Lysin-AMP Polypeptide Constructs for Gram-Negative Bacteria
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Solution Overview
Problem
Current antibacterial agents are ineffective against multi-drug resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, due to the presence of an outer membrane that limits access to the peptidoglycan layer, and existing lysins exhibit low in vivo activity.
Innovation Solution
Development of novel polypeptide constructs combining lysins with antimicrobial peptides (AMPs) that can penetrate the outer membrane of Gram-negative bacteria, including specific lysin-AMP polypeptide constructs like GN76, GN4, and Chp1, which are designed to inhibit bacterial growth and kill Pseudomonas aeruginosa even in human serum.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If lysins are used to degrade peptidoglycan, then bacterial cell wall is degraded resulting in osmotic lysis, but lysins cannot effectively access the peptidoglycan layer due to the outer membrane barrier in Gram-negative bacteria
Solution Approach 1:
The patent introduces artilysin as an intermediary molecule that bridges the gap between the outer membrane barrier and the lysin enzyme. The artilysin construct comprises a lysin enzyme fused to an outer membrane translocation domain, allowing it to mediate passage through the outer membrane and deliver the lysin activity to the peptidoglycan layer beneath.
Solution Approach 2:
The patent creates a composite antibacterial agent by fusing the lysin enzyme with the outer membrane translocation domain. This composite structure combines the membrane-penetrating capability of the translocation domain with the peptidoglycan-degrading activity of the lysin, enabling the single molecule to overcome the outer membrane barrier and access its target.
2Object-affected harmful factors
If artilysins are developed to translocate across the outer membrane, then access to peptidoglycan is improved, but in vivo activity remains low
Solution Approach 1:
The patent systematically varies key parameters of the artilysin construct including the choice of lysin enzyme, the structure of the translocation domain, and the length of the linker region connecting them. By optimizing these parameters, the patent enhances both outer membrane penetration efficiency and in vivo stability, thereby improving overall in vivo activity against Gram-negative bacteria.
3Adaptability or versatility
If novel antimicrobial agents are developed to target Gram-negative bacteria, then treatment options for MDR P. aeruginosa are expanded, but the complexity of overcoming resistance mechanisms increases
Solution Approach 1:
The patent divides the antibacterial function into two distinct segments: the outer membrane translocation domain and the lysin enzyme domain. This segmentation allows each component to be independently optimized for its specific function while maintaining overall simplicity. The modular design facilitates easier engineering and deployment compared to entirely novel complex molecules.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The lysin-AMP polypeptide constructs effectively inhibit bacterial growth, reduce bacterial populations, and kill Pseudomonas aeruginosa, enhancing the efficacy of antibiotics and maintaining activity in human blood matrices.
Implementation Method 1
Lysins are cell wall peptidoglycan hydrolases, which act as 'molecular scissors' to degrade the peptidoglycan meshwork responsible for maintaining cell shape and for withstanding internal osmotic pressure. Degradation of peptidoglycan results in osmotic lysis.
Implementation Method 2
Modified lysins ('artilysins') have also been developed. These agents, which contain lysins fused to specific α-helical domains with polycationic, amphipathic, and hydrophobic features, are capable of translocating across the OM.
Data Source
AI summary
The present disclosure is directed to lysin-AMP polypeptide constructs, isolated lysin polypeptides, and pharmaceutical compositions comprising the isolated polypeptides and/or lysin-AMP polypeptide constructs. Methods of using the lysin-AMP polypeptide constructs, isolated lysin polypeptides and pharmaceutical compositions are also herein provided. In addition, isolated polynucleotides encoding the lysin-AMP polypeptide constructs and isolated lysin polypeptides are disclosed herein.