Yves Briers

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria.

One of the last untapped reservoirs in nature for the identification of new anti-microbials is bacteriophages, the natural killers of bacteria. Lytic bacteriophages encode peptidoglycan (PG) lytic enzymes able to degrade the PG layer in different steps of their infection cycle. Endolysins degrade the bacterial cell wall at the end of the infection cycle, causing lysis of the host to release the viral progeny. Recombinant endolysins have been successfully applied as anti-bacterial agent against antibiotic-resistant Gram-positive pathogens. This has boosted the study of these enzymes as new anti-microbials in different fields (e.g. medical, food technology). A key example is the recent development of endolysin-based anti-bacterials against Gram-negative pathogens in which the exogenous application of endolysins is hindered by the outer membrane (OM). These novel anti-microbials, termed Artilysin®s, are able to pass through the OM and reach the PG where they exert their action. In addition, mycobacteria whose cell wall is structurally different from both Gram-positive and Gram-negative bacteria have also been reported to be inhibited by mycobacteriophage-encoded endolysins. Endolysins and endolysin-based anti-microbials can be considered as ideal candidates for an alternative to antibiotics for several reasons: (1) their unique mode of action and activity against bacterial persisters (independent of an active host metabolism), (2) their selective activity against both Gram-positive and Gram-negative pathogens (including antibiotic resistant strains) and mycobacteria, (3) the limited resistance development reported so far. The present review summarizes and discusses the potential applications of endolysins as new anti-microbials.

Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes

Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96xa0% DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus “Kp15virus” within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between l-alanine and d-glutamic acid.

Synthetic biology of modular endolysins

Endolysins and their derivatives have emerged in recent years as a novel class of antibacterials, which have now entered the clinical phases. Their rapid mode-of-action and proteinaceous nature differentiates them from any other class of antibiotics. A key feature of endolysins is their modularity and the opportunities that emerge thereof to customize properties such as specificity, activity, stability and solubility. Extensive protein engineering efforts have expanded the activity spectrum to (pan)drug-resistant Gram-negative bacteria or have improved the activity against Gram-positive pathogens. In addition, specific cell wall binding domains derived from endolysins are exploited for the development of diagnostics.

Inhibitory and bactericidal effect of Artilysin ® Art-175 against colistin-resistant mcr-1 -positive Escherichia coli isolates

The number of mcr-1-positive isolates has increased tremendously since the first report of the plasmid-mediated colistin– resistance mechanism [1]. Isolates with the transferable gene have been identified in (food) animals, various types of meat, vegetables and humans in more than 30 countries [2]. Consequently, the Chinese government has forbidden the use of colistin as a growth promotor for the veterinary sector. The European Medicines Agency now suggests to classify colistin in a higher-risk category and to restrict the use in animals to a maximum 5 mg/ population correction unit [2]. However, in humans, colistin is predominantly used as a last resort parenteral drug against highly resistant bacteria, such as carbapenem-resistant Enterobacteriaceae and Acinetobacter spp. A total ban on colistin in human health is thus not conceivable, but there is an increasing risk that pan– drug-resistant bacteria will emerge. As such, the development of new agents effective against resistant Gram-negative pathogens is important. Artilysin®s are new, engineered, enzyme-based experimental therapeutics active against Gram-negative and Gram-positive pathogens [3]. Specifically, Artilysin® Art-175 consists of an outer membrane (OM) destabilizing peptide fused to an endolysin, which is a peptidoglycan hydrolase encoded by a bacterial virus. Upon application, the OM is first locally destabilized by the peptide, after which Art175 is transferred through the OM and the endolysin moiety extensively degrades the peptidoglycan layer, resulting in membrane bulging and osmotic cell lysis [4]. Based on high clinical impact and technical feasibility, endolysin-based antibacterials, such as Artilysin®s, have recently been classified as antibiotic alternatives with the highest potential as therapeutics [5]. The bactericidal effect of Artilysin® Art-175 against Gramnegative pathogens has already been demonstrated for resistant and persistent cells of Pseudomonas aeruginosa and Acinetobacter baumannii. Moreover, neither resistance development through genetic alterations, nor cross-resistance with existing antibiotic resistance mechanisms has been observed [3,4]. However, both colistin and Art-175 target anionic lipopolysaccharide (LPS) molecules, which leads to a local disturbance in the OM of Gramnegative bacteria. Furthermore, colistin resistance is caused by phosphoethanolamine substitutions on the phosphate groups of the lipid A moiety of the LPS molecules, caused by the mcr-1– encoded phosphoethanolaminotransferase. Hence, cross-resistance could potentially occur. The objectives of this study, therefore, were (i) to evaluate the potential of Art-175 against isolates that have high risk of developing pan-drug resistance; and (ii) to exclude a potential cross–resistance between colistin and Art-175. During a period of three months in 2016, faeces and organ samples from diarrheic swine and cattle from Bavarian farms were monitored for the presence of Escherichia coli. The species of the obtained isolates (n = 19) were confirmed by MALDI-TOF MS fingerprinting (Microflex LT, Bruker Daltonik, Bremen, Germany) using the MALDI Biotyper software, version 4.0.0.1. Confirmed isolates were tested for colistin susceptibility by broth microdilution (Sifin Diagnostics, Berlin, Germany) according to the guidelines of the CLSI. Eleven isolates were found to be colistin-resistant (Table 1). Plasmid DNA was isolated from all colistin-resistant isolates and analysed by polymerase chain reaction (PCR) for the presence of the mcr-1

