Bacteriophages, or phages, are viruses that infect bacteria, at the end of their life 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.
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, and also showed enhanced bactericidal activity with MIC (minimum inhibitory concentration) values of 62.5, 100, 500 and 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 Triton X–100 as a surfactant. Unlike true lipases, LysB–D29 has higher enzymatic activity in the absence of Triton X–100 and hence does not require interfacial activation. Moreover, some LysB homologs with varying degrees 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 a range of substrates (C4–C16), with catalytic preference for para–nitrophenyl laurate (C12). The mycolylarabinogalactan esterase activity for hydrolysis of mycolylarabinogalactan–peptidoglycan complex as substrate for the LysB–His6 enzymes was confirmed by mass spectrometry. Extracellular application of LysB–His6 enzymes against Mycobacterium smegmatis resulted in marginal antibacterial activity but combining the enzymes with half MIC (1 μg/ml) of colistin (outer membrane permealizer) enhanced the antibacterial activity. 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 and disruption in the cell envelope morphology was observed. The immobilized preparations further exhibited enhanced storage stability compared to the free enzymes. The mycolylarabinogalactan esterase Lysin B (LysB) is produced by mycobacteriophages that infect mycobacterial cells that possess a unique cell wall structure with a thick mycolic acid layer. 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 modelling of 30 LysB proteins differing in their similarity to LysB–D29.
- Hatti-Kaul, Rajni, handledare
- Holst, Olle, Biträdande handledare
- Dishisha, Tarek, Biträdande handledare
|2019 okt. 9
|Published - 2019 okt. 9
Place: Lecture Hall B, Kemicentrum, Sölvegatan 39A, Lund University, Faculty of Engineering LTH
Name: Drulis-Kawa, Zuzanna
Affiliation: University of Wroclaw, Poland
- Annan industriell bioteknik
- Biokemi och molekylärbiologi