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

Author summary Pathogenic microorganisms are living at the expense of their host organisms and immediate killing of the host may be disadvantageous. Therefore, many bacterial or fungal pathogens developed an arsenal of small-secreted proteins during the colonization to modulate their host for instance to suppress its defense reactions. This allows a biotrophic phase, at least for some time. Some higher eukaryotic pathogens also live at the expense of their hosts but are often predators. In this case, quick killing is followed by digestion. In the case of predatory fungi, one could expect a similar situation, quick killing followed by digestion. However, the genome of such fungi encodes many putative small-secreted proteins, and we show here that one of them indeed appears to be secreted into the host and contributes to virulence. The protein is produced in the trapping devices of the fungus and especially in the penetration peg right after entering the nematode. Heterologous expression in Caenorhabditis elegans reduced the lifespan of the worms. This protein is to our knowledge the first characterized small-secreted protein in the predatory relationship between fungi and nematodes. 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.

Automated summary

Pathogenic microorganisms have different strategies to interact with their hosts, some modulate the host to obtain nutrients while others quickly kill and consume the host for energy. Predatory fungi produce a large number of small-secreted proteins to regulate the predation process, which is crucial for the predatory relationship between fungi and nematodes.