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

Phage-borne depolymerases decrease Klebsiella pneumoniae resistance to innate defense mechanisms

Klebsiella pneumoniae produces capsular polysaccharides that are a crucial virulence factor protecting bacteria against innate response mechanisms of the infected host. Simultaneously, those capsules are targeted by specific bacteriophages equipped with virion-associated depolymerases able to recognize and degrade these polysaccharides. We show that Klebsiella phage KP32 produces two capsule depolymerases, KP32gp37 and KP32gp38, with a high specificity for the capsular serotypes K3 and K21, respectively. Together, they determine the host spectrum of bacteriophage KP32, which is limited to strains with serotype K3 and K21. Both depolymerases form a trimeric beta-structure, display moderate thermostability and function optimally under neutral to alkaline conditions. We show that both depolymerases strongly affect the virulence of K. pneumoniae with the corresponding K3 and K21 capsular serotypes. Capsule degradation renders the otherwise serum-resistant cells more prone to complement-mediated killing with up to four log reduction in serum upon exposure to KP32gp37. Decapsulated strains are also sensitized for phagocytosis with a twofold increased uptake. In addition, the intracellular survival of phagocytized cells in macrophages was significantly reduced when bacteria were previously exposed to the capsule depolymerases. Finally, depolymerase application considerably increases the lifespan of Galleria mellonella larvae infected with K. pneumoniae in a time- and strain-dependent manner. In sum, capsule depolymerases are promising antivirulence compounds that act by defeating a major resistance mechanism of K. pneumoniae against the innate immunity

Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their efficacy against Pseudomonas aeruginosa biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants

Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with “phiKMV-like viruses”. Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae

The cytotoxicity of 3-bromopyruvate in breast cancer cells depends on extracellular pH

Although the anti-cancer properties of 3BP have been described previously, its selectivity for cancer cells still needs to be explained. In the work reported here we characterized the kinetic parameters of radiolabelled [14C]-3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0 the affinity of cancer cells for 3BP transport, correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the proton motive force and is decreased by MCTs inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. Here, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1 in 3BP uptake and the importance of CD147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acid. This is a typical phenotype of tumor microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies.FEDER (Fundo Europeu deDesenvolvimento Regional), through POFC (Programa Operacional Factores de Competitividade) – COMPETE, and by Portuguese National Funds from FCT (Fundac¸˜ao para a Ciˆencia e Tecnologia) in the scope of the project PEst-OE/BIA/U14050/2014. JAS [grant number SFRH/BD/76038/2011] received a fellowship from the Portuguese government from the FCT through FSE (Fundo Social Europeu) and POPH (Programa Operacional Potencial Humano)

Targeting biofilms using phages and their enzymes

"Available online 10 March 2021"The complex biofilm architecture composed of extracellular polymeric structures (EPS) provides a protective shield to physiologically diverse bacterial cells immersed in its structure. The evolutionary interplay between bacteria and their viruses (phages) forced the latter ones to develop specific strategies to overcome the biofilm defensive barriers and kill sessile cells. Phages are equipped with a wide panel of enzyme-degrading EPS macromolecules which together are powerful weapons to combat biofilms. Antibiofilm performance can be achieved by combining phages or phage-borne enzymes with other antimicrobials such as antibiotics. Nevertheless, a variety of enzymes encoded in phage genomes still need to be explored. To advance in biofilm control strategies we must deepen the understanding of the biofilm biology itself, as well as discover and better exploit the unlimited antibacterial potential of phages.JA was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. PG was supported by grants from Innovative Training Networks (ITN) Marie Skłodowska-Curie Actions H2020-MSCA-ITN2018. Reference 813439, and PID2019-105311RB-I00 (MICIU/AEI/FEDER, UE, Spain). ZDK was supported by the National Science Centre of Poland (Narodowe Centrum Nauki), grant numbers 2016/21/B/NZ6/01157 and 2017/26/M/NZ1/00233.info:eu-repo/semantics/publishedVersio

