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TitleMolecular studies on bacteriophage endolysins and their potential to control gram-negative bacteria
Author(s)Oliveira, Hugo Alexandre Mendes
Advisor(s)Azeredo, Joana
Kluskens, Leon
Issue date21-May-2014
Abstract(s)Bacteriophages are viruses that specifically infect bacterial hosts to reproduce. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that impedes their release into the environment. Consequently, bacteriophages encode hydrolytic enzymes, called endolysins, to digest the peptidoglycan and cause bacteriolysis. In contrast to their extensively studied counterparts, active against Gram-positives, endolysins from bacteriophages from a Gram-negative background remain less explored. This knowledge gap is largely due to their limited potential as an antimicrobial, which is related to the presence of an impermeable outer membrane in Gram-negatives that blocks the exogenous endolysin action. The experimental work developed in the scope of this thesis aimed at developing efficient strategies to potentiate the endolysin action against these pathogens. An extensive in silico analysis was performed to provide new insights about endolysins structure and function and bacteriophage-endolysin-host ecology. It was possible to identify and analyze 723 putative endolysins sequences from 5 distinct bacteriophages families, infecting 64 different bacterial genera. These endolysins are tremendously diverse in terms of enzymatic function (24 different enzymatic and 13 binding domains), architecture arrangements (89 different types with either globular or modular design) and length (72 to 578 amino acid residues). Three different novel endolysins (Lys68, ABgp46 and PVP-SE1gp146) were studied in detail. Biochemical characterization of Lys68 (from a Salmonella-infecting bacteriophage) showed that it is highly thermostable, withstanding temperatures up to 100°C, and able to refold to its original conformation upon thermal denaturation. Lys68 was able to lyse a wide panel of Gram-negative bacteria in combination with outer membrane permeabilizers. While the Lys68/EDTA combination could only inactivate Pseudomonas strains, the use of citric or malic acid as permeabilizer broadened and increased its antibacterial effect. Particularly against Salmonella, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions after 2 hours, respectively. During an acid-promoted effect, weak acids permeabilized the lipopolysaccharide of most bacteria to Lys68, which retained a relative high activity under these acidic conditions. In case of EDTA, its chelation effect was only observed against Pseudomonas membranes, where ionic interactions are crucial stabilizing forces. The endolysin ABgp46 (from an Acinetobacter-infecting bacteriophage) was shown to naturally inactivate 1 log of certain Acinetobacter strains. Tests in the presence of a number of weak acids (citric, malic, lactic, benzoic and acetic acid) resulted in a powerful antibacterial effect when combined with Abgp46. Higher bactericidal activity was consistently obtained when ABgp46 was combined with citric and malic acid, reducing all planktonic Cronobacter, Klebsiella and E. coli O157 (reductions of 1 to 3 logs) and Pseudomonas, Acinetobacter and Salmonella (reduction of more than 4 logs) species tested. It can be speculated that the major weak acid differences observed are related to their acid dissociated constant, that seems to favor compounds (with lower pKa values) that tend be more ionized. The same combinations did not have significant antibacterial activity when applied against Pseudomonas and Acinetobacter biofilms. To enhance the activity of endolysins against Gram-negative cells, modified endolysins where constructed by fusing PVP-SE1gp146 (from a Salmonella-infecting bacteriophage) with different LPS-destabilizing peptides of polycationic, hydrophobic and amphipathic nature. This strategy resulted in an improvement of the activity of the modified endolysin compared to the native one (1 log reduction on Pseudomonas and Salmonella cells was obtained). The bactericidal activity of all modified variants was increased further in the presence of EDTA. A polycationic nonapeptide was the most efficient tag (maximum reduction of 5 logs). With a different purpose, attempts of increasing the endolysin (in this case Lys68) action against Listeria monocytogenes cells, by inserting species-specific peptidoglycan-binding peptides, did not result in a higher activity. From the moment their genetic identity became known, endolysins have sparked the interest as alternatives for existing antibiotics. Here it was shown that endolysins can be used to kill not only Gram-positive, but also Gram-negative bacterial pathogens. These obtained results underline the great potential of using an endolysin-based strategy for prevention and/or control of Gram-negative pathogens in foodstuff, food processing surfaces, veterinary and medical applications.
TypeDoctoral thesis
DescriptionThesis for PhD degree in Chemical and Biological Engineeering
AccessOpen access
Appears in Collections:BUM - Teses de Doutoramento
CEB - Teses de Doutoramento / PhD Theses

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