Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus . It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A . fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species. Fungal cell wall associated exopolymer GAG was first identified from Aspergillus niger decades ago and has been well characterized in terms of its biosynthetic mechanism, pathological and biological functions in Aspergillus fungi, especially the human pathogen A . fumigatus . With the accomplishment of diverse fungal genomes, the conserved GAG biosynthetic gene cluster is found to patchily distribute in fungal species beyond Aspergilli. However, until this study, the contribution of the clustered genes to GAG production has not been investigated in any non- Aspergillus fungi. We performed gene deletions and functional characterizations of GAG in an insect pathogenic fungus Metarhizium robertsii , and found that the partially acetylated GAG can be produced by the fungus to contribute to fungal virulence during topical infection of insect hosts. This exopolymer can be produced on germ tubes, mycelia and appressoria to mediate cell adherence, cuticle penetration, biofilm formation, mycelium aggregation in liquid culture and in particular, appressorium mucilaginous matrix formation. In addition, it was found that loss of GAG production could alter appressorium protein expressions including the enzymes involved in degrading insect cuticles. Our data advance the understanding of GAG biosynthesis and functions in an animal pathogenic fungus that is highly divergent from Aspergilli.