Ability of phages to infect Acinetobacter calcoaceticus-Acinetobacter baumannii complex species through acquisition of different pectate lyase depolymerase domains

Bacteriophages are ubiquitous in nature and represent a vast repository of genetic diversity, which is driven by the endless coevolution cycle with a diversified group of bacterial hosts. Studying phage-host interactions is important to gain novel insights into their dynamic adaptation. In this study, we isolated 12 phages infecting species of the Acinetobacter baumannii-Acinetobacter calcoaceticus complex which exhibited a narrow host range and similar morphological features (podoviruses with short tails of 9-12 nm and isometric heads of 50-60 nm). Notably, the alignment of the newly sequenced phage genomes (40-41 kb of DNA length) and all Acinetobacter podoviruses deposited in Genbank has shown high synteny, regardless of the date and source of isolation that spans from America to Europe and Asia. Interestingly, the C-terminal pectate lyase domain of these phage tail fibers is often the only difference found among these viral genomes, demonstrating a very specific genomic variation during the course of their evolution. We proved that the pectate lyase domain is responsible for phage depolymerase activity and binding to specific Acinetobacter bacterial capsules. We discuss how this mechanism of phage-host co-evolution impacts the tail specificity apparatus of Acinetobacter podoviruses.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI01–0145-FEDER-006684) and the Project PTDC/BBB-BSS/6471/2014 (POCI-01–0145-FEDER-016678). This work was also supported by BioTecNorte operation (NORTE-01–0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. We acknowledge Dr. Lenie Dijkshoorn (Leiden Medical Center) for the provision of some strains (LUH or RUH designations). AFM was performed at i3s- Instituto de Investigação e Inovação para a Sa ude at the Biointerfaces and Nanotechnology platform. SS is an FCT Investigator (IF/01413/ 2013). HO and ARC acknowledge FCT for grants SFRH/BPD/ 111653/2015 and SFRH/BPD/94648/2013 respectively. The authors declare that they have no competing financial interests.info:eu-repo/semantics/publishedVersio

The structure and function of human IgA subclasses

An unusual structural feature of human immunoglobulin A (IgA) is the heterogeneity of the molecular forms, with a characteristic distribution in various body fluids. Serum IgA is largely monomeric, but in external secretions it exists as S-IgA - a dimer consisting of two IgA molecules bound together by J chain and attached to secretory piece (SC). Both in serum and secretions IgA occurs in two isotypic forms, IgA1 and IgA2. IgA2 exists as two known allotypes, namely IgA2m(1) and IgA2m(2), with a form IgA2(n) possibly representing a third allotype. The major difference between the IgA subclasses is an absence of 13-aminoacid segment in the hinge region of IgA2 that is found in IgA1 molecules. This truncated hinge region in IgA2 molecules renders them resistant to at least two families of IgA1-specific bacterial proteases, which presumably is advantageous to IgA2 antibody function at mucosal surfaces. Further and profound structural difference between the iα1 and α2 chains concerns the distribution and composition of the oligosaccharide side chains. The IgA1 contains two N-linked glycosylation sites (Asn263 in the CH2 domain and Asn459 in the tail piece) as well as nine potential O-linked glycosylation sites in the hinge region. All three IgA2 variants lack these O-linked sugars, but they have two extra N-linked glycosylation sites (Asn166 in the CH1 and Asn337 in the CH2). The IgA2m(2) and IgA2(n) allotypes have a fifth potential N-linked side in the CH1 (Asn211) domain. The variations in the glycosylation structure of the different form of IgA play a significant role in determining antibodies conformation and assembly, receptors (TCR, ASGP-R) binding and t1/2. Here we review current knowledge concerning the relationship of the structure of human IgA1 to the IgA2 isotype, the polymeric IgA and secretory IgA structures, and IgA function