Screening and Functional Analyses of Novel Cecropins from Insect Transcriptome

Abstract

Antibiotic resistance is a significant and growing threat to global public health. However, antimicrobial peptides (AMPs) have shown promise as they exhibit a broad spectrum of antibacterial activities with low potential for resistance development. Insects, which inhabit a wide range of environments and are incredibly diverse, remain largely unexplored as a source of novel AMPs. To address this, we conducted a screening of the representative transcriptomes from the 1000 Insect Transcriptome Evolution (1KITE) dataset, focusing on the homologous reference genes of Cecropins, the first identified AMPs in insects known for its high efficiency. Our analysis identified 108 Cecropin genes from 105 insect transcriptomes, covering all major hexapod lineages. We validated the gene sequences and synthesized mature peptides for three identified Cecropin genes. Through minimal inhibition concentration and agar diffusion assays, we confirmed that these peptides exhibited antimicrobial activities against Gram-negative bacteria. Similar to the known Cecropin, the three Cecropins adopted an alpha-helical conformation in membrane-like environments, efficiently disrupting bacterial membranes through permeabilization. Importantly, none of the three Cecropins demonstrated cytotoxicity in erythrocyte hemolysis tests, suggesting their safety in real-world applications. Overall, this newly developed methodology provides a high-throughput bioinformatic pipeline for the discovery of AMP, taking advantage of the expanding genomic resources available for diverse organisms.

Keywords: 1KITE; Cecropin; antimicrobial peptide; genomics; insect; transcriptomics.

Figure 1

The Maximum Likelihood tree of identified novel Cecropins. The sequences obtained from varied insect orders are shaded in different colors. The sequences identified in this study are marked in red. The genes for antimicrobial activity assays, namely Peyn-cec, Pvul-cec, and Tpen-cec, are highlighted in bold.

Figure 2

Nucleic and amino acid sequences encoding Peyn-cec, Pvul-cec, Tpen-cec, and Hcec-cecB. Nucleotide positions are indicated by numbers. Putative signal peptides are enclosed in boxes. The synthesized mature peptides are indicated by underscores, and the stop codon is indicated by an asterisk symbol (*).

Figure 3

Hydrophobicity/hydrophilicity analysis and three-dimensional structure of the synthesized peptides of Peyn-cec, Pvul-cec, Tpen-cec, and Hcec-cecB. (A) The Kyte and Doolittle hydropathicity score. A higher score indicates higher hydrophobicity. (B) The helical wheel projection. Residues are numbered from the N-terminus. Circles: hydrophilic residues; diamonds: hydrophobic residues; triangles: potentially negatively charged residues; pentagons: potentially positively charged residues. Hydrophobicity is also color coded. Green: the most hydrophobic residues. Yellow: zero hydrophobicity. Red: the most hydrophilic residues. Light blue: the potentially charged residues. (C) Theoretical three-dimensional structure projection of peptides.

Figure 4

The circular dichroism (CD) spectra of peptides in different environments. Different colors represent different peptides. Red line: Peyn-cec; Orange line: Pvul-cec; Green line: Tpen-cec; Deepsky; Blue line: Hcec-cecB. The peptides were dissolved in 10 mM phosphate-buffered saline (PBS, pH 7.4) (A); 20 mM sodium dodecyl sulfate (SDS) (B); and 50% trifluoroethanol (TFE) (C).

Figure 5

Antimicrobial activities against E. coli (A), P. aeruginosa (B), S. aureus (C), and M. luteus (D). 1: Ampicilin; 2: Peyn-cec; 3: Pvul-cec; 4: Tepn-cec; 5: Hcec-cecB; 6: PBS (negative control). A total of 20 µg of peptide solution or ampicillin was added into each hole.

Figure 6

Membrane permeabilization of E. coli by Peyn-cec, Pvul-cec, and Hcec-cecB. E. coli (1 × 10⁶ CFU/mL) were incubated with 4 µg/mL of antimicrobial peptides for 1 h at 37 °C. The bacteria were stained with propidium iodide (PI) and visualized under an optical microscope (A) and a fluorescence microscope (B).