Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens.

The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.

ß-xylosidases and α-L-arabinofuranosidases: accessory enzymes for arabinoxylan degradation.

Arabinoxylan (AX) is among the most abundant hemicelluloses on earth and one of the major components of feedstocks that are currently investigated as a source for advanced biofuels. As global research into these sustainable biofuels is increasing, scientific knowledge about the enzymatic breakdown of AX advanced significantly over the last decade. This review focuses on the exo-acting AX hydrolases, such as α-arabinofuranosidases and ß-xylosidases. It aims to provide a comprehensive overview of the diverse substrate specificities and corresponding structural features found in the different glycoside hydrolase families. A careful review of the available literature reveals a marked difference in activity between synthetically labeled and naturally occurring substrates, often leading to erroneous enzymatic annotations. Therefore, special attention is given to enzymes with experimental evidence on the hydrolysis of natural polymers.

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 42.6 kb 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.

A theoretical and experimental proteome map of Pseudomonas aeruginosa PAO1.

A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spots were visualized, and 181 protein spots were characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same protein may be detected in two or more neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks.

Characterization of two ß-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides.

Xylooligosaccharides have strong bifidogenic properties and are increasingly used as a prebiotic. Nonetheless, little is known about the degradation of these substrates by bifidobacteria. We characterized two recombinant ß-xylosidases, XylB and XylC, with different substrate specificities from Bifidobacterium adolescentis. XylB is a novel ß-xylosidase that belongs to the recently introduced glycoside hydrolase family 120. In contrast to most reported ß-xylosidases, it shows only weak activity on xylobiose and prefers xylooligosaccharides with a degree of polymerization above two. The remaining xylobiose is efficiently hydrolyzed by the second B. adolescentis ß-xylosidase, XylC, a glycoside hydrolase of family 43. Furthermore, XylB releases more xylose from arabinose-substituted xylooligosaccharides than XylC (30% and 20%, respectively). The different specificities of XylB, XylC, and the recently described reducing-end xylose-releasing exo-oligoxylanase RexA show how B. adolescentis can efficiently degrade prebiotic xylooligosaccharides.

The lysis cassette of bacteriophage ϕKMV encodes a signal-arrest-release endolysin and a pinholin.

The lysis cassette of Pseudomonas aeruginosa phage ϕKMV encodes a holin, endolysin, Rz and Rz1 in the canonical order. It has a tight organization with a high degree of overlapping genes and is highly conserved (between 96 and 100% identity at the protein level) among several other members of the "phiKMV-like viruses." The endolysin KMV45 exhibits characteristics as expected for a signal-arrest-release (SAR) endolysin, whereas the holin KMV44 is a typical pinholin. KMV45 is initially secreted as an inactive, membrane-anchored endolysin, which is subsequently released by membrane depolarization driven by the pinholin KMV44. The SAR domain of KMV45 is necessary for its full enzymatic activity, suggesting a refolding of the catalytic cleft upon release from the membrane. The physical proximity of the catalytic glutamic acid residue close to SAR domain suggests an alternative activation mechanism compared to the SAR endolysin of phages P1, ERA103 and 21. Expression of KMV44 leads to a quick cell lysis when paired with SAR endolysin KMV45, but not with the cytoplasmic phage λ endolysin, indicating the membrane depolarizing function of KMV44 rather than the large hole-making function characteristic of classical holins.

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 (10(4) and 10(6) 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.

The kalimantacin/batumin biosynthesis operon encodes a self-resistance isoform of the FabI bacterial target.

BatG is a trans-2-enoyl-ACP reductase, encoded in the kalimantacin/batumin (kal/bat) biosynthesis operon. It is not essential for the production of the kal/bat secondary metabolite. Instead, BatG is an isoform of FabI, conferring full resistance to target bacteria. It also complements FabI in its role in fatty acid biosynthesis. The identification of FabI as the antibacterial target is important to assess clinical potential of the kalimantacin/batumin antibiotics against Staphylococcus aureus.

Substrate specificity of three recombinant α-L-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides.

Bifidobacterium adolescentis possesses several arabinofuranosidases able to hydrolyze arabinoxylans (AX) and AX oligosaccharides (AXOS), the latter being bifidogenic carbohydrates with potential prebiotic properties. We characterized two new recombinant arabinofuranosidases, AbfA and AbfB, and AXH-d3, a previously studied arabinofuranosidase from B. adolescentis. AbfA belongs to glycoside hydrolase family (GH) 43 and removed arabinose from the C(O)2 and C(O)3 position of monosubstituted xylose residues. Furthermore, hydrolytic activity of AbfA was much larger towards substrates with a low amount of arabinose substitutions. AbfB from GH 51 only cleaved arabinoses on position C(O)3 of disubstituted xyloses, similar to GH 43 AXH-d3, making it to our knowledge, the first reported enzyme with this specificity in GH 51. AbfA acted synergistically with AbfB and AXH-d3. In combination with AXH-d3, it released 60% of arabinose from wheat AX. Together with recent studies on other AXOS degrading enzymes from B. adolescentis, these findings allowed us to postulate a mechanism for the uptake and hydrolysis of bifidogenic AXOS by this organism.

