Calcineurin signaling pathway influences Aspergillus niger biofilm formation by affecting hydrophobicity and cell wall integrity

Background: Biofilms, as a kind of fixed-cell community, can greatly improve industrial fermentation efficiency in immobilized fermentation, but the regulation process is still unclear, which restricts their application. Ca²⁺ was reported to be a key factor affecting biofilm formation. However, the effect of Ca²⁺ on biofilm structure and microbiology was yet only studied in bacteria. How Ca²⁺-mediated calcineurin signaling pathway (CSP) alters biofilm formation in bacteria and fungi has rarely been reported. On this basis, we investigated the regulation of CSP on the formation of biofilm in Aspergillus niger.

Results: Deletion of the key genes MidA, CchA, CrzA or CnaA in the CSP lowered the Ca²⁺ concentration in the mycelium to a different extent, inhibited the formation of A. niger biofilm, reduced the hydrophobicity and adhesion of spores, destroyed the cell wall integrity of hyphae, and reduced the flocculation ability of hyphae. qRT-PCR results showed that the expression of spore hydrophobic protein RodA, galactosaminogalactan (GAG) biosynthesis genes (uge3, uge5, agd3, gtb3), and α-1,3-glucan biosynthesis genes (ags1, ags3) in the ∆MidA, ∆CchA, ∆CrzA, ∆CnaA strains were significantly down-regulated compared with those of the wild type (WT). In addition, the transcription levels of the chitin synthesis gene (chsB, chsD) and β-1,3-glucan synthesis gene (FksA) were consistent with the change in chitin and β-1,3-glucan contents in mutant strains.

Conclusion: These results indicated that CSP affected the hydrophobicity and adhesion of spores, the integrity of mycelial cell walls and flocculation by affecting Ca²⁺ levels in mycelium, which in turn affected biofilm formation. This work provides a possible explanation for how CSP changes the formation of A. niger biofilm, and reveals a pathway for controlling biofilm formation in industrial immobilized fermentation.