity is conserved in other fungal species. Liquid cultures of the indicated S. pombe (A) and C. albicans (B) strains were serially diluted and spotted on YES or YPD agar plates, respectively, containing the indicated concentrations of micafungin (MF), caspofungin (CF), or calcofluor white (CFW). CLP1 is the S. pombe homolog of CDC14. (C) Steady-state kinetic analysis of CaCdc14 (1–427) and CaCdc14 (1–427AARA) using the Yen1 phosphopeptide substrate. kcat values were 0.092 (1–427) and 0.015 (1–427AARA) sec−1. Data are means from three independent trials and error bars are standard deviations. Liquid cultures of the indicated C. albicans strains were serially diluted and spotted on YPD agar plates containing the indicated concentrations of micafungin (D) or caspofungin (E). Plates were incubated at 30°C for 3–5 days (until mature colonies were visible in the wild-type strain). Images are representative of three independent trials.
Figure 4
Figure 4
Reduced Cdc14 activity causes elevated CWI signaling and perturbs cell wall gene expression in C. albicans. (A) Phosphorylation of Mkc1 (pMkc1) was detected in whole cell extracts from mid-log phase cultures of the indicated C. albicans strains by anti-p44/p42 MAP kinase immunoblotting. Saturated cultures were back-diluted to OD600 = 0.01 and grown to mid-log phase. A portion of the untreated culture was collected and the remainder was treated with 50 ng/ml micafungin (MF) for 30 min. Anti-PSTAIR is a loading control. (B) pMkc1 chemiluminescence signals from (A) were normalized to PSTAIR and plotted as a fold-increase above the untreated wild-type signal. Values are means from three independent experiments and error bars are standard deviations. Unpaired T-tests assuming equal variance comparing pMkc1 level in each strain to wild-type were used to determine statistical significance – *p < 0.05; ns, not significant (p > 0.05). (C–F) Relative mRNA levels of selected cell wall stress-responsive genes in mid-log phase cultures of C. albicans strains either untreated (−) or treated (+) with 50 ng/ml MF for 1 h were measured by qRT-PCR. Transcript levels were normalized to RDN18 and set relative to the untreated wild-type sample. Data are averages of at least five biological trials and error bars are standard deviations. Outliers were identified by Grubbs test and removed if detected. Unpaired T-tests assuming equal variance were used to compare (1) the basal (untreated) expression in each strain with wild-type (†p < 0.05) and (2) untreated with MF-treated samples of each strain. If a significant increase was observed with MF treatment, then the average fold-increase was added above the bar and an additional T-test was performed to determine if fold-increase was significantly different than wild-type fold-increase (*p < 0.05). ns, not significant (p > 0.05).
Figure 5
Figure 5
Reduced Cdc14 activity impairs C. albicans septum assembly and cell separation. (A–D) Transmission electron micrographs of C. albicans yeast form cells of the indicated strains undergoing septation in mid-log phase liquid cultures. PS, primary septum; SS, secondary septum. (E) Light microscopy images of log phase cultures of the indicated strains.
Figure 6
Figure 6
Reduced Cdc14 activity impairs hyphal development on solid medium. (A) The indicated C. albicans strains were grown on Spider medium agar plates at 37°C for 5 days. Plates were imaged before and after vigorous washing of unattached cells off the surface with water. After washing, agar plugs were removed and cross sections were imaged to illustrate depth of invasive growth. (B) Pieces of the agar plugs from (A) were manually ground into small pieces, which were then mounted on a microscopy slide, compressed under a cover slip, and images of embedded cells captured by DIC microscopy.
Figure 7
Figure 7
Loss or reduction of Cdc14 function impairs pathogenesis of C. albicans. (A) G. mellonella larvae or (B) immunosuppressed BALB/c mice were infected with 2 × 105 or 5 × 105 C. albicans cells of the indicated strains, respectively, or mock-uninfected. p-values were determined using a log-rank (Mantel–Cox) test comparing the survival distribution of hosts infected with wild-type C. albicans to each experimental strain. (C–E) Histopathological analysis of tissue sections from mouse organs harvested after completion of experiment in (B). In (C) fungal cells were stained with periodic acid-Schiff (PAS) in representative tissue sections to illustrate morphology. In (D,E) liver and lung tissues, respectively, were stained with H&E. All images were acquired at 200x magnification, except the CDC14/Δ sample in (D), which was imaged at 20x to illustrate the necrotic lesion with surrounding inflammation. The cdc14Δ/Δ liver section in (D) illustrates one of the rare lesions [associated with the cluster of fungal cells shown in (C)] found in mice infected with cdc14 mutant strains. In (D,E) the following shock-related lesions (lacking invading fungal cells) in mice infected with CDC14/CDC14 and CDC14/Δ strains are labeled: endothelial leakage characterized by hepatic (D) or pulmonary alveolar (E) edema (a), multifocal hemorrhage and congestion (b), and intravascular microthrombi (c). Recruited inflammatory cells (d) and necrotic lesions (e) are also labeled. H&E-stained liver and lung sections of an uninfected mouse can be found in Supplementary Figure S8C for comparison.
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