Characterization of Pseudomonas-infecting bacteriophages: the search for virion-associated biofilm-degrading enzymes

The biofilm mode of growth represents an important bacterial survival strategy, providing the enclosed bacterial cells with an increased resistance to antimicrobial agents and to the host immune system. Biofilm formation has been implicated in more than 60% of all bacterial infections where they form a source of recurrent infections. Moreover, it is one of the many bacterial resistance mechanisms to bacterial viruses, (bacterio)phages, their natural predators. In their co-evolutionary arms race, particular phages have developed the ability to tunnel through these biofilms. These phage posses an enzymatic activity in their virion-associated tail spikes or fibers which degrades the bacterial exopolysaccharides (EPS), one of the main constituting components of the biofilm matrix. These phages form a promising tool with regard to the renewed interest for phage therapy.The Pseudomonas infecting phages and their associated tail spikes or fibers form the main objective of this dissertation. Based on the formation of the typical plaque morphology with increasing opaque-looking halo zones, phages with potential virion-associated EPS depolymerase activity were selected and subjected to an in-depth characterization of their biofilm degradative properties. The 283,757 bp dsDNA genome of Pseudomonas fluorescens phage OBP possesses strong sequence similarity to the genome of Pseudomonas phage EL. Comparison of the genomic organization of the φKZ-related phages assembled in syntenic genomic blocks interspersed with hyperplastic regions, supports the proposed division in the EL-like viruses and the phiKZ-like viruses within a larger subfamily. Identification of putative early transcriptional promoters scattered throughout the hyperplastic regions, explains several features of the φKZ-related genome organization (existence of genomic islands) and evolution (multi-inversion in hyperplastic regions). Using Hidden Markov modeling typical conserved core genes encoding the portal protein, the injection needle, and two polypeptides with similarity to the 3 -5 exonuclease domain and the polymerase domain of the T4 DNA polymerase, respectively, were identified. Two putative OBP paralog families possibly coding for the abundant proteinaceous fibers which are attached to the OBP tail structure, were annotated. While the N-terminal domains of the peptidoglycan degrading proteins and of one of these tail fiber modules are conserved, the observation of C-terminal catalytic domains typical for the different genera supports the further subdivision of the φKZ-related phages in the two separate genera. The Pseudomonas putida phages AF and φ15 were selected in collaboration with Prof. V.N. Krylov (Laboratory for Genetics of Bacteriophages, Russian Academy of Medical Sciences, Russia) as they form expanding halo zones around the zone of infection. Although φ15 and AF are related to different genera - the T7-like viruses and the epsilon15-like viruses and BPP-1-like viruses , respectively - of the Podoviridae family, both phages possess a closely related EPS-degrading tail spike protein which assembles in a SDS-resistant trimer. This tail spike protein appears to be the sole viral structural component which determines host specificity, since both phages share an identical host spectrum on a library of 53 P. putida strains. Moreover, their EPS substrate is easily modified/lost upon resistance development since all isolated infection-resistant strains are also resistant to the formation of halo zones. The in vitro degradation of single-species P. putida biofilms displays a time- and dose-dependent response upon phage inoculation independent of the bacterial strain, phage or age of the pre-grown biofilm. Killing of the associated planktonic cultures occurred in parallel with, but was always more pronounced than the biofilm disintegration. However, the degree of biofilm disintegration and planktonic killing in response to a particular phage is dependent on the bacterial strain involved and/or age of the pre-grown biofilms. No correlation was noted between the phage susceptibility of the biofilm environment and the relative biofilm forming capacity (RBFC) of the specific strain or its susceptibility of its associated planktonic culture. Generally, 24 h after phage inoculation a re-growth of the planktonic culture and biofilm environment was observed which suggest bacterial resistance development to phages. Application of purified tail spike proteins or UV-inactivated phage particles could not disintegrate 24 h pre-grown PpG1 biofilms.The Pseudomonas aeruginosa infecting phages of the phiKMV-like viruses form around their plaques a halo zone which increases in diameter over the course of time. LKA1 only infects strain PAO1 and encodes one tail spike protein Gp49 which forms SDS-resistant trimers and degrades the B-band of the PAO1 lipopolysaccharides. Infection analysis of outer membrane PAO1 mutants indicated that the formation of halo zones upon LKA1 infection is mediated by this tail spike endorhamnosidase activity and is independent of EPS degradation. Thus, LKA1 represents an exception to the current rule of thumb which correlates halo formation and EPS degradation. Despite in vitro antibacterial activity in planktonic cultures of the LKA1 tail spike protein, no in vitro biofilm degradation or killing of its associated planktonic culture was noted. Application of LKA1 phage particles resulted only in a minor biofilm degradation, while a clear time- and dose-dependent killing of the planktonic culture was observed. The other phiKMV-like viruses which contain a tail spike region encoding four proteins, Gp38-41, infect and form halo zones on the alginate-producing strain Pa573. These phages display a fairly broad host spectrum infecting 29% of the P. aeruginosa library. Type IV pili serve as an essential primary receptor, while the AlgC function - involved in alginate biosynthesis is necessary for halo formation. It is expected that Gp38 functions as a versatile adapter which connects the host interacting proteins, Gp40 and 41, to the viral particle. Degradation of the PAO1k and the mucoid Pa573 biofilms by the φKMV-like phage PT-6 appeared to be time- and dose-dependent and decreases with increasing age of the pre-grown biofilms. The planktonic P. aeruginosa cultures were more susceptible for phage infection than their associated biofilms, but both show a re-growth 24 h after inoculation. PT-6-mediated degradation of biofilms formed by twelve susceptible P. aeruginosa strains could be clustered in three groups (no effect, only killing of the associated planktonic culture, combined biofilm degradation and planktonic killing). Some correlation was observed between the RBFC or the clustering of the P. aeruginosa strains and the biofilm/planktonic susceptibility for phage PT-6. No degradation of pre-grown biofilms was observed in the absence of bacterial lysis but with enzymatic active tail spike proteins. The vertical evolution of phages is clearly marked by horizontal transfer of gene(s) (segments) coding for host cell-interacting protein(s) (domains). As such, tail spikes or fibers contain a conserved N-terminal head-binding domain. Horizontal exchange of their C-terminal catalytic domain permits a rapid adaptation of phages to the continually changing host cell surface receptors in the ongoing co-evolutionary arms race with their bacterial hosts. Phages with associated EPS-degrading tail spikes or fibers are able to affect two main constituents of the biofilm environment, the bacterial cells and their EPS molecules. Although, no biofilm degradation is observed in the absence of bacterial lysis, the EPS-degrading tail spikes/fibers are thought to facilitate movement of the phage particles through the biofilm. Probably, the world-wide phage collection contains substantial numbers of phages with not yet recognized EPS-degrading activity. The present data further underscores the need for careful selection of phages for therapeutic purposes. Good phage amplification characteristics in combination with proper EPS depolymerases form a prerequisite, but the final outcome will depend on the environmental conditions and the specific strains involved.nrpages: 212status: publishe