Phosphoregulation of Nap1 plays a role in septin ring dynamics and morphogenesis in Candida albicans

Abstract

Nap1 has long been identified as a potential septin regulator in yeasts. However, its function and regulation remain poorly defined. Here, we report functional characterization of Nap1 in the human-pathogenic fungus Candida albicans. We find that deletion of NAP1 causes constitutive filamentous growth and changes of septin dynamics. We present evidence that Nap1's cellular localization and function are regulated by phosphorylation. Phos-tag gel electrophoresis revealed that Nap1 phosphorylation is cell cycle dependent, exhibiting the lowest level around the time of bud emergence. Mass spectrometry identified 10 phosphoserine and phosphothreonine residues in a cluster near the N terminus, and mutation of these residues affected Nap1's localization to the septin ring and cellular function. Nap1 phosphorylation involves two septin ring-associated kinases, Cla4 and Gin4, and its dephosphorylation occurs at the septin ring in a manner dependent on the phosphatases PP2A and Cdc14. Furthermore, the nap1Δ/Δ mutant and alleles carrying mutations of the phosphorylation sites exhibited greatly reduced virulence in a mouse model of systemic candidiasis. Together, our findings not only provide new mechanistic insights into Nap1's function and regulation but also suggest the potential to target Nap1 in future therapeutic design.

Importance: Septins are conserved filament-forming GTPases involved in a wide range of cellular events, such as cytokinesis, exocytosis, and morphogenesis. In Candida albicans, the most prevalent human fungal pathogen, septin functions are indispensable for its virulence. However, the molecular mechanisms by which septin structures are regulated are poorly understood. In this study, we deleted NAP1, a gene encoding a putative septin regulator, in C. albicans and found that cells lacking NAP1 showed abnormalities in morphology, invasive growth, and septin ring dynamics. We identified a conserved N-terminal phosphorylation cluster on Nap1 and demonstrated that phosphorylation at these sites regulates Nap1 localization and function. Importantly, deletion of NAP1 or mutation in the N-terminal phosphorylation cluster strongly reduced the virulence of C. albicans in a mouse model of systemic infection. Thus, this study not only provides mechanistic insights into septin regulation but also suggests Nap1 as a potential antifungal target.

FIG 1

nap1Δ/Δ cells exhibit constitutive filamentous and invasive growth. (A) WT (BWP17; all strains used in this study are listed in Table S1 in the supplemental material) (47), nap1Δ/Δ (HZX01), and NAP1-reintegrated (HZX02) cells were grown on GMM plates and incubated at 30°C for 5 days and in liquid YPD at 30°C or 37°C overnight. Size bars, 5 µm throughout the figures. (B) Cells were spotted onto GMM plates and incubated at 30°C for 5 days. The spots were photographed before and after washing the plate with water. (C) Agar sections underneath the colonies were examined and photographed under a microscope. Arrows indicate fungal filaments.

FIG 2

Nap1 subcellular localization and its interaction with septins. (A) An overnight culture of cells expressing Nap1-GFP (HZX02) was grown in GMM at 30°C for yeast cells or in GMM plus 20% serum at 37°C for hyphal induction before differential interference contrast (DIC) and fluorescence microscopy. Cells at different stages of growth are shown. (B) Co-IP of Cdc3 and Gin4 with Nap1. Cell extracts were prepared from log-phase yeast cells of HZX05 (NAP1-HA CDC3-Myc) and HZX06 (NAP1-HA GIN4-Myc) and subjected to IP using HA antibody (αHA), followed by WB with αHA and αMyc. HZX03 (NAP1-HA) was included as a negative control. (C) Mislocalization of Nap1-GFP in septin mutants. Yeast cells of HZX18 (cdc10Δ/Δ NAP1-GFP) and HZX19 (cdc11Δ/Δ NAP1-GFP) were imaged as described for panel A. (D) Septin localization and organization defects in nap1Δ/Δ cells. Log-phase yeast cells of HZX27 (WT; CDC3-GFP) and HZX26 (nap1Δ/Δ CDC3-GFP) were examined for morphology and Cdc3-GFP localization. Arrows show septin defects. (E) Gin4 localization and organization defects in nap1Δ/Δ cells. Log-phase yeast cells of HZX48 (WT; GIN4-GFP) and HZX47 (nap1Δ/Δ GIN4-GFP) were examined for morphology and Gin4-GFP localization.

