Chimeric Antibacterial Polypeptides for Resistant Gram-Negative Bacteria
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Solution Overview
Problem
Current antimicrobial treatments are ineffective against antibiotic-resistant Gram-negative bacteria due to the bacterial adaptation and the outer membrane barrier that prevents access to peptidoglycan-degrading enzymes.
Innovation Solution
A chimeric polypeptide combining a muralytic domain for degrading bacterial cell walls with a membrane traversing domain to cross the outer membrane, allowing the enzymatic activity to reach the peptidoglycan layer, thereby lysing the bacteria.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotics are used to treat bacterial infections, then bacterial growth is inhibited, but antibiotic-resistant bacteria develop rendering treatments ineffective
Solution Approach 1:
The invention divides the antimicrobial function into two separate domains: a membrane traversing domain (MTD) that handles outer membrane penetration, and a muralytic domain (MD) that performs cell wall degradation. This segmentation allows each domain to be optimized independently, with the MTD ensuring delivery across the outer membrane barrier and the MD providing broad-spectrum cell wall degradation activity against resistant bacteria.
Solution Approach 2:
The invention creates a composite polypeptide structure combining two functional domains with different properties: the MTD (derived from proteins like BPI, T4 lysozyme, or phage tail fibers) that provides membrane interaction and penetration capability, and the MD (from lysozymes, endolysins, or autolysins) that provides enzymatic cell wall degradation. This composite structure overcomes bacterial resistance by delivering the muralytic activity directly to the peptidoglycan layer through the outer membrane.
2Reliability
If peptidoglycan-degrading enzymes are applied to Gram-negative bacteria, then cell wall degradation occurs, but the outer membrane prevents enzyme access to the peptidoglycan layer
Solution Approach 1:
The membrane traversing domain acts as an intermediary that bridges the outer membrane barrier and the peptidoglycan layer. The MTD contains hydrophobic regions that interact with and traverse the outer membrane, while the muralytic domain extends into the periplasmic space to access and degrade the peptidoglycan. This intermediary structure enables the enzymatic activity to reach its target despite the protective outer membrane.
Solution Approach 2:
The invention adds a spatial dimension to enzyme delivery by creating a polypeptide with an extended structure that spans from the outer membrane surface through the periplasmic space to the peptidoglycan layer. The MTD provides the transmembrane dimension, while the MD provides the periplasmic extension dimension, creating a three-dimensional delivery pathway that overcomes the two-dimensional barrier of the outer membrane.
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 chimeric polypeptide effectively reduces the colony-forming units of Gram-negative bacteria by at least 50% in a CFU drop assay, demonstrating its antibacterial efficacy against resistant strains.
Implementation Method 1
a muralytic domain comprising a sequence of amino acids 737-875 of SEQ ID NO:2, or a variant thereof having at least 90% sequence identity to amino acids 737-875 of SEQ ID NO:2
Implementation Method 2
a membrane traversing domain comprising a sequence selected from the group consisting of: amino acids 16-39 of SEQ ID NO:4; amino acids 242-264 of SEQ ID NO:15; amino acids 242-271 of SEQ ID NO:17; amino acids 220-406 of SEQ ID NO:19; amino acids 220-400 of SEQ ID NO:21; amino acids 220-885 of SEQ ID NO:23
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A
AI summary
Provided herein are antibacterial compositions and methods of making and using the compositions.