Extraction and identification of extracellular mucilage proteins from the conidia of Hirsutella satumaensis

Abstract:

[Background] The surface mucilage layer of conidia plays an important role in the interaction between insects and fungi. Hirsutella satumaensis is a host-specific entomopathogenic fungus, and its conidia are covered with a mucilage layer. However, the composition of the mucilage remains unclear. [Objective] The extraction method of mucilage proteins from the conidia of H. satumaensis was optimized, and the extracted proteins were identified by mass spectrometry. [Methods] The extracellular mucilage was eluted with a low concentration of dithiothreitol (DTT) and the extraction conditions were optimized. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the extracted proteins concentrations and propidium iodide (PI) to detect the permeability of conidial membrane. Finally, the proteins from extracellular mucilage were identified by peptide enzymatic hydrolysis, LC-MS/MS, and protein comparative analysis. [Results] The optimized extraction condition was as follows: extraction of the conidia at a concentration of 5×10⁷ cells/mL with 2 mmol/L DTT at 4 ℃ for 24 h, and the cell membrane integrity was not affected after extraction. Seventy-seven proteins were identified from the extracellular mucilage of H. satumaensis conidia, most of which were small molecular secretory proteins, including unannotated proteins, chitinase, lipase, and other insect cuticle-degrading enzymes. [Conclusion] The extracellular mucilage of H. satumaensis has rich proteins involved in host recognition and penetration, which is associated with adhesion and recognition of the host insect.