LYSINS AS POTENTIAL ANTIBIOTICS: IDENTIFICATON OF CANDIDATE LYSINS WITH PROPOSED ANTIMICROBIAL PEPTIDE-LIKE PROPERTIES THAT TARGET PSEUDOMONAS AERUGINOSA

Abstract

Introduction The discovery of bacteriophages that have specific antimicrobial activity against bacteria was prior to the turn of the 20th century. More recently, viral components of bacteriophages, such as lysin, have been described for their lytic activity against antibiotic resistant bacteria. Given the widespread antibiotic resistance in the most common bacterial pathogens, this project aims to better understand how lysins kill bacteria and to identify new lysin candidates that may function as better antimicrobials. Due to an almost infinite number of bacteriophages on the planet, obtaining specific lysins for application in clinical research for use against antibiotic resistant pathogens requires further understanding of their structure and function. Lysins have demonstrated antimicrobial activity in the laboratory setting, and when applied in animal models of infection. Lysins are enzymes that are produced and assembled inside a host bacterium during phage infection. These enzymes typically degrade the bacterial cell wall, with access from the interior of the infected cell. We hypothesized that the C-terminus of lysins from Gram-negative phages will have more antimicrobial peptide properties than Gram-positive phage lysins, which should promote enhanced killing of Gram-negative bacteria. Lysins that can disrupt the outer membrane can gain access to the cell wall from both sides of the membrane. Methods Using the PhaLP database of predicted phage proteins, we recovered all lysin proteins from the model Gram-negative and Gram-positive pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The total number of lysins was reduced by removing all duplicate lysin sequences, as identical lysins were common. Next, we examined the hydrophobicity, net charge, as well as the arginine to tryptophan frequency in the C-terminal 50 amino acids of all lysins, to determine if there was a difference between lysins from Gram-negative and Gram-positive bacteria.

Results This analysis indicated that the C-terminal portion of lysins from P. aeruginosa contained more hydrophobic amino acids, higher net positive charge and a higher ratio of lysin/arginine amino acids when compared to S. aureus lysins. Secondary structure analysis of priority lysins had predicted alpha helical structure, which is characteristic of antimicrobial peptides. Conclusion By examining the C-terminal domain of phage lysins, we identified more features of antimicrobial peptides in lysins that originate in phages that kill Gram-negative bacteria. Two primary lysins were identified as the lead candidates, as well as six additional candidate lysins for future experimental research to test their antimicrobial efficiency. This work supports and extends the observations that lysins contain domains in their C-terminus that might function to disrupt the outer membrane of Gram-negative bacteria, thereby enhancing the antimicrobial activity of phage lysins. With this potentially new domain, lysins can access the cell wall from the interior or exterior of a Gram-negative bacterium, and therefore should result in stronger antimicrobial activity. This research has identified antimicrobial enzyme candidates to combat the inherently antibiotic resistant Gram-negative pathogen, P. aeruginosa.