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

توصيف إنزيمات إزالة البلمرة المشفرة بالبكتيريا الانتقائية للكلبسيلة الرئوية الرئيسية عديد السكاريد الخارجي المحفظي
Authors: George Blundell-Hunter; Mark C. Enright; David Negus; Matthew J. Dorman; Gemma E. Beecham; Derek J. Pickard; Phitchayapak Wintachai; +4 Authors

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

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.

Related Organizations
Keywords

capsule depolymerase, Genome, Viral, 0601 Biochemistry and Cell Biology, Microbiology, Gene, whole-genome sequencing (WGS), Host Specificity, Capsule Depolymerase, Cellular and Infection Microbiology, Enterobacteriaceae, bacteriophage, jumbo phage, alternative antibacterial therapy, Biochemistry, Genetics and Molecular Biology, Klebsiella, Virology, Genetics, Escherichia coli, Humans, Bacteriophages, Alternative Antibacterial Therapy, Bacteriophage, Molecular Biology, Biology, Bacterial Capsules, Ecology, Bacteria, Life Sciences, Bacterial Physiology and Genetics, Ecology and Evolution of Viruses in Ecosystems, Thailand, QR1-502, capsular polysaccharide, Klebsiella Infections, Klebsiella pneumoniae, Whole-genome Sequencing (Wgs), Jumbo Phage, FOS: Biological sciences, Environmental Science, Physical Sciences, Protein Aggregation and Biopharmaceutical Stability, Capsular polysaccharide, 0605 Microbiology

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
26
Top 10%
Average
Top 10%
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