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7xa0%) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7–49.5xa0% identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29–98xa0% homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5xa0h at pHxa04–10 and are stable at 60xa0°C for 30xa0min, but are sensitive to chloroform, with no remaining infective particles after 24xa0h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (Pxa0<xa00.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki

Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB_AbaS_Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME_AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB_PmaS_IMEP1 and Pseudomonas phages vB_Pae_Kakheti25, vB_PaeS_SCH_Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB_AbaS_Loki was deposited in the European Nucleotide Archive (ENA) under the accession number LN890663.

Synthetic biology of modular proteins

ABSTRACT The evolution of natural modular proteins and domain swapping by protein engineers have shown the disruptive potential of non-homologous recombination to create proteins with novel functions or traits. Bacteriophage endolysins, cellulosomes and polyketide synthases are 3 examples of natural modular proteins with each module having a dedicated function. These modular architectures have been created by extensive duplication, shuffling of domains and insertion/deletion of new domains. Protein engineers mimic these natural processes in vitro to create chimeras with altered properties or novel functions by swapping modules between different parental genes. Most domain swapping efforts are realized with traditional restriction and ligation techniques, which become particularly restrictive when either a large number of variants, or variants of proteins with multiple domains have to be constructed. Recent advances in homology-independent shuffling techniques increasingly address this need, but to realize the full potential of the synthetic biology of modular proteins a complete homology-independent method for both rational and random shuffling of modules from an unlimited number of parental genes is still needed.

Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria

Bacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport in Escherichia coli. In total, we constructed twelve Innolysins fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity against E. coli reducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using an E. coli fhuA deletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor on E. coli is needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria. IMPORTANCE The extensive use of antibiotics has led to the emergence of antimicrobial resistant bacteria responsible for infections causing more than 50,000 deaths per year across Europe and the US. In response, the World Health Organization has stressed an urgent need to discover new antimicrobials to control in particular Gram-negative bacterial pathogens, due to their extensive multi-drug resistance. However, the outer membrane of Gram-negative bacteria limits the access of many antibacterial agents to their targets. Here, we developed a new approach, Innolysins that enable endolysins to overcome the outer membrane by exploiting the binding specificity of phage receptor binding proteins. As proof of concept, we constructed Innolysins against E. coli using the endolysin and the receptor binding protein of phage T5. Given the rich diversity of phage receptor binding proteins and their different binding specificities, our proof of concept paves the route for creating an arsenal of pathogen specific alternative antimicrobials.