Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-β-defensin family

Abstract

Out of the 14 avian β-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian β-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to β-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed β-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-β-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

Keywords: NMR structure; alarmin; avian egg; avian influenza virus; defensin.

Fig. 1.

Amino acid sequences of Gga-AvBD11 and of the 2 derived peptides. Gga-AvBD11 precursor (accession NP_001001779) is a 104-amino acid protein encoded by 3 exons (residues overlapping the splice sites at exons 1 and 2 and exons 2 and 3 are given in green in the sequence). The signal peptide and the mature chain are highlighted in gray and purple, respectively. The position of cysteine residues and the spacing between cysteines are specified. The sequences of [1–40]Gga-AvBD11 and [41–82]Gga-AvBD11, delimited by the junction of exons 2 and 3 and further named N-ter and C-ter peptides, are highlighted in cyan and pink.

Fig. 2.

Multiple sequence alignment of AvBD11s. Protein sequences of AvBD11 of various bird species were retrieved from National Center for Biotechnology Information (NCBI) and Ensembl databases, aligned with Clustal, and drawn with JalView software (54). Sequences were ordered according to the classification of the International Ornithological Committee (IOC) World Bird List v9.2 (https://www.worldbirdnames.org/). Cysteine residues are highlighted in yellow. Other amino acids are colored according to the identity score, from light gray (low identity) to dark gray (high identity).

Fig. 3.

Sequence, cysteines bonding pattern, and 3D structure of (A) N-ter peptide [1–40]Gga-AvBD11 in cyan, (B) C-ter peptide [41–82]Gga-AvBD11 in pink, and (C) the full-length [1–82]Gga-AvBD11 in purple. In each locant from Top to Bottom are represented the peptide sequence with the cysteines bonding pattern in yellow, then the elements of secondary structure, and, finally, the 3D structure with disulfide bridges shown in yellow (drawn with PYMOL) (55).

Fig. 4.

Functional characterization of Gga-AvBD11 ([1–82]), its derived N-ter ([1–40]), and C-ter ([41–82]) peptides, and the combination [1–40]+[41–82]. (A) Antibacterial activities against L. monocytogenes and S. enterica sv. Enteritidis. Inhibition zones are shown as a function of molecule concentrations. Data are presented as means ± SEM of at least 4 independent experiments performed in duplicates. Representative pictures of inhibition zones are shown in Insets above the bar charts. (B) Antiparasitic activities against E. tenella sporozoites. (B, Top) Data represent means ± SEM of sporozoite viability for at least 3 independent experiments performed in duplicates. (B, Bottom) Scanning electron microscopy analysis of E. tenella sporozoites under control conditions or treated with Gga-AvBD11. (Scale bar, 1 µm.) (C) Relative ATP production as a measure for treatment/virus-induced cytotoxic or cytopathogenic effects in noninfected or H1N1 virus-infected CLEC213. Relative ATP production rates for treated and/or infected cells were calculated as fold changes with reference to mean control values (nontreated cells) set to 1. Shown are means ± SEM from a minimum of 2 independent experiments with 6 replicate samples. (D) H1N1 growth kinetics in Gga-AvBD11–treated CLEC213. CLEC213s were infected at an MOI of 0.01 and viral titers (PFU/mL) in cell culture supernatants at 12 h, 24 h, and 48 h pi were determined by conventional plaque assay on MDBK cells. Shown are means ± SEM from 1 experiment with duplicate samples. (E) Effects on invasion and viability of the human non–small-cell lung cancer cell line NCI-H460. Data are presented as the percentage of invasive cells or cell viability compared to those of control cells without any molecule added. Results are expressed as means ± SEM of at least 5 independent experiments performed at least in duplicates. (B–E) Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.