FIG 3

Cell cycle-dependent phosphorylation of Nap1. (A) Log-phase yeast cells expressing Nap1-HA (HZX03) were subjected to IP with αHA. The IP product was divided into two halves, and one was treated with λPP. Nap1 phosphorylation was analyzed using Phos-tag SDS-PAGE followed by αHA WB. (B) G₁ cells of HZX03 were released into YPD at 30°C for growth. Aliquots were harvested at intervals to derive a budding index and for Phos-tag analysis. The intensity of Nap1 bands was quantified using ImageJ, and the Phos/Unphos ratio of Nap1 at each time point was calculated.

FIG 4

Phosphorylation of Nap1 is crucial for its localization and function. (A) WT Nap1 (HZX02), Nap1-10A (HZX07), or Nap1-10E (HZX08) was expressed as a GFP fusion protein in nap1Δ/Δ cells. Colony morphology on GMM plates (top) and cell morphology in liquid YPD at 30°C (middle) were examined. Nap1 localization was visualized by fluorescence microscopy (bottom). (B) Co-IP of Cdc3 with WT Nap1, Nap1-10A, or Nap1-10E. Cell extracts were prepared from log-phase yeast cells of HZX15 (NAP1-Myc CDC3-GFP), HZX13 (nap1-10A-Myc CDC3-GFP), and HZX14 (nap1-10E-Myc CDC3-GFP). Nap1 was immunoprecipitated with αMyc. Nap1 and Cdc3 in the IP products were detected with αMyc and αGFP WB, respectively. (C) The intensity of protein bands shown in panel B was determined by using ImageJ, and the ratio of Cdc3 to Nap1 in each lane was calculated. Student’s t test was performed. Error bars represent standard errors.

FIG 5

Phos-tag WB analysis of WT Nap1, Nap1-10A, and Nap1-10E. (A) Nap1-HA (HZX03), Nap1-10A-HA (HZX09), and Nap1-10E-HA (HZX10) were immunoprecipitated from log-phase yeast cells with αHA. Half of each IP product was treated with λPP before Phos-tag gel electrophoresis and αHA WB. (B) Phos-tag WB analysis of Nap1-HA in WT (HZX03), cdc10Δ/Δ (HZX20), and cdc11Δ/Δ (HZX21) cells. Experiments were done as described for panel A. (C) Phos-tag WB comparison of Nap1-10A in WT and cdc10Δ/Δ cells. Experiments were done as described for panel A using log-phase yeast cells of HZX09 (nap1-10A-HA) and HZX22 (nap1-10A-HA cdc10Δ/Δ). (D) Phos-tag WB analysis of Nap1 phosphorylation in kinase and phosphatase mutants. Experiments were done as described for panel A using log-phase yeast cells of HZX03 (NAP1-HA), HZX41 (NAP1-HA rts1Δ/Δ), HZX42 (NAP1-HA cdc14Δ/Δ), HZX43 (NAP1-HA cla4Δ/Δ), and HZX44 (NAP1-HA gin4-KD). (E) Septin localization in cla4Δ/Δ yeast cells. G₁ yeast cells of HZX45 (cla4Δ/Δ CDC3-GFP) were released into GMM for growth at 30°C, and cells at different stages of growth were examined for morphology and septin localization.

FIG 6

Genetic interaction between nap1Δ/Δ and cdc10Δ/Δ mutants and independence of nap1Δ/Δ pseudohyphal growth on Swe1. (A and B) Cells of the indicated genotypes were grown overnight in liquid YPD at 30°C before imaging.

FIG 7

Septin ring dynamics in nap1Δ/Δ cells. FRAP analysis of Cdc3-GFP in the septin ring in WT (HZX27) and nap1Δ/Δ (HZX26) yeast cells with a small bud, a large bud with a single ring, and a large bud with split rings (cell images are shown in Fig. S3 in the supplemental material). Fluorescence intensity was measured at 1-min intervals after photobleaching of the septin ring. Average values taken from 5 cells of each budding stage were used to generate the curves. Student’s t tests were performed, and error bars represent standard errors.

FIG 8

NAP1 mutants exhibited reduced virulence. (A) Doubling times of strains. Cells were grown in YPD at 37°C, and optical density at 600 nm was measured at regular intervals. Doubling times were calculated by averaging optical density values measured during the log phase of growth from 3 independent experiments. Error bars represent 1 standard deviation from the mean. (B) CFU/kidney of infected mice. Two mice for each C. albicans strain were sacrificed 48 h or 5 h postinfection to determine CFU/kidney. (C) Survival curve of mice (n = 8 per strain) injected with 1 × 10⁶ cells with the indicated genotype.