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.

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 β-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.

The rise of antibiotic-resistant Klebsiella pneumoniae, a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36), from the Siphoviridae family. To gain insights into the catalytic and structural features of depoKP36, we have recombinantly produced this protein of 93.4 kDa and showed that it is able to hydrolyze a crude exopolysaccharide of a K. pneumoniae host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical K. pneumoniae strains, representing the K63 type, and significantly inhibited Klebsiella-induced mortality of Galleria mellonella larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of Klebsiella strains. The activity of this enzyme was retained in a broad range of pH values (4.0–7.0) and temperatures (up to 45 °C). Consistently, the circular dichroism (CD) spectroscopy revealed a highly stability with melting transition temperature (Tm) = 65 °C. In contrast to other phage tailspike proteins, this enzyme was susceptible to sodium dodecyl sulfate (SDS) denaturation and proteolytic cleavage. The structural studies in solution showed a trimeric arrangement with a high β-sheet content. Our findings identify depoKP36 as a suitable candidate for the development of new treatments for K. pneumoniae infections.

The high specificity of bacteriophages is driven by their receptor-binding proteins (RBPs). Many Klebsiella bacteriophages target the capsular exopolysaccharide as the receptor and encode RBPs with depolymerase activity. The modular structure of these RBPs with an N-terminal structural module to attach the RBP to the phage tail, and a C-terminal specificity module for exopolysaccharide degradation, supports horizontal transfer as a major evolutionary driver for Klebsiella phage RBPs. We mimicked this natural evolutionary process by the construction of modular RBP chimeras, exchanging N-terminal structural modules and C-terminal specificity modules. All chimeras strictly follow the capsular serotype specificity of the C-terminal module. Transplanting chimeras with a K11 N-terminal structural RBP module in a Klebsiella phage K11 scaffold results in a capsular serotype switch and corresponding host range modification of the synthetic phages, demonstrating that horizontal transfer of C-terminal specificity modules offers Klebsiella phages an evolutionary highway for rapid adaptation to new capsular serotypes. IMPORTANCE The antimicrobial resistance crisis has rekindled interest in bacteriophage therapy. Phages have been studied over a century as therapeutics to treat bacterial infections, but one of the biggest challenges for the use of phages in therapeutic interventions remains their high specificity. In particular, many Klebsiella phages have a narrow spectrum constrained by the high diversity of exopolysaccharide capsules that shield access to the cells. In this work, we have elaborated how Klebsiella phages deal with this high diversity by exchanging building blocks of their receptor-binding proteins.

Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding. NMR spectroscopy and mass spectrometry analyses of degraded LPS (OSA) fragments show an O5 serotype-specific polysaccharide lyase specificity. LKA1gp49 reduces virulence in an in vivo Galleria mellonella infection model and sensitizes P. aeruginosa to serum complement activity. This enzyme causes biofilm degradation and does not affect the activity of ciprofloxacin and gentamicin. This is the first comprehensive report on LPS-degrading lyase derived from a Pseudomonas phage. Biological properties reveal a potential towards its applications in antimicrobial design and as a microbiological or biotechnological tool.

Bacteriophages (phages or bacterial viruses) are the most abundant biological entities in our planet; their influence reaches far beyond the microorganisms they parasitize. Phages are present in every environment and shape up every bacterial population in both active and passive ways. They participate in the circulation of organic matter and drive the evolution of microorganisms by horizontal gene transfer at unprecedented scales. The mass flow of genetic information in the microbial world influences the biosphere and poses challenges for science and medicine. The genetic flow, however, depends on the fate of the viral DNA injected into the bacterial cell. The archetypal notion of phages only engaging in predatorprey relationships is slowly fading. Because of their varied development cycles, environmental conditions, and the diversity of microorganisms they parasitize, phages form a dense and highly complex web of dependencies, which has important consequences for life on Earth. The sophisticated phage-bacteria interplay includes both aggressive action (bacterial lysis) and “diplomatic negotiations” (prophage domestication). Here, we review the most important mechanisms of interactions between phages and bacteria and their evolutionary consequences influencing their biodiversity.