The small-secreted cysteine-rich protein CyrA is a virulence factor participating in the attack of Caenorhabditis elegans by Duddingtonia flagrans

Abstract

Nematode-trapping fungi (NTF) are a diverse and intriguing group of fungi that live saprotrophically but can switch to a predatory lifestyle when starving and in the presence of nematodes. NTF like Arthrobotrys oligospora or Duddingtonia flagrans produce adhesive trapping networks to catch and immobilize nematodes. After penetration of the cuticle, hyphae grow and develop inside the worm and secrete large amounts of hydrolytic enzymes for digestion. In many microbial pathogenic interactions small-secreted proteins (SSPs) are used to manipulate the host. The genome of D. flagrans encodes more than 100 of such putative SSPs one of which is the cysteine-rich protein CyrA. We have chosen this gene for further analysis because it is only found in NTF and appeared to be upregulated during the interaction. We show that the cyrA gene was transcriptionally induced in trap cells, and the protein accumulated at the inner rim of the hyphal ring before Caenorhabditis elegans capture. After worm penetration, the protein appeared at the fungal infection bulb, where it is likely to be secreted with the help of the exocyst complex. A cyrA-deletion strain was less virulent, and the time from worm capture to paralysis was extended. Heterologous expression of CyrA in C. elegans reduced its lifespan. CyrA accumulated in C. elegans in coelomocytes where the protein possibly is inactivated. This is the first example that SSPs may be important in predatory microbial interactions.

Fig 1. CyrA is a secreted cysteine-rich protein induced during trap formation.

(A) Scheme of CyrA. The 155 amino acid long protein contains a 21 amino acid long signal peptide and a cysteine-rich region with eight cysteine residues. (B) The CyrA-LccC expressing D. flagrans strain sNH08 and the WT were grown on LNA with 1 mM ABTS for 48 hours. (C) Quantitative real time PCR analysis of cyrA expression in D. flagrans hyphae grown on LNA and in hyphae and traps co-cultivated for 24 h with C. elegans. The three biological replicates are displayed individually, and the error bar indicates the standard deviation of three technical replicates, P-value = 0.0342. The expression was normalized to actin. (D) Spatial analysis of cyrA expression using a transcriptional reporter assay. The h2b-mCherry reporter was expressed under the constitutive h2b (sNH14) or the cyrA promoter (sNH21), respectively. Pictures of traps and captured nematodes were taken after 24 h of co-incubation. Scale bar = 100 μm. Pictures of uninduced mycelium were taken after 24 h at 28°C. Scale bar = 10 μm. (E) The mean fluorescence of single nuclei of the reporter strains in traps (ind.) and vegetative mycelium (unind.) was measured in different pictures taken with the same settings using ImageJ (arbitrary units). cyrA(p) uninduced n = 39, cyrA(p) induced n = 48, h2b(p) uninduced n = 30, h2b(p) induced n = 30. P-value h2b(p) induced vs. cyrA(p) induced < 0.0001.

Fig 2. Localization of CyrA and different vesicles and organelles in traps of D. flagrans.

(A) The CyrA-mCherry expressing strain sNH25 was co-incubated with C. elegans on LNA slides for 24 h at 28°C to induce traps. The cell wall was stained with calcofluor white (CFW). (B) Visualization of vesicles and organelles through GFP fusion proteins. Clathrin coated vesicles (ClaH-GFP, strain sNH66), exosomes (BroA-GFP, sNH52), endosomes (RabA-GFP, sNH29), peroxisomes (GFP-SKL, sNH16), the ER (GFP-KDEL, sNH22) and nuclei (H2B-mCherry, sNH14). Scale bars = 10 μm. (C) BroA-GFP and RabA-GFP, respectively, were expressed in the CyrA-mCherry expressing strain. Scale bars = 10 μm. (D) cyrA-mCherry was expressed under the constitutive oliC-promoter (sNH25) but the localization of the fusion protein in speckles was only apparent in traps (arrow). In the surrounding vegetative mycelium, CyrA-mCherry localized in vacuolar structures. (E) CyrA was expressed without its signal peptide sequence fused to GFP (strain sNH41). (F) The 21 amino acid long signal peptide of CyrA was fused to GFP (strain sNH42) and the construct was expressed under the constitutive oliC promoter.

Fig 3. CyrA accumulates at the infection bulb during the attack.

(A) The cyrA(p)::cyrA::mCherry expressing strain sNH65 was co-incubated for 24 h at 28°C with C. elegans Ban126 (his-72p::his-72::GFP). The enlargement shows the CyrA-mCherry localization in the infection bulb. The arrow points to the entry point of the fungus, above the infection bulb. (B) Time course of infection bulb formation (arrow) and accumulation of CyrA-GFP (strain sNH30). Pictures of C. elegans captured by D. flagrans were taken every minute and assembled into a movie sequence (S1 Movie). Pictures of the indicated time points are displayed. Scale bars = 10 μm.

Fig 4. Brefeldin A treatment inhibits penetration and CyrA accumulation at the infection bulb.

