Phage–derived Endolysins as Potential Antibacterials: A Study of Peptidoglycan Hydrolase and Mycolylarabinogalactan Esterase Enzymes

Bacteriophages, or phages, are viruses that infect bacteria, at the end of their lytic cycle produce a set of enzymes called endolysins to lyse host cells from within facilitating the release of the viral progeny. Due to their lytic activity, endolysins have gained great interest as potential antibacterials targeting both Gram–positive and –negative bacteria, especially in the actual context of
increasing rates of antibiotics resistance. This approach relies on the observation that, external application of recombinant endolysins (enzybiotics) can efficiently lyse target bacteria from without. The current thesis explores the potential of two groups of endolysins, peptidoglycan hydrolase and mycolylarabinogalactan esterase as potential antibacterials. The peptidoglycan hydrolases hydrolyze glycosidic and amide bonds in the peptidoglycan layer of the bacterial cell wall, while mycolylarabinogalactan esterases hydrolyze the ester bond between mycolylarabinogalactan and peptidoglycan in mycobacterial cell wall. The current thesis approach was accomplished through development of novel strategies for immobilization, increasing the spectrum of activity, improving stability and characterization of novel enzymes. Different strategies for immobilization of the well–known peptidoglycan hydrolase, lysozyme from T4 bacteriophage and its
antibacterial activity was studied. Immobilization of the T4 lysozyme (T4Lyz) to wound dressing gauze in a single facile binding step was achieved through engineering the endolysin with a cellulose binding module (CBM) as a fusion tag. T4Lyz–CBM–immobilized gauze retained antibacterial activity against Gram–positive Micrococcus lysodeikticus (3.8 Log10 reduction) and Gram–negative Escherichia coli and Pseudomonas mendocina with 1.59 and 1.39 Log10 reduction, respectively. In another approach, the antibacterial activity and storage stability of the T4Lyz as well as Hen Egg White Lysozyme (HEWL)
were enhanced via covalent immobilization to tailored positively charged aminated cellulose nanocrystals (Am–CNC). Am–CNC–lysozyme conjugates retained muralytic activity of 86.3% and 78.3% for HEWL and T4Lyz, respectively. Am–CNC–T4Lyz conjugates also showed enhanced bactericidal activity with MIC (minimum inhibitory concentration) values of 62.5, 100, 500 and 625 μg/ml against M. lysodeikticus, Corynebacterium sp., E. coli and P. mendocina, respectively. The Log10 reduction of the tested bacteria occurred in a relatively shorter time as confirmed by time kill study using Alamarblue® as metabolic indicator dye. Transmission electron microscopy revealed altered membrane morphology of the cells treated with the conjugates. The immobilized preparations further exhibited enhanced storage stability at 4 and 22 °C.
The second part of the study dealt with lysin B (LysB), a mycolylarabinogalactan esterase produced by mycobacteriophages that infect mycobacterial cells, which possess a unique cell wall structure with a thick mycolic acid layer. In this work, the genome database of mycobacteriophages was explored to find and categorize LysB enzymes based on similarity to LysB–D29, the only LysB with available crystal structure. Comparative structural analysis of some novel mycobacteriophage LysB enzymes resulted in homology modeling of 30 LysB proteins different in their similarity to LysB–D29. Structure alignment showed that LysB enzymes are not true lipases due to the lack of the lid domain which was confirmed by testing the esterase activity of LysB–D29 against para–nitrophenyl butyrate (pNPB) in presence and absence of surfactant. Our results showed that unlike true lipases, LysB–D29 has higher enzymatic activity in the absence of Triton X–100 as a surfactant and hence doesn’t require interfacial
activation. Moreover, some LysB homologs with different degree of similarity to LysB–D29 were cloned and recombinantly expressed in E. coli BL 21 (DE3) expression host. Characterization of their kinetic parameters for the hydrolysis of para–nitrophenyl ester substrates showed LysB–His6 enzymes to be active against range of substrates (C4–C16), with a catalytic preference for para–nitrophenyl laurate (C12). Moreover, LysB–His6 enzymes have the highest catalytic activity at 37°C, and some divalent metal ions e.g. Ca2+ and Mn2+ enhance the catalytic activity. The mycolylarabinogalactan esterase activity for
hydrolysis of mycolylarabinogalactan––peptidoglycan complex as substrate for the LysB–His6 enzymes was confirmed by LC/MS. Extracellular application of LysB–His6 against Mycobacterium smegmatis resulted in marginal antibacterial activity. However, combining LysB–His6 enzymes with half MIC (1 μg/ml) of colistin (outer membrane permealizer) enhanced the antibacterial activity of LysB–His6 enzymes against M. smegmatis.

Ensuring good health and well–being is one of the 17 sustainable development goals adopted by United Nations Member states. Sustainability of mankind is dependent to a great extent on our ability to prevent and cure diseases. The current dissemination of antibiotic resistance puts the future efficacy of current antibiotics under question. The misuse and overuse of existing antibiotics has led to the evolution of superbugs that are resistant to nearly all available antibiotics. Indeed, catastrophic scenarios are predicted indicating severe human and economic losses if we fail in finding new treatments with tens of million deaths per year and costs ascending to trillions of USD by 2050. Moreover, this threat is also associated with a very limited pipeline of new effective therapies from the pharmaceutical industry. Concerted efforts are thus required to tackle antimicrobial resistance and to discover new antibiotics and alternatives.
Among the various alternatives are bacteriophage derived enzymes, endolysins.
Bacteriophages or simply phages are abundant in the environment and are
considered as the natural enemy of bacteria and can help in eradicating pathogenic bacteria. The phages inject their own genetic code into a bacterial cell, turning it into a phage factory until the virus progeny bursts out of the cell by the action of the endolysins on the bacterial cell envelope. Endolysins have rapid onset of action and high potency (i.e. active at a very low concentration), and do not provoke resistance. Despite their efficiency, endolysins are active mainly against Gram–positive bacteria. The high lipid content in the outer layer of both Gram–negative and mycobacteria protects them from the action of endolysins making them ineffective. Therefore, new strategies are being developed to extend the action of endolysins against Gram– negative and mycobacteria, for example binding of endolysins to tailored nanoparticles or using compounds that destabilize the outer layer of bacterial cell
wall to grant access to the endolysins. This thesis presents studies on different endolysins with potential antibacterial activity. The well–known endolysin from T4 bacteriophage was genetically modified to allow it to bind easily to a wound dressing gauze with retention of significant antibacterial activity. The same enzyme was also bound to biodegradable cellulose nanocrystals and used to kill both Gram–positive and –negative bacteria. Furthermore, new endolysins produced by bacteriophages infecting mycobacteria were identified in databases, and some of them were produced by recombinant DNA and tested for their activity to be a foundation for their application against the pathogenic Mycobacterium tuberculosis that causes the lung disease, tuberculosis.