Electron Donor Cytochrome b ₅ Is Required for Hyphal Tip Accumulation of Sterol-Rich Plasma Membrane Domains and Membrane Fluidity in Aspergillus fumigatus

Abstract

The electron donor cytochrome b5 (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 lack of Aspergillus fumigatus (A. 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 CybE locates at the endoplasmic reticulum by its C-terminus with two transmembrane regions. Therefore, lack of the C-terminus of CybE is able to phenocopy a cybE deletion. 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 correspond to tardiness of hyphal extension and hypersensitivity to low temperature in cybE deletion mutant. Additionally, overexpressing another electron donor-heme-independent P450 reductase (CPR) significantly rescued growth defects and recovered SRD accumulation in deletion of cybE almost to the wild-type level, suggesting CybE maintaining the growth and deposition of SRDs in hyphal tips attributes to its nature as an electron donor. Protein pull-down assays revealed that CybE probably participates in 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. 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 A. fumigatus is conducive to design new antifungal approach. The electron donor cytochrome b5 (CybE) plays a crucial role in maintaining the normal growth of A. fumigatus, however, the potential mechanism remains elusive. Herein, we characterized the molecular features of CybE and found the C-terminus with two transmembrane domains are required for its 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 b5 protein-CybE in A. fumigatus. CybE maintains the normal growth probably via supporting two crucial physiological processes, the SRD accumulation in hyphal tips and membrane fluidity. Therefore, our finding reveals the mechanisms underlying the regulatory effect of CybE on A. fumigatus growth and indicates that inhibition of CybE might be an effective approach for alleviating A. fumigatus infection.

Iron Deprivation Modulates the Exoproteome in Paracoccidioides brasiliensis

“…A very relevant finding in the present work was the upregulation of PbCyb5, secreted in response to Fe deprivation. Cyb5 and related enzymes are involved in multiple cellular processes, including sterol biosynthesis, maintenance of cell membranes, and azole resistance (Misslinger et al, 2017;Zhang et al, 2021). Cyb5 is also related to the regulation of Fe homeostasis in S. cerevisiae (Dap1) and A. fumigatus (CybE) (Craven et al, 2007;Misslinger et al, 2017).…”

Pig vaccination strategies based on enterotoxigenic Escherichia coli toxins

“…As F4/LT/STb and F18/STa/STb/Stx2e are the predominant ETEC pathotypes causing PWD in weaned pigs [ 57 ], Lu T. et al (2020) recently explored a novel epitope- and structure-based vaccinology platform called multiepitope-fusion-antigen (MEFA) for vaccine development [ 68 ]. As MEFA-based vaccine does not carry somatic antigens (somatic proteins, LPS), it is less likely to cause associated side effects.…”

Multifunctional in vitro, in silico and DFT analyses on antimicrobial BagremycinA biosynthesized by Micromonospora chokoriensis CR3 from Hieracium canadense

“…CYP51 is one of the members of the heme-containing cytochrome-P450 oxidoreductase enzymes found in the outer membranes of the endoplasmic reticulum of the cells of fungi and other eukaryotes, including humans 58 . It works by catalyzing the methylation of lanosterol 14α in order to biosynthesize ergosterol, which is involved in the structural integrity and permeability of the cell membrane, without which the fungal cells do not survive 59 . Therefore, it can be a potential candidate for both systematic and topical clinical antifungal drugs 60 .…”