Montano-Silva, P. | bioRxiv Science

bioRxiv2026

Are hyaluronic acid synthases widely encoded in fungi?

Hyaluronic acid (HA) is a biologically versatile polysaccharide synthesized by vertebrates and several microbial pathogens. To date, Cryptococcus neoformans CPS1p is the only reported hyaluronic acid synthase (HAS) in fungi, which is functionally related to bacterial HASs. Considering the phylogenetic and biochemical connection between chitin synthases (CHSs), essential for fungal cell wall synthesis, and HASs, it is reasonable to hypothesize the latter might be more common in fungi than expected. In this work, a comprehensive in silico survey of putative HASs in the Fungal Tree of Life was carried out. 68 putative HASs, mainly in Basidiomycota, were found, although other AI-inferred HASs were found among Ascomycota. Global fold and arrangement of essential amino acids were shared by all kingdoms HASs; however, fungal HASs showed additional exclusive conserved sequence signatures. Moreover, fungal HASs bore an only 3-helices transmembranal pore and their gating loop, which regulates the entrance of substrates to the catalytic site, was directly connected to an also exclusive intrinsically disordered (IDR) C-terminus. Phylogenetically, fungal HASs were found in a clade different to that of bacterial, animal and viral HASs, and all HASs shared the same ancestor with class VI CHSs. The atypical features of fungal HASs could influence the size and biological role of the HA they synthesize and also highlight potential regulatory differences among HASs at the gating loop configuration level. ImportanceDespite the report of CPS1p, the hyaluronic acid synthase (HAS) of Cryptococcus neoformans, the diversity, structural features and biochemical assets of fungal HASs remain unknown. Here, 68 putative fungal HASs were identified, mainly among Basidiomycota. Although their fold is similar to that of already characterized HASs, their transmembranal pore, integrated by only 3 helices, and their atypical gating loop configuration, suggest they could be also differently regulated, influencing size and function of HA they synthesize.

biochemistry↗

bioRxiv2024

Cell wall-resident PIR proteins show an inverted architecture in Neurospora crassa, but keep their role as wall stabilizers

Proteins with internal repeats (PIRs) are the second most abundant class of fungal cell wall resident proteins. In yeasts, PIRs preserve the wall stability under stressful conditions. They are characterized by conserved N-terminal amino acid sequences repeated in tandem (PIR domains), and a Cys-rich C-terminal domain. Despite PIRs have been inferred in several filamentous fungi genomes, they have not been studied beyond yeasts. In this work, PIRs diversity, evolution and biological role, focused on a new PIRs class, were addressed. Bioinformatic inference of PIRs in fungi indicated they were an innovation in Ascomycota. Predicted PIRs clustered in two main groups: classical yeasts PIRs (N-terminal PIR domains; C-terminal Cys-rich domain), and PIRs from filamentous fungi with an inverted architecture (N-terminal Cys-rich domain; C-terminal PIR domains), which could harbor additional GPI-signals. As representatives of the second group, Neurospora crassa (Nc) PIR-1 (NCU04033) and PIR-2 (NCU07569) were studied. Confocal microscopy of eGFP-labeled PIR-1 and PIR-2 revealed they accumulate in apical plugs; additionally, PIR-1 requires the Kex2 processing site for correct maturation, and its predicted C-terminal GPI modification signal resulted functional. Moreover, Nc {Delta}pir-1 and {Delta}pir-2 single mutants showed a growth rate similar to that of Nc WT, but the double mutant Nc {Delta}pir-1/{Delta}pir-2 grew significatively slower. Similarly, Nc {Delta}pir-1 and Nc {Delta}pir-2 were mildly sensitive to calcofluor white, although Nc {Delta}pir-1/{Delta}pir-2 double mutant was severely impaired. Despite the inverted architecture of PIR-1 and PIR-2, they resulted in cell wall stabilizers as classical yeast PIRs.

microbiology↗