Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC

Abstract

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical β-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

The membrane activity of the amphibian Temporin B peptide analog TB_KKG6K sheds light on the mechanism that kills Candida albicans

“…Octenidine yielded an IC90 of 1 µM with 1×10 4 cells/mL and 2 µM with 1×10 6 cells/mL. As described previously (14), the peptide PCγ C-terminal (amino acid sequence: CGGASCRG; MW 709.8 Da), which derives from the C-terminal part of the Penicillium chrysogenum antifungal protein C (PAFC), was found to be inactive against C. albicans. No fungal growth inhibition at the administered peptide concentration (0-32 µM) was detected.…”

“…TB_KKG6K, and the PCγ C-terminal (amino acid sequence: CGGASCRG; MW 709.8 Da; 14) were synthesized on solid phase, using standard protocols for the Fmoc chemistry and were then purified by reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by electrospray ionization-mass spectrometry (ESI-MS) as described previously (7, 14). For fluorescence based analyses, the TB analog was synthesized and conjugated to the 6-aminohexanoic acid linker first and then to the green fluorophore fluorescein isothiocyanate (FITC) as described (74).…”

“…The IC 90 of the TB analog, octenidine (N,Nʹ-(1,10 decanediyldi-1[4H]-pyridinyl-4-ylidene)-bis-(1-octanamine) dihydrochloride), Schülke and Mayr GmbH, Vienna, Austria), and PCγ C-terminal (14) was determined for C. albicans using broth microdilution assays carried out in 96-well microtiter plates (Nunclon Delta, Thermo Fisher Scientific, Waltham, MA, USA). The assay was performed with 1×10 4 cells/mL and 1×10 6 cells/mL in 0.05×PDB.…”

The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans

“…Peptide synthesis and protein expression. TB_KKG6K and PCg C-terminal were synthesized on solid phase, using standard protocols for the 9-fluoroenylmethoxy chemistry and were then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed by electrospray ionization-mass spectrometry (ESI-MS) as described previously (7,14). For fluorescence-based analyses, the TB analog was synthesized and conjugated to the 6-aminohexanoic acid linker first and then to the green fluorophore FITC as described elsewhere (77).…”

Development of a FungalBraid Penicillium expansum‐based expression system for the production of antifungal proteins in fungal biofactories

“…Similar to other CRPs, AFPs contain a conserved γ‐core motif (Yount and Yeaman, 2004 ) and are coded with a signal peptide (SP) at the N‐termini that includes a pre‐sequence involved in AFP secretion to the extracellular space and a pro‐sequence (SP‐pro sequence) that has been predicted to inactivate the protein until cleavage (Marx et al ., 1995 ). AFPs exhibit potent antifungal activity and different mechanisms of action against opportunistic human, animal, plant and foodborne pathogenic fungi (Marx et al ., 2008 ; Hegedüs and Marx, 2013 ; Delgado et al ., 2016 ; Tóth et al ., 2020a ; Czajlik et al ., 2021 ; Martínez‐Culebras et al ., 2021 ).…”