Insights into the Alcyoneusvirus Adsorption Complex

Abstract

The structures of the Caudovirales phage tails are key factors in determining the host specificity of these viruses. However, because of the enormous structural diversity, the molecular anatomy of the host recognition apparatus has been elucidated in only a number of phages. Klebsiella viruses vB_KleM_RaK2 (RaK2) and phiK64-1, which form a new genus Alcyoneusvirus according to the ICTV, have perhaps one of the most structurally sophisticated adsorption complexes of all tailed viruses described to date. Here, to gain insight into the early steps of the alcyoneusvirus infection process, the adsorption apparatus of bacteriophage RaK2 is studied in silico and in vitro. We experimentally demonstrate that ten proteins, gp098 and gp526–gp534, previously designated as putative structural/tail fiber proteins (TFPs), are present in the adsorption complex of RaK2. We show that two of these proteins, gp098 and gp531, are essential for attaching to Klebsiella pneumoniae KV-3 cells: gp531 is an active depolymerase that recognizes and degrades the capsule of this particular host, while gp098 is a secondary receptor-binding protein that requires the coordinated action of gp531. Finally, we demonstrate that RaK2 long tail fibers consist of nine TFPs, seven of which are depolymerases, and propose a model for their assembly.

Exploring the enzymatic activity of depolymerase gp531 from Klebsiella pneumoniae jumbo phage RaK2

“…Our previous study ( Noreika et al., 2023 ) has provided valuable insights into the role of gp531 in the infection process of the jumbo bacteriophage RaK2. We have demonstrated that RaK2 gp531 functions as a tail fiber protein, which is an integral component of the phage's adsorption complex.…”

“…The isolation of bacteriophage RaK2, K. pneumoniae KV-3 propagation, as well as the construction and purification of recombinant gp531 were performed according to previously described methods ( Noreika et al., 2023 ; Šimoliunas et al., 2013 ).…”

“…After a brief centrifugation (10,000 rpm, 8 min), the resulting insoluble precipitate was removed, and the supernatant was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography–mass spectrometry (HPLC-MS). Enzymatic hydrolysis of the CPS of whole cells was performed following the procedure described previously ( Noreika et al., 2023 ).…”

“…In K. pneumoniae -infecting viruses, CPS-degrading depolymerases are usually virion-associated and function as the enzymatic receptor-binding proteins (RBPs). Such RBPs specifically recognize and then cleave the cellular capsule to allow the virus particle to reach its secondary receptors, typically located on the outer membrane ( Bertozzi Silva et al., 2016 ; Fernandes and São-José, 2018 ; Knecht et al., 2020 ; Knirel et al., 2020 ; Noreika et al., 2023 ). Just like other phage depolymerases, which target LPS or exopolysaccharides, K-type specific enzymes encoded by K. pneumoniae bacteriophages are classified as lyases or hydrolases ( Squeglia et al., 2020 ).…”

“…In our previous study on the adsorption complex of alcyoneusviruses, we investigated the biological function of gp531, a depolymerase domain-containing protein encoded by the unique Klebsiella jumbo phage RaK2 ( Noreika et al., 2023 ). Our findings revealed that RaK2 gp531 is a component the long tail fiber and functions as a homotrimer and is essential for the recognizing and attaching to K. pneumoniae KV-3 cells.…”