Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio

Abstract

The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1–3 (a novel lymphocyte antigen 6 (Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1–3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1–3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. We also showed that recombinant proteins Ly2.1–3 (rLy2.1–3) were able to bind to the Gram-negative bacteria A. hydrophila, E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1–3 could specifically bind to LPS. Finally, we found that Ly2.1–3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1–3 can function as AMPs.

A cell atlas of microbe-responsive processes in the zebrafish intestine

“…Removal of the microbiome had cluster-specific impacts on several immune regulators. For example, microbiome elimination resulted in significantly diminished expression of the putative LPS-binding molecule and antimicrobial peptide ly97.2 (Liu et al, 2017;Wang et al, 2016) in goblet cell clusters one and two, as well as the inflammation mediators irg1l (Hall et al, 2014;van Soest et al, 2011) andlect2l (Gonc ¸alves et al, 2012) in goblet cell cluster one. In contrast, GF growth led to diminished expression of interferon alpha inducible protein 27 (IFI27) orthologs across all goblet cells, whereas absence of the microbiome exclusively attenuated expression of CCL19 orthologs in cluster one cells (Figure 3D).…”

A Cell Atlas of Microbe-Responsive Processes in the Zebrafish Intestine

“…Removal of the microbiome had cluster-specific impacts on several immune regulators. For example, microbiome elimination resulted in significantly diminished expression of the putative LPS-binding molecule and antimicrobial peptide ly97.2 (Liu et al, 2017;Wang et al, 2016), as well as the inflammation mediators irg1l (Hall et al, 2014;van Soest et al, 2011) and lect2l (Gonçalves et al, 2012) in goblet cell clusters 1 and 2.…”

“…Zebrafish larvae have emerged as a valuable tool to identify key regulators of host-microbe interactions (Brugman, 2016;Flores et al, 2020;López Nadal et al, 2020). Zebrafish embryos develop within a protective chorion that shields them from environmental microbes up to forty-eight hours post fertilization (hpf).…”

Improving the Activity of Antimicrobial Peptides Against Aquatic Pathogen Bacteria by Amino Acid Substitutions and Changing the Ratio of Hydrophobic Residues

“…To determine whether AMPs are cytotoxic to murine RAW264.7, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed as described by Cui et al (2016) . The MTT assay can be used to detect cellular activity ( Liu et al, 2017 ). RAW264.7 cells were suspended in serum-free DMEM, and 100 μL cell suspension (1 × 10 6 cells/mL) was placed in a 96-well plate and cultured at 37°C with 5% CO 2 for 2 h. AMPs were added, producing final concentrations of 12.5, 25, and 50 μg/mL.…”