Isolation and purification of a new kalimantacin/batumin-related polyketide antibiotic and elucidation of its biosynthesis gene cluster.

Kal/bat, a polyketide, isolated to high purity (>95%) is characterized by strong and selective antibacterial activity against Staphylococcus species (minimum inhibitory concentration, 0.05 microg/mL), and no resistance was observed in strains already resistant to commonly used antibiotics. The kal/bat biosynthesis gene cluster was determined to a 62 kb genomic region of Pseudomonas fluorescens BCCM_ID9359. The kal/bat gene cluster consists of 16 open reading frames (ORF), encoding a hybrid PKS-NRPS system, extended with trans-acting tailoring functions. A full model for kal/bat biosynthesis is postulated and experimentally tested by gene inactivation, structural confirmation (using NMR spectroscopy), and complementation. The structural and microbiological study of biosynthetic kal/bat analogs revealed the importance of the carbamoyl group and 17-keto group for antibacterial activity. The mechanism of self-resistance lies within the production of an inactive intermediate, which is activated in a one-step enzymatic oxidation upon export. The genetic basis and biochemical elucidation of the biosynthesis pathway of this antibiotic will facilitate rational engineering for the design of novel structures with improved activities. This makes it a promising new therapeutic option to cope with multidrug-resistant clinical infections.

Comparative analysis of the widespread and conserved PB1-like viruses infecting Pseudomonas aeruginosa.

We examined the genetic diversity of lytic Pseudomonas aeruginosa bacteriophage PB1 and four closely related phages (LBL3, LMA2, 14-1 and SN) isolated throughout Europe. They all encapsulate linear, non-permuted genomes between 64 427 and 66 530 bp within a solid, acid-resistant isometric capsid (diameter: 74 nm) and carry non-flexible, contractile tails of approximately 140 nm. The genomes are organized into at least seven transcriptional blocks, alternating on both strands, and encode between 88 (LBL3) and 95 (LMA2) proteins. Their virion particles are composed of at least 22 different proteins, which were identified using mass spectrometry. Post-translational modifications were suggested for two proteins, and a frameshift hotspot was identified within ORF42, encoding a structural protein. Despite large temporal and spatial separations between phage isolations, very high sequence similarity and limited horizontal gene transfer were found between the individual viruses. These PB1-like viruses constitute a new genus of environmentally very widespread phages within the Myoviridae.

Biochemical characterization of malate synthase G of P. aeruginosa.

BACKGROUND: Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen. RESULTS: Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5 degrees C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 microM), Km acetyl CoA (12 microM) and Vmax (16.5 micromol/minutes/mg enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source. CONCLUSION: The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa.

Identification and comparative analysis of the structural proteomes of phiKZ and EL, two giant Pseudomonas aeruginosa bacteriophages.

Giant bacteriophages phiKZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phiKZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The phiKZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201phi2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.

Plant cell walls: Protecting the barrier from degradation by microbial enzymes.

Plant cell walls are predominantly composed of polysaccharides, which are connected in a strong, yet resilient network. They determine the size and shape of plant cells and form the interface between the cell and its often hostile environment. To penetrate the cell wall and thus infect plants, most phytopathogens secrete numerous cell wall degrading enzymes. Conversely, as a first line of defense, plant cell walls contain an array of inhibitors of these enzymes. Scientific knowledge on these inhibitors significantly progressed in the past years and this review is meant to give a comprehensive overview of plant inhibitors against microbial cell wall degrading enzymes and their role in plant protection.

The adsorption of Pseudomonas aeruginosa bacteriophage phiKMV is dependent on expression regulation of type IV pili genes.

Pseudomonas aeruginosa bacteriophage phiKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host (P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore phiKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage phiKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1DeltapilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage phiKMV infectivity.

The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144.

The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD(KZ)), originating from Pseudomonas aeruginosa bacteriophage varphiKZ, has been examined using a fusion protein of PBD(KZ) and green fluorescent protein (PBD(KZ)-GFP). A fluorescence recovery after photobleaching analysis of bound PBD(KZ)-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10(7)M(-1) for the PBD(KZ)-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD(KZ)-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

Genetic and physical map of broad host range cosmid pRG930cm

We hereby present the complete sequence and annotation of pRG930cm, a spectinomycin/streptomycin/chloramphenicol-resistant cosmid vector. pRG930cm (17,256 bp; GenBank Accession No.: FM174471) has a broad host range, and is stably maintained by a number of Gram-negative bacteria including Pseudomonas spp, Escherichia coli, Agrobacterium tumefaciens and Azorhizobium caulinodans ORS571. pRG930cm is already widely used and its sequence will aid efficient construction and analysis of cosmid libraries.

A procedure for systematic identification of bacteriophage-host interactions of P. aeruginosa phages.

Immediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these - often uncharacterized - proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32x10(6) independent clones), was screened against early proteins (gp1 to 9) of phiKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage-host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to phiKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa.

Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials.

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.