Mining Amphibian and Insect Transcriptomes for Antimicrobial Peptide Sequences with rAMPage

Abstract

Antibiotic resistance is a global health crisis increasing in prevalence every day. To combat this crisis, alternative antimicrobial therapeutics are urgently needed. Antimicrobial peptides (AMPs), a family of short defense proteins, are produced naturally by all organisms and hold great potential as effective alternatives to small molecule antibiotics. Here, we present rAMPage, a scalable bioinformatics discovery platform for identifying AMP sequences from RNA sequencing (RNA-seq) datasets. In our study, we demonstrate the utility and scalability of rAMPage, running it on 84 publicly available RNA-seq datasets from 75 amphibian and insect species—species known to have rich AMP repertoires. Across these datasets, we identified 1137 putative AMPs, 1024 of which were deemed novel by a homology search in cataloged AMPs in public databases. We selected 21 peptide sequences from this set for antimicrobial susceptibility testing against Escherichia coli and Staphylococcus aureus and observed that seven of them have high antimicrobial activity. Our study illustrates how in silico methods such as rAMPage can enable the fast and efficient discovery of novel antimicrobial peptides as an effective first step in the strenuous process of antimicrobial drug development.

Associating Biological Activity and Predicted Structure of Antimicrobial Peptides from Amphibians and Insects

“…A total of 1137 putative AMPs were identified by rAMPage, and 88 peptides were selected for synthesis using three selection criteria: “Species Count”, “Insect Peptide”, or “AMPlify Score” (see Methods). The shortlist of 88 putative AMPs include 21 peptides described previously [ 22 ]. We investigated, in three independent experiments per peptide, the antimicrobial activity of the 88 peptides against Escherichia coli ATCC 25922, a clinical strain of Salmonella enterica serovar Enteritidis, and Staphylococcus aureus ATCC 29213.…”

“…In the present study, we characterized 51 AMP candidates originally derived from a bioinformatics scan of RNA-seq data from amphibians and insects using rAMPage [ 22 ]. Fourteen of these had high antimicrobial activity to at least one of the bacterial species tested.…”

“…Peptide sequences were discovered using the Rapid Antimicrobial Peptide Annotation and Gene Estimation (rAMPage) pipeline v1.0 [ 22 ], which is publicly available on Github ( , accessed on 14 February 2021). rAMPage is a homology-based pipeline that uses RNA-seq reads as input to generate putative AMPs for downstream in vitro testing.…”

“…Here, we studied the relationship between predicted AMP structure and observed antimicrobial activity. Specifically, we used ColabFold to predict the 3D structure of a list of 88 putative amphibian and insect AMPs discovered using rAMPage, a homology-based bioinformatic pipeline for in silico discovery of AMPs using RNA-seq reads [ 22 ]. We examined the antimicrobial activity of these peptides against two Gram-negative and one Gram-positive bacteria in the WHO’s list of priority pathogens [ 23 ].…”

The Expression of Antibacterial Peptide Turgencin A in Pichia pastoris and an Analysis of Its Antibacterial Activity

“…They consist of between 10 and 50 amino acids and are now candidates for addressing the challenges associated with antibiotic resistance [ 8 , 9 ]. Antimicrobial peptides are derived from a wide range of sources, and as many as 3000 have been identified and isolated from mammals [ 10 ], amphibians [ 11 ], marine animals [ 12 ], insects [ 13 ], and plants [ 14 , 15 ]. Antimicrobial peptides can rupture bacteria by forming ion channels in cell membranes [ 16 ], piercing pores, and promoting membrane disintegration.…”