Author:

Keywords:

bacteriophage, endolysins, antibacterial activity, Gram-negative bacteria

Abstract:

Bacteriophages, viruses infecting bacteria, disrupt the bacterial cell wall at the end of their replication cycle to release newly produced virions. The major constituent of the bacterial cell wall is the peptidoglycan. To degrade this rigid layer, bacteriophages encode peptidoglycan hydrolases, called endolysins, that hydrolyze specific bonds in the peptidoglycan. This dissertation specifically focuses on endolysins, isolated from phages infecting Gram-negative bacterial species, including Pseudomonas aeruginosa, Salmonella Typhimurium, Escherichia coli, Klebsiella pneumoniae and Citrobacter rodentii. Most of these bacteria are opportunistic pathogens that are of increasing concern in hospitals due to their high intrinsic and acquired antibiotic resistance. In a first part of this study, we extend the pool of available endolysins from Gram-negative origin and analyze their potential and applicability as alternative antibacterial agents for antibiotics to combat these Gram-negative pathogens. The Gram-negative outer membrane prevents exogenously applied endolysins from reaching the peptidoglycan layer and protects bacteria against their lytic activity. We therefore evaluate in the second part of this dissertation an approach that allows the endolysin to efficiently destabilize the outer membrane and subsequently reach the peptidoglycan. This approach consists of the fusion of a set of outer membrane-permeabilizing antimicrobial peptides to the endolysin to allow for an autonomous interaction with the outer membrane. To sketch the background, this dissertation starts with an overview of the literature concerning bacteriophage endolysins (history and structural diversity), antimicrobial peptides (types and mode of action) and outer membrane diversity present among Gram-negative bacteria.From an in silico analysis of fully sequenced phage genomes, a selection of fifteen interesting candidate endolysins is made (Chapter 4). Six single-domain (Chapter 5) and three modular (Chapter 6) endolysins with the highest maximal muralytic activity under physiological conditions, are selected for extensive characterization on biochemical (pH-dependency, enzymatic activity, activity upon heating) and antibacterial level. In this way, we aim to prove their lytic role and to reveal enzyme-specific characteristics interesting from an application perspective. In silico, the single-domain endolysins consist of a catalytic domain, whereas the modular ones feature an N-terminal peptidoglycan binding domain and a C-terminal catalytic domain, hitherto a unique property present in a few endolysins from Gram-negative origin. In addition, the predicted peptidoglycan binding domains are experimentally verified.The modular endolysins in this study are shown to be enzymatically more active than the single-domain endolysins, an observation that was translated into their in vitro antibacterial activity. Of all tested endolysins, the modular endolysin from Pseudomonas fluorescens phage OBP, OBPgp279, shows the highest muralytic and antibacterial activity, followed by PVP-SE1gp146, the endolysin from Salmonella Enteritidis phage PVP-SE1. The peptidoglycan binding domain present in modular endolysins accounts for their strong lytic action since the contribution of this domain (38 to 56 %) to the total enzymatic activity is considerable. In addition, the enzymatic activity is consistent for the different Gram-negative bacterial species due to their conserved peptidoglycan (A1gamma chemotype). This characterization also revealed various interesting biochemical properties. OBPgp279 shows intrinsic antibacterial activity on P. aeruginosa PAO1 (± 1 log unit), probably by destabilizing the Pseudomonas outer membrane. PVP-SE1gp146 remains active up to temperatures of 90°C with 60 % residual enzymatic activity after 40 minutes. This last property makes the enzyme a potential candidate as antibacterial component in hurdle technology for food preservation. At the start of the second part, OBPgp279 and PVP-SE1gp146, the two most promising endolysins, are selected to evaluate the proposed fusion approach for passage of the outer membrane (Chapter 7). The N-terminal fusion of a polycationic PK peptide (KRKKRKKRK) composed of lysine and arginine residues, turns out to be the most effective fusion to improve the antibacterial activity of both endolysins. The highest activity is reached for P. aeruginosa with maximal 2.61 log units. Addition of minor EDTA concentrations enhances activity and extends the activity range with E. coli (maximal 1.70 log units) and S. Typhimurium (maximal 0.91 log units). This fused PK peptide is believed to compete with the Achilles’ heel of the outer membrane: the stabilizing divalent cations. A double N-terminal fusion of this promising PK peptide with other antimicrobial peptides only increases the antibacterial activity of OBPgp279 against E. coli to maximal 2.22 log units (for PP-PK double fusion), but is detrimental for the activity against other Gram-negative species. Analysis for the impact of the N-terminal PK fusion on endolysin characteristics reveals a protein-dependent inhibition of the enzymatic activity (with 52 to 94 %), a reduced heat resistance and a switch in pH-dependency to slightly more alkaline values (Chapter 8). Due to a more hydrophobic outer membrane, the antibacterial efficacy of the PK fusion is limited for Enterobacteriaceae. Additionally, the PK fusion also confers biofilm-degrading activity to PVP-SE1gp146. Extension of the linker length between the PK peptide and endolysin partly reconstitutes the reduced muralytic activity due to the PK peptide fusion, leading to an improved antibacterial activity against Pseudomonads and Enterobacteriaceae. Switching the PK peptide to the C-terminal end does not improve activity. These data nicely illustrate that endolysins can be turned into effective anti-Gram-negative compounds by an N-terminalfusion approach and subsequent optimization of the linker length.In the last part, we evaluate the PK-fused endolysin approach on an in vitro human keratinocyte monolayer and an in vivo Caenorhabditis elegans model. PK-PVP-SE1gp146 is able to protect the keratinocyte monolayer from a P. aeruginosa PA14 infection (Chapter 9). In addition, PK-PVP-SE1gp146 improves the survival of PA14-infected C. elegans with 60 % after five days of treatment (Chapter 10). These results prove the in vitro and in vivo applicability of the PK-fused endolysin approach against P. aeruginosa, offering promising perspectives towards prophylactic and therapeutic applications in human health and veterinary and towards microbial decontamination purposes in the food industry.