White matter alterations in glaucoma and monocular blindness differ outside the visual system

The degree to which glaucoma has effects in the brain beyond the eye and the visual pathways is unclear. To clarify this, we investigated white matter microstructure (WMM) in 37 tracts of patients with glaucoma, monocular blindness, and controls. We used brainlife.io for reproducibility. White matter tracts were subdivided into seven categories ranging from those primarily involved in vision (the visual white matter) to those primarily involved in cognition and motor control. In the vision tracts, WMM was decreased as measured by fractional anisotropy in both glaucoma and monocular blind subjects compared to controls, suggesting neurodegeneration due to reduced sensory inputs. A test-retest approach was used to validate these results. The pattern of results was different in monocular blind subjects, where WMM properties increased outside the visual white matter as compared to controls. This pattern of results suggests that whereas in the monocular blind loss of visual input might promote white matter reorganization outside of the early visual system, such reorganization might be reduced or absent in glaucoma. The results provide indirect evidence that in glaucoma unknown factors might limit the reorganization as seen in other patient groups following visual loss

The T7-Related Pseudomonas putida Phage ϕ15 Displays Virion-Associated Biofilm Degradation Properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy

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

Fighting bacteriophage infection : mechanisms of bacterial resistance

In man-made and natural environments, there is a continuous ongoing interaction between phages and their bacterial hosts, a co-evolutionary arms race between two competing organisms which contributes enormously to their diversity. During continuous cycles of co-evolution, phage-resistant bacterial hosts emerge aiming at preservation of their bacterial lineages. For every step in the phage infection cycle, bacteria have evolved various defense mechanisms, passive or active, to evade phage propagation and subsequent spreading of phage progeny in the surrounding environment. However, when facing this selective pressure imposed by the host, phages have developed different strategies to subvert these defense systems in order to thrive in these new bacterial populations. Knowledge of these phage-host dynamics represents a vital tool for phage therapeutic purposes in which the emergence of phage-resistant bacterial pathogen forms a notable disadvantage. In contrast, in the fermentation industries, bacteriophages themselves pose a contamination problem, which can be relieved by selection of phage-insensitive bacterial strains

Complete genome sequence of the giant pseudomonas phage lu11

The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage ϕRSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.status: publishe

Analysis of outer membrane permeability of Pseudomonas aeruginosa and bactericidal activity of endolysins KZ144 and EL188 under high hydrostatic pressure

The parameters influencing outer membrane permeability of Pseudomonas aeruginosa PAO1 under high hydrostatic pressure were quantified and optimized, using fusion between a specific A1 gamma peptidoglycan-binding domain and green fluorescent protein (PBD-GFP). Based on the obtained data, optimal conditions were defined to assess the synergistic bactericidal action between high hydrostatic pressure and peptidoglycan hydrolysis by bacteriophage-encoded endolysins KZ144 and EL188. Under high hydrostatic pressure, both endolysins show similar inactivation of P. aeruginosa as the commonly used hen egg white lysozyme or slightly higher inactivation in the case of EL188 at 150 and 200 MPa. The partial contribution of pressure to the bacterial inactivation increases with higher pressure, while the partial contribution of the enzymes is maximal at the onset pressure of outer membrane permeabilization for the PBD-GFP protein (175 MPa). This study's results demonstrate the usefulness of this approach to determine optimal synergy for hurdle technology applications.status: publishe

Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL

Pseudomonas aeruginosa bacteriophage endolysins KZ144 (phage phi KZ) and EL188 (phage EL) are highly lytic peptidoglycan hydrolases (210 000 and 390 000 units mg(-1)), active on a broad range of outer membrane-permeabilized Gram-negative species. Site-directed mutagenesis indicates E115 (KZ144)and E155 (EL188) as their respective essential catalytic residues. Remarkably, both endolysins have a modular structure consisting of an N-terminal substrate-binding domain and a predicted C-terminal catalytic module, a property previously only demonstrated in endolysins originating from phages infecting Gram-positives and only in an inverse arrangement. Both binding domains contain conserved repeat sequences, consistent with those of some peptidoglycan hydrolases of Gram-positive bacteria. Fusions of these domains with green fluorescent protein immediately label all outer membrane-permeabilized Gram-negative bacteria tested, isolated P. aeruginosa peptidoglycan and N-acetylated Bacillus subtilis peptidoglycan, demonstrating the broad range of peptidoglycan-binding capacity by these domains. Specifically, A1 chemotype pepticloglycan and fully N-acetylated glucosamine units are essential for binding. Both KZ144 and EL188 appear to be a natural chimeric enzyme, originating from a recombination of a cell wall-binding domain encoded by a Bacillus or Clostridium species and a catalytic domain of an unknown ancestor

Identification of EPS-degrading activity within the tail spikes of the novel Pseudomonas putida phage AF

We report the study of phage AF, the first member of the canonical lambdoid phage group infecting Pseudomonas putida. Its 42.6kb genome is related to the "epsilon15-like viruses" and the "BPP-1-like viruses", a clade of bacteriophages shaped by extensive horizontal gene transfer. The AF virions display exopolysaccharide (EPS)-degrading activity, which originates from the action of the C-terminal domain of the tail spike (Gp19). This protein shows high similarity to the tail spike of the T7-like P. putida-infecting phage φ15. These unrelated phages have an identical host spectrum and EPS degradation characteristics, designating the C-terminal part of Gp19 as sole determinant for these functions. While intact AF particles have biofilm-degrading properties, Gp19 and non-infectious AF particles do not, emphasizing the role of phage amplification in biofilm degradation.status: publishe