Specificity and diversity of Klebsiella pneumoniae phage-encoded capsule depolymerases

Abstract

Klebsiella pneumoniae is an opportunistic pathogen with significant clinical relevance. K. pneumoniae-targeting bacteriophages encode specific polysaccharide depolymerases with the ability to selectively degrade the highly varied protective capsules, allowing for access to the bacterial cell wall. Bacteriophage depolymerases have been proposed as novel antimicrobials to combat the rise of multidrug-resistant K. pneumoniae strains. These enzymes display extraordinary diversity, and are key determinants of phage host range, however with limited data available our current knowledge of their mechanisms and ability to predict their efficacy is limited. Insight into the resolved structures of Klebsiella-specific capsule depolymerases reveals varied catalytic mechanisms, with the intra-chain cleavage mechanism providing opportunities for recombinant protein engineering. A detailed comparison of the 58 characterised depolymerases hints at structural and mechanistic patterns, such as the conservation of key domains for substrate recognition and phage tethering, as well as diversity within groups of depolymerases that target the same substrate. Another way to understand depolymerase specificity is by analyzing the targeted capsule structures, as these may share similarities recognizable by bacteriophage depolymerases, leading to broader substrate specificities. Although we have only begun to explore the complexity of Klebsiella capsule depolymerases, further research is essential to thoroughly characterise these enzymes. This will be crucial for understanding their mechanisms, predicting their efficacy, and engineering optimized enzymes for therapeutic applications.

Keywords: Klebsiella pneumoniae; capsular polysaccharide depolymerases; capsule; depolymerase; phage.

Figure 1. Representation of CPS removal by phage-derived depolymerases

K. pneumoniae is surrounded by a capsule polysaccharide (CPS) layer (structural representation shown in blue) serving to protect from antibiotics and the immune system. The CPS is composed of various sugars, with the K2 variant illustrated in the figure. Phages can encode depolymerases to target and degrade the CPS for removal. These depolymerases are usually found on the phage tailspike or tail fiber proteins. Two structurally characterised depolymerases, K2-2 (8IQ5) and DpK2 (7LZJ), are depicted in the figure on the tailspike or tail fibre. PDB entries correct at time of writing.

Figure 2. Structure illustrations and TM-scores of eight structurally characterised depolymerases

(A) The tertiary structures for K2 depolymerases Depo32, DpK2 and K2-2, K1 depolymerase NTUH-K2044-K1-1 ORF34 (K1-1 ORF34 for short), K21 depolymerase KP32gp38, K47 depolymerase P560dep, K64 depolymerase K64-ORF41 and K63 depolymerase KP34gp57 (which was deposited as a truncated monomer). All structures sourced from PDB, with IDs shown in parentheses below. Proteins are oriented NTD to CTD from top to bottom and grouped by depolymerase mechanism. Green box highlights K2 depolymerases. (B) Table displaying the TM-score (template modelling score, which ranges from 0-1. Score <0.3 indicates likely unrelated structures and score >0.5 indicates a high probability of proteins with the same fold [65], calculated using TM-align [66]) between these depolymerases. The amino acid sequence identities are listed in parentheses under the TM-score (calculated using EMBOSS Stretcher [67]). Each alignment score is calculated using the depolymerase in the top (cascading) row as the template and cell colours are based on TM-score significance.

Figure 3. Polysaccharide depolymerase mechanisms of action

Depolymerase mechanisms employed by hydrolases are shown in A (retaining) and B (inverting). The mechanism employed by lyases is shown in C. (A) Capsule polysaccharide cleavage by the retaining mechanism needs a gap of 5-6Å between the carboxyl sidechains of Asp/Glu catalytic residues, within the depolymerase active site (shown by dashed arrows). The acid-base reaction, requiring the presence of water, results in net retention of anomeric stereochemistry (circled in red) at the cleavage site. (B) The inverting mechanism requires a gap of 10Å between the carboxyl sidechains of Asp/Glu catalytic residues (shown by dashed arrows). The process results in inversion of anomeric stereochemistry (circled in red). (C) The lytic mechanism of polysaccharide cleavage does not require a water molecule, unlike the hydrolytic mechanisms. The uronic acid allows electron localisation on the carboxyl group following C5 hydrogen abstraction by the catalytic base within the depolymerase active site (shown here as an arginine residue in blue). The C4 linkage is broken, resulting in the introduction of a double bond (circled in red) to the oligosaccharide product.

Figure 4. Structures of similar Klebsiella capsule polysaccharides

Schematic representations of capsule polysaccharide repeat unit structures were sourced from K-PAM (https://www.iith.ac.in/K-PAM/k_antigen.html) and are arranged side-by-side in similar pairs. Similar structures with depolymerases known to target both are highlighted by a green outline. Similar structures that have been proposed previously [50] are highlighted in yellow. A glycan key is included.

Figure 5. Structures and cleavage sites of Klebsiella capsule polysaccharides

Schematic representations of capsule polysaccharide repeat unit structures were sourced from K-PAM (https://www.iith.ac.in/K-PAM/k_antigen.html) with the exception of K108 sourced from [68]. Identified cleavage sites (see Table 3) of CPS structures shown by a red arrow. A glycan key is included. * 2-Acetylation on the Fucose of K54 appears every other repeat unit.

Figure 6. Phylogenetic analysis of experimentally validated Klebsiella CPS depolymerases and the phages which encode them

(A) Phylogenetic tree of phages encoding experimentally validated depolymerases. The tree was generated using all phage CDS in VICTOR, a method for the genome-based phylogeny and classification of prokaryotic viruses [69,70]. Phages encoding depolymerases discussed in the text are highlighted: K21 phages (grey), K64 phages (blue), K57 phages (red), K2 phages (green), K1 phages (yellow), and phage K64-1 (purple) that encodes K1, K21 and K64 depolymerases. Depolymerase names are listed alongside their encoding phages for easier interpretation (B) Phylogenetic tree of 58 experimentally validated Klebsiella phage-encoded CPS depolymerases, and the E. coli-targeting phage K1F depolymerase as an outgroup. The entire depolymerase sequence was used and aligned using T-Coffee, a structure-based consensus alignment. Capsule specificity determines the node colours (legend shown).