Characterization of lytic bacteriophages infecting Pseudomonas aeruginosa and their peptidoglycan and exopolysaccharide degrading enzymes

Antibiotic resistance of bacterial pathogens is an emerging problem worldwide.
As multidrug resistant organisms fail to respond to antimicrobial therapy, infections become more severe and cause more complications which in turn leads to longer illnesses (Gould, 2006; Haecker, 2009; Hübner et al., 2012). The aerobic Gram-negative bacterium Pseudomonas aeruginosa is one of the most important and dangerous microbes inhabiting the hospital environment. It has the ability to adapt to and thrive in many ecological niches, from water and soil environments to plant and animal tissues. P. aeruginosa also possesses a wide array of virulence factors, that do not only cause extensive tissue damage, but also interfere with the human immune system (Dzierżanowska, 2008). As an opportunistic pathogen it is particularly known for causing endogenous infections in immune-deficient individuals (AIDS, cystic fibrosis, cancer) and presents resistance to many antibiotics (Cornelis et al., 2008).

During chronic infections populations of P. aeruginosa undergo characteristics evolutionary adaptations, including reduction of virulence factors production and transition from planktonic form to biofilm-associated lifestyle. The biofilm play an important role in evolution of high-level antibiotic resistance and protects the embedded bacteria from antimicrobial agents applied from without. Moreover, during prolonged infections coexistence of divergent phenotypic lineages of Pseudomonas within patients was observed, which makes accurate diagnosis and treatment even more challenging (Winstanley et al., 2016).

For these reason, new antibacterial therapies are urgently needed. With hurdles in the antibiotic research and development pipelines, especially against Gram-negative pathogens, the scientific community is exploring the development of alternative forms of antimicrobial therapies. The idea of using bacteriophages as natural parasites of bacteria is well known. Bacteriophages have co-evolved intimately with their host for three billion years, and therefore highly efficient antibacterial mechanisms have emerged, granting them unique advantages over classical antibiotics to kill bacteria. Indeed, phage cocktails are commonly applied as alternative or as supportive treatments simultaneously with antibiotics in Eastern Europe, as part of standard care. Positive results are routinely obtained with the eradication of Escherichia, Pseudomonas, Proteus, Klebsiella and Staphylococcus clinical strains from various kinds of purulent wounds (Slopek et al., 1981; Wright et al., 2009; Maura and Debarbieux, 2011). These viruses developed also the ability to tunnel through bacterial biofilms by employment of specific enzymes, which degrade the bacterial exopolysaccharides (EPS), one of the main component of the biofilm matrix. Such phages can be identified by the appearance of a halo zone around the phage plaques, which results from the enzymatic degradation of bacterial EPS without phage infection (Azeredo et al., 2008). Furthermore, bacteriophages can kill bacteria with peptidoglycan degrading enzymes, endolysins and virion-associated peptidoglycan hydrolases (VAPGH), that disrupt specific bonds in the peptidoglycan, a major component of bacterial cell wall. The use of bacteriophages and their recombinantly manufactured proteins offers a great opportunity to bypass antibiotic therapy hurdles. This dissertation specifically focuses on bacteriophages and their two types of enzymes: the polysaccharide depolymerases and peptidoglycan degrading enzymes, endolysins and VAPGHs, active against P. aeruginosa.

In first part of this study we focused on characterization of four newly isolated bacteriophages KT28, KTN6, KTN4 and PA5oct, lytic towards P. aeruginosa. Their basic biology was evaluated, including morphology, host surface receptors, host range, stability and infection process. All phages were stable in a range of temperatures, pH and in presence of chloroform. KT28, KTN6 and KTN4 had relatively broad host range, in contrast to PA5oct. Host cell receptors were different for each type of phage, and included LPS, type IV pili and flagella. Furthermore, the phage genomes were sequenced and analyzed in depth. Unwanted genes or mobile elements were not found, which supports obligatory lytic nature of these bacteriophages. Functional genome analysis was supported with ESI-MS/MS analysis of phage proteome and RNA seq. With the use of comparative genomics, the protein-sharing network was constructed, that revealed phage evolution and homology. KTN6 and KT28 belong to Pbunavirus, KTN4 is a Phikzvirus and PA5oct is a unique giant virus. Finally phage antibacterial activity was evaluated in vitro using a novel Airway Surface Liquid model on non-CF and CF epithelial cells lines, in an effort to mimic in vivo conditions of the respiratory tract, developed at the laboratory of Prof. B. Harvey (Department of Molecular Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin, Ireland). Phages KTN4 and PA5oct presented substantial antibacterial activity, reducing bacterial load from 2.5 to 7 logs, depending on the P. aeruginosa strain used.

Second part of this dissertation focuses on phage-encoded PG degrading enzymes (KT28 gp49, KT28 gp41 and its domain, KTN6 gp46, PA5oct gp214, PA5oct gp250, KTN4 gp48) and polysaccharide depolymerases (LKA1 gp49, LUZ7 gp56 and their domains). Their secondary and tertiary structure was analyzed in depth based
on available homology and crystal structures. The corresponding recombinant proteins have been produced and their bactericidal activity and biofilm eradication potential was evaluated. Among the PG degrading enzymes included in this study, the strongest antibacterial activity presented KTN6 gp46, a globular endolysin. Research of polysaccharide depolymerases was complicated, time consuming and required the greatest effort to obtain bioactive, recombinant and purified protein. LKA1 gp49 and its domain reduced EPS slime surrounding bacterial cell. In the future, their biofilm degradation activity should be further evaluated with more specific and quantitative biofilm assays.

This research presents the potential of bacteriophages and their enzymes, which can be considered for therapy or industrial purposes. However, further research is required to analyze e.g. antibacterial activity in details, stability, dose, toxicity or structure. Furthermore, in vivo animal studies would show the true power of this alternative therapy.