Studies On The Molecular Design And Function Of Peptide From Small Open Reading Frames In Ciona Intestinalis

Antibiotic resistance has become a global public health problem, thus there is aneed to develop a new class of antibiotics. Natural antimicrobial peptides have got anincreasing attention as potential therapeutic agents.Antimicrobial peptides are smallcationic peptides with broad antimicrobial activity and play an important role in theinnate host defense mechanisms of most living organisms including plants, insects,amphibians and mammals against microbial attack.Even though they possess adifferent mode of action compared to traditional antibiotics, antimicrobial peptidescould not go into the drug markets because of problems in application such as toxicity,susceptibility to proteolysis, manufacturing cost, size, and molecular size.Nevertheless,antimicrobial peptides can be new hope in developing novel, effective and safetherapeutics without antibiotic resistance. Thus, it is necessary to discover newantimicrobial sources in nature and study their structures and physicochemicalproperties more in depth.Though being able to encode various kinds of bioactive peptides, small openreading frames (sORFs) are poorly annotated in many genomic data. The present studywas conducted to evaluate the potential of sORFs in encoding antimicrobial peptides(AMPs) in the basal chordate model Ciona intestinalis.About4.8M genomic sequencewas first retrieved for sORFs mining by the program sORFfinder, then the sORFs weretranslated into amino acid sequences for AMP prediction via CAMP server, andthereafter, ten putative AMPs were selected for expression and antimicrobial activityvalidation. In total, over180peptides deduced from the sORFs were predicted to beAMPs. Among the ten tested peptides, six were found to have significant expressed sequence tag matches, providing strong evidence for gene expression;five were provedto be active against the bacterial strains. These results indicate that many sORFs inC.Intestinalis genome contain AMP information. This work can serve as an importantinitial step to investigate the role of sORFs in the innate defense of C. Intestinalis.In the present study, a19-aa linear cationic α-helical peptide identified fromCiona intestinalis was used as the framework to study the role of each structuralelement on the antimicrobial activity. A series of N-truncated, C-terminally amidatedand amino acid-substituted analogs were synthesized, and their antimicrobial activitywas examined. C-terminal amidation increased the antibacterial activity against mosttested strains. Deletion of the N-terminal random coil region also could increase theantibacterial activity. The substitution of serine for alanine increased activity againstE.coli, S.mutan and M. tetragenus by2~8fold, whereas substitution of proline led toan apparent decline of the activity. Increasing the net positive charge by lysinesubstitution only improved the activity against partial strains. A systemic analysis onthe amphipathicity of our analogs showed that no significant linear relationshipbetween antibacterial activity and amphipathicity could be found, which indicated thata balance of these factors should be required for retention of high antimicrobial activity.Our findings provide important information in designing new antimicrobial peptideswith promising activity.