Electron Donor Cytochrome b5 Is Required for Hyphal Tip Accumulation of Sterol-Rich Plasma Membrane Domains and Membrane Fluidity in Aspergillus fumigatus

The electron donor cytochrome b(5) (CybE/Cyb5) fuels the activity of the ergosterol biosynthesis-related P450 enzymes (P450s) by providing electrons to P450s to promote ergosterol biosynthesis. Previous studies reported that a lack of Aspergillus fumigatus CybE reduces the proportion of ergosterol in total sterols and induces severe growth defects. However, the molecular characteristics of CybE and the underlying mechanism for CybE maintaining A. fumigatus growth remain poorly understood. Here, we found that the C terminus of CybE, with two transmembrane domains, is located at the endoplasmic reticulum. Therefore, a strain lacking the C terminus of CybE shows a defective growth phenotype similar to that of a cybE deletion strain. Notably, cybE deletion reduced the accumulation of the sterol-rich plasma membrane domains (SRDs; the assembly platform of polarity factors/cell end markers and growth machinery) in hyphal tips and decreased membrane fluidity, which corresponds to tardiness of hyphal extension and hypersensitivity to low temperature in the cybE deletion mutant. Additionally, overexpressing another electron donor, a heme-independent P450 reductase (cytochrome P450 reductase [CPR]), significantly rescued growth defects and recovered SRD accumulation in the cybE deletion mutant almost to the wild-type level, suggesting that CybE maintaining the growth and deposition of SRDs in hyphal tips is attributed to its nature as an electron donor. Protein pulldown assays revealed that CybE probably participates in the metabolism and transfer of lipids, construction of cytoskeleton, and mitochondria-associated energy metabolism to maintain the SRD accumulation in hyphal tips, membrane fluidity, and hyphal extension. The findings in this study give a hint that inhibition of CybE may be an effective strategy for resisting the infection of the human pathogen A. fumigatus. IMPORTANCE Investigating the knowledge of the growth regulation in the human opportunistic pathogen Aspergillus fumigatus is conducive to designing a new antifungal approach. The electron donor cytochrome b, (CybE) plays a crucial role in maintaining the normal growth of A. fumigatus; however, the potential mechanism remains elusive. Here, we characterized the molecular features of CybE and found that the C terminus with two transmembrane domains is required for its endoplasmic reticulum (ER) localization and functions. In addition, we demonstrated that CprA, an electron donor-heme-independent P450 reductase, provides a reciprocal function for the missing cytochrome b(5) protein-CybE in A. fumigatus. CybE maintains the normal growth probably via supporting two crucial physiological processes, the sterol-rich plasma membrane domain (SRD) accumulation in hyphal tips and membrane fluidity. Therefore, our findings reveal the mechanisms underlying the regulatory effect of CybE on A. fumigatus growth and indicate that inhibition of CybE might be an effective approach for alleviating A. fumigatus infection.

Automated summary

The electron donor cytochrome b(5) plays a crucial role in promoting ergosterol biosynthesis and maintaining the growth of Aspergillus fumigatus. Inhibition of CybE may be an effective strategy for resisting the infection of A. fumigatus.