Capsular specificity in temperate phages of Klebsiella pneumoniae is driven by diverse receptor-binding enzymes

Understanding host-range determinants in temperate bacteriophages is critical for elucidating phage-host co-evolution and advancing phage therapies.We analysed 3,900 Klebsiella genomes from diverse ecological niches to identify prophage-encoded proteins mediating capsule tropism. We applied a genome-wide association study (GWAS) correlating prophage protein clusters (from 8,105 prophages) with confidently assigned bacterial K-loci. GWAS identified high-confidence predictors for 16 out of 35 most diverse K-loci, of which 14 were receptor-binding proteins (RBPs) belonging to classical depolymerases ( n = 6), SGNH hydrolases which deacetylate polysaccharides ( n = 6), and structurally novel RBPs ( n = 2). When we relaxed the filtering thresholds, we identified 26 putative depolymerases, of which 12 were deemed as strong predictions against 10 K-types. In parallel, 50 depolymerases manually found in prophages from the representative subset of 99 bacterial isolates, together with an additional 10 depolymerases based on GWAS predictions, were prepared as recombinant proteins and tested on a Klebsiella reference panel of 119 K-types. Most predicted prophage depolymerases (34/60) failed to yield soluble products, 6 were not active on the K-types panel, and 5/14 targeted a different K-type than their bacterial host, highlighting the unpredictability of prophages as a source of functional enzymes. A comparison of GWAS-predicted enzymes, from the manual search and from virulent phages showed that depolymerase specificity was often difficult to infer from sequence or structure alone. Our findings reveal that Klebsiella prophages encode structurally diverse RBPs, suggesting temperate phages may rely not only on depolymerisation but also on capsule modification—such as deacetylation—for infection. This suggests capsule diversity in K. pneumoniae may be substantially underestimated, with implications for phage specificity, competition and vaccine design.