(A) D. flagrans expressing cyrA(p)::cyrA::mCherry (sNH65) was co-incubated with C. elegans on thin LNA slides for 24 h at 28°C. A 1 cm square was cut from the agar and placed upside down onto a 5 μl drop of 50 μg/ml BFA. A hypha after 60 and 90 min. (B) CyrA-mCherry localization in the trap after BFA treatment. (C) Observation of an infection site in the DIC channel (enlargement in (D)). The red box indicates the penetration site. (E) Localization of CyrA-mCherry after BFA treatment and penetration. The red box indicates the penetration site. (F, G) Time course of infection bulb formation and CyrA-mCherry distribution in the presence (F) and the absence (G) of BFA. (H) Enlargement of the formed infection bulbs after 90 min. The arrow points to the entry point of the fungus. Scale bars = 10 μm.

Fig 5. The exocyst component exoA is necessary for CyrA accumulation at the infection bulb.

(A) Scheme of the deletion strategy for exoA. The gene was deleted via homologous recombination by flanking the hygromycin resistance cassette (hph) with 1 kb upstream (LB) and downstream (RB) flanks of exoA. The LB was used as a probe. (B) Southern-blot of genomic DNA of the ΔexoA-deletion mutant (strain sNH60) and WT using the probe indicated in (A). (C) D. flagrans wildtype and the ΔexoA-deletion strain were grown for 7 days at 28°C on PDA. (D) Virulence assay with the D. flagrans wildtype (WT), the ΔexoA-deletion mutant, the complementation strain (Re) and the ΔcyrA-deletion mutant. The strains were co-incubated with a mixed C. elgans N2 population on thin LNA-slides for 24 h at 28°C. After trap formation the N2 worms were washed off the slides and a synchronized population of Ban126 L4-larvae was added to the traps just before the microscopical observation. Pictures of single traps were taken every 5 minutes for 20 hours. The time from the capturing event to full paralysis of the nematodes was taken. The statistical significance was calculated using a student’s t-test (*** = p-value< 0.0001 n [WT] = 35, n [ΔexoA] = 34, n [Re] = 17, n [ΔcyrA] = 27). The error bar indicates the standard deviation. (E) Visualization of CyrA-mCherry in wild type (sNH65), the ΔexoA-mutant strain (sNH60) and the complementation strain (ΔexoA+exoA). The red box indicates the penetration area and the arrow points to the penetration site. (S2 and S3 Movies) (F) Enlargement of the interaction zone of the cyrA-mCherry expressing ΔexoA-mutant strain in (E). Initially, CyrA accumulates at the infection site after 10 minutes (star). The infection bulb is established after 30 minutes but CyrA does not accumulate in the bulb, instead some signals are visible at the outer rim of the entrance site. After 60 min trophic hyphae have colonized the nematode but the fusion protein is not visible. Scale bars = 10 μm.

Fig 6. The cyrA gene is required for full virulence.

(A) Scheme of the deletion strategy. The wild type cyrA-gene locus (upper panel) is replaced by the 1.8 kb hygromycin resistance cassette resulting in the ΔcyrA-mutant locus (lower panel). Southern-blot analysis using a 1 kb fragment from the LB as probe and the restriction enzyme PstI for the digestion of the genomic DNA. (B) Confirmation of the ΔcyrA deletion using Southern-blot analysis. Genomic DNA was digested with PstI, and the LB was used as probe. (C) The D. flagrans WT and the ΔcyrA-deletion strain (sNH11) were grown for seven days at 28°C on PDA. (D) Virulence assay with wild type (WT), the ΔcyrA-mutant strain and the strain re-complemented with the cyrA ORF (sNH27) under its native promoter (Rec.). Multiple traps were observed and the time from the capturing event to full paralysis was taken. Error bars indicate the standard deviation. A student’s t-test was performed for statistical analysis (*** = p-value < 0.0001; n [WT] = 29 n [KO] = 26 n [Rec] = 52).

Fig 7. CyrA accumulates in coelomocytes in C. elegans.

The cyrA gene fused to scarlet was expressed with (A, C, strain KIT02) and without (B, strain KIT04) its signal peptide sequence under the heat inducible promoter hsp-16.48. Expression was induced by heat shock at 37°C for 1 hour. (C) CyrA-Scarlet expressed with SP in the coelomocyte uptake deficient C. elegans strain GS2478 (strain KIT26). Scale bars = 100 μm. (D) CyrA-Scarlet was co-expressed with the coelomocyte marker unc-122(p)::GFP. Co-localization is shown in the enlargements.

Fig 8. CyrA expression in the C. elegans hypodermis reduces the life span.

(A) The cyrA gene fused to GFP was expressed with (strain KIT01) and without its signal peptide sequence (strain KIT23) in C. elegans N2 and GS2478 (Δcup-8, strains KIT31/KIT29) under the hypodermal and adult specific promoter of the col-19 gene. Scale bars = 100 μm. (B) Survival curves of N2 and GS2478 expressing cyrA with (strain KIT22/KIT28) and without SP sequence (strain KIT18/KIT30). The empty vector (EV) was expressed in both strains as a control. Lifespan assays were performed on NGM plates with 150 mM FudR at 20°C. P—values log-rank-test N2: EV v.s. no SP = 0.0001; EV v.s. +SP = 1.2E-06; P—values GS2478: EV v.s. no SP = 0.9; EV v.s. +SP = 0.5.