Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.

George Blundell-Hunter,Supayang P Voravuthikunchai,Peter W Taylor,David Negus,Matthew J Dorman,Phitchayapak Wintachai,Nicholas R Thomson,Mark C Enright,Gemma E Beecham,Derek J Pickard

doi:10.3389/fcimb.2021.686090

Abstract

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41–348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

Highlights

Extracellular polysaccharide and polypeptide capsules are major virulence determinants of Gram-positive and Gram-negative pathogens, aiding colonisation of mucosal surfaces and protecting invasive bacteria from immune recognition and killing by cellular and humoral immune mechanisms during infection of susceptible hosts
Sixty-two phage were isolated, encompassing a variety of distinct host range patterns (Supplementary Table 2); 28 of these were chosen for further study, based on divergent morphology, host range, genome size and sequence similarity, and were ordered into seven groups based on a high degree (>95%) of nucleotide sequence identity (Table 2)
All lytic plaques on fully-susceptible host strains were surrounded by halos after overnight incubation at 37°C followed by 3-4 days storage at 4°C, indicating diffusion of soluble capsule depolymerase into the surrounding bacterial lawn (Adams and Park, 1956)

Summary

Introduction

Extracellular polysaccharide and polypeptide capsules are major virulence determinants of Gram-positive and Gram-negative pathogens, aiding colonisation of mucosal surfaces and protecting invasive bacteria from immune recognition and killing by cellular and humoral immune mechanisms during infection of susceptible hosts. They are key contributors to the enormous diversity of bacterial surfaces and provide the interface >with their immediate external environment (Roberts, 1996; Mostowy and Holt, 2018). A depolymerase from an environmental bacterium was shown to prevent lethal infection in mice infected with a highly virulent strain of Bacillus anthracis (Negus et al, 2015)

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Conclusion