Cordycepin and the entomopathogenic fungus Cordyceps militaris

Cordyceps militaris is a widespread entomopathogenic fungus found in Europe, Asia, and North America, with a large number of insect hosts, predominantly lepidopteran larvae (caterpillars). This species is well known for its production of the nucleoside analogue cordycepin (3’-deoxyadenosine). Co-produced with its protector molecule pentostatin, cordycepin is a polyadenylation inhibitor, via its active modified form as cordycepin triphosphate. Pentostatin protects cordycepin from degradation to 3’-deoxyinosine by inhibition of the enzyme adenosine deaminase. It has been shown to have anti-inflammatory effects and hence has been the subject of much pharmacological research. Until recently, very little was known about the role of cordycepin in the ecology of the fungus, or why its production is favoured by natural selection. There are also gaps in the understanding of the process of infection of the host by the fungus, which is directly followed by sexual development and the formation of stromata bearing sexual fruiting bodies and spores (ascospores). Understanding these areas could have implications for biological control of insect pests. Indeed, the related species Beauveria bassiana and Metarhizium anisopliae have been used as bioinsecticides, precluding the use of harmful chemical insecticides. Culture degeneration is a phenomenon defined previously as a reduction in the production of cordycepin by C. militaris. Experiments comparing a degenerated strain of an isolate of C. militaris with its parental control strain were performed, involving the use of gene expression analysis and metabolomics. Reduced cordycepin production in the degenerated strain was shown to be accompanied by declines in sexual development-related gene expression, and reduced production of other metabolites involved in the citrate cycle and purine metabolism. This suggested a link between cordycepin production, primary metabolism, and sexual development. We hypothesised that the production of cordycepin by C. militaris aids the infection of the insect by suppression of the host immune system, and that pentostatin, by providing molecular protection, enhances this effect. In a caterpillar infection assay system involving the injection of spores into the model species Galleria mellonella (greater wax moth) caterpillars, the lower cordycepin-producing degenerated strain was shown to produce a significantly-decreased pathogenic response, marked by reduced fungal growth in the host. When spores of the degenerated strain were supplemented by cordycepin and pentostatin, fungal emergence rates and levels significantly increased, restoring the infection performance of the degenerated strain to that of the parental control. Assays of insect gene expression were also performed, and cordycepin was demonstrated to suppress the upregulation of immune response genes in both Drosophila melanogaster Schneider 2 cells and G. mellonella haemolymph cells. Pentostatin enhanced the effects of low cordycepin concentrations in both models. These findings support the hypothesis that cordycepin has an important role in aiding insect infection by the fungus via immune suppression, and that the effect of cordycepin on host cell responses is maintained by pentostatin. Biosynthesis genes (Cns genes) for cordycepin and pentostatin are located in the same gene cluster. We hypothesised that cordycepin-pentostatin co-production was a rare trait, and its evolution had been resultant partly due to horizontal gene transfer between different species. This was due to the lack of cordycepin in other Cordyceps species, and genetic evidence of its production only found previously in two other, distantly-related species. Bioinformatics work involving tBLASTn searches through the sequenced genomes of over two and a half thousand fungal species uncovered evidence of homologous Cns gene clusters in five new species. This together with consideration of protein structures suggests that the development of cordycepin-pentostatin co-production has occurred by convergent evolution involving duplication and subfunctionalisation of genes involved in the purine synthesis pathway, and/or through horizontal gene transfer