Assessing the effects of domain truncation on the antibacterial activity of endolysins
(2026) KBTM05 20261Biotechnology
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
- Abstract
- Tuberculosis and nontuberculous mycobacterial infections remain a major global health challenge due to rising antimicrobial resistance, prolonged treatment regimens, and the complex, poorly permeable structure of the mycobacterial cell envelope. This envelope limits antibiotic penetration and contributes to intrinsic drug tolerance. Bacteriophage-derived lytic enzymes, including Lysin A (LysA) and Lysin B (LysB), are emerging as a potential alternative due to their ability to degrade essential components of the mycobacterial cell envelope.
This study evaluated recombinant chimeric enzymes, referred to as mycolysins, containing truncated LysA, full-length LysB, outer membrane permeabilizers (OMPs), protein transduction domains (PTDs) and... (More) - Tuberculosis and nontuberculous mycobacterial infections remain a major global health challenge due to rising antimicrobial resistance, prolonged treatment regimens, and the complex, poorly permeable structure of the mycobacterial cell envelope. This envelope limits antibiotic penetration and contributes to intrinsic drug tolerance. Bacteriophage-derived lytic enzymes, including Lysin A (LysA) and Lysin B (LysB), are emerging as a potential alternative due to their ability to degrade essential components of the mycobacterial cell envelope.
This study evaluated recombinant chimeric enzymes, referred to as mycolysins, containing truncated LysA, full-length LysB, outer membrane permeabilizers (OMPs), protein transduction domains (PTDs) and variable linker sequences. The aim was to assess how truncation and modular construct design affected antimicrobial activity, expression, and solubility. Constructs were expressed in Escherichia coli BL21(DE3), followed by initial screening against Mycobacterium smegmatis using OD600 measurements and resazurin microtiter assays. Selected enzymes were further purified, analysed by SDS-PAGE and tested against pathogenic mycobacteria, including M. tuberculosis H37Ra, M. avium, M. intracellulare and M. abscessus.
Several constructs were successfully expressed, but expression did not consistently result in soluble or active protein after scale-up. Solubility, aggregation during purification, protein loss during desalting and assay interference were major limitations. Activity against M. smegmatis was generally weak after purification, whereas selected constructs showed measurable inhibition against pathogenic mycobacterial strains. Three enzymes exhibited relatively low MIC values against M. tuberculosis H37Ra, ranging from 68 to 137 µg/mL. These results suggest that LysA truncation and chimeric mycolysin design can be compatible with retained antimicrobial activity, but further optimization of expression, purification, stability and quantitative antimicrobial testing is required before therapeutic potential can be reliably assessed. (Less) - Popular Abstract
- Mycobacteria are bacteria that can cause serious and long-lasting infections, such as tuberculosis and lung infections caused by nontuberculous mycobacteria. These infections are often difficult to treat, partly because mycobacteria have a thick, waxy outer layer that prevents many antibiotics from entering and killing the cell. With antibiotic resistance becoming a global issue, new treatment strategies are needed to fight these infections.
This thesis investigated engineered enzymes called mycolysins. Mycolysins are inspired by proteins from bacteriophages, which are viruses that naturally infect bacteria. Mycolysins are designed to damage the protective outer layer of mycobacteria and may therefore be useful as future antibacterial... (More) - Mycobacteria are bacteria that can cause serious and long-lasting infections, such as tuberculosis and lung infections caused by nontuberculous mycobacteria. These infections are often difficult to treat, partly because mycobacteria have a thick, waxy outer layer that prevents many antibiotics from entering and killing the cell. With antibiotic resistance becoming a global issue, new treatment strategies are needed to fight these infections.
This thesis investigated engineered enzymes called mycolysins. Mycolysins are inspired by proteins from bacteriophages, which are viruses that naturally infect bacteria. Mycolysins are designed to damage the protective outer layer of mycobacteria and may therefore be useful as future antibacterial treatments.
In this project, several mycolysin variants were produced in laboratory bacteria, purified, and tested against different mycobacterial species, including clinically relevant strains. Some variants showed promising antibacterial activity, particularly against tuberculosis-related mycobacteria. However, many of the enzymes were difficult to produce in a soluble and pure form. This is an important challenge to overcome, because reliable production and purification are necessary before the enzymes can be tested more thoroughly and developed further.
The results suggest that mycolysins have potential as enzyme-based antibacterial tools against mycobacterial infections. However, more work is needed to improve their production, stability, and activity before they can be considered for medical use. In the future, they may also be combined with other therapies to make treatments more effective against difficult mycobacterial pathogens. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/student-papers/record/9236430
- author
- Jamal, Husam Naseer Jamal LU
- supervisor
- organization
- course
- KBTM05 20261
- year
- 2026
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Endolysins, Antimycobacterial agents, Biotechnology
- language
- English
- id
- 9236430
- date added to LUP
- 2026-06-12 14:40:30
- date last changed
- 2026-06-12 14:40:30
@misc{9236430,
abstract = {{Tuberculosis and nontuberculous mycobacterial infections remain a major global health challenge due to rising antimicrobial resistance, prolonged treatment regimens, and the complex, poorly permeable structure of the mycobacterial cell envelope. This envelope limits antibiotic penetration and contributes to intrinsic drug tolerance. Bacteriophage-derived lytic enzymes, including Lysin A (LysA) and Lysin B (LysB), are emerging as a potential alternative due to their ability to degrade essential components of the mycobacterial cell envelope.
This study evaluated recombinant chimeric enzymes, referred to as mycolysins, containing truncated LysA, full-length LysB, outer membrane permeabilizers (OMPs), protein transduction domains (PTDs) and variable linker sequences. The aim was to assess how truncation and modular construct design affected antimicrobial activity, expression, and solubility. Constructs were expressed in Escherichia coli BL21(DE3), followed by initial screening against Mycobacterium smegmatis using OD600 measurements and resazurin microtiter assays. Selected enzymes were further purified, analysed by SDS-PAGE and tested against pathogenic mycobacteria, including M. tuberculosis H37Ra, M. avium, M. intracellulare and M. abscessus.
Several constructs were successfully expressed, but expression did not consistently result in soluble or active protein after scale-up. Solubility, aggregation during purification, protein loss during desalting and assay interference were major limitations. Activity against M. smegmatis was generally weak after purification, whereas selected constructs showed measurable inhibition against pathogenic mycobacterial strains. Three enzymes exhibited relatively low MIC values against M. tuberculosis H37Ra, ranging from 68 to 137 µg/mL. These results suggest that LysA truncation and chimeric mycolysin design can be compatible with retained antimicrobial activity, but further optimization of expression, purification, stability and quantitative antimicrobial testing is required before therapeutic potential can be reliably assessed.}},
author = {{Jamal, Husam Naseer Jamal}},
language = {{eng}},
note = {{Student Paper}},
title = {{Assessing the effects of domain truncation on the antibacterial activity of endolysins}},
year = {{2026}},
}