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Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types.

ABSTRACT: Klebsiella pneumoniae is an important human pathogen causing opportunistic nosocomial and community-acquired infections. A major public health concern regarding K. pneumoniae is the increasing incidence of multidrug-resistant strains. Here, we isolated three novel Klebsiella bacteriophages, KN1-1, KN3-1 and KN4-1, which infect KN1, KN3 and K56, and KN4 types respectively. We determined their genome sequences and conducted a comparative analysis that revealed a variable region containing capsule depolymerase-encoding genes. Recombinant depolymerase proteins were produced, and their enzymatic activity and specificity were evaluated. We identified four capsule depolymerases in these phages that could only digest the capsule types of their respective hosts. Our results demonstrate that the activities of these capsule depolymerases were correlated with the host range of each phage; thus, the capsule depolymerases are host specificity determinants. By generating a capsule mutant, we demonstrate that capsule was essential for phage adsorption and infection. Further, capsule depolymerases can enhance bacterial susceptibility to serum killing. The discovery of these phages and depolymerases lays the foundation for the typing of KN1, KN3, KN4 and K56 Klebsiella and could be useful alternative therapeutics for the treatment of K. pneumoniae infections.

SUBMITTER: Pan YJ

PROVIDER: S-EPMC6465236 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types.

Pan Yi-Jiun YJ Lin Tzu-Lung TL Chen Yi-Yin YY Lai Peng-Hsuan PH Tsai Yun-Ting YT Hsu Chun-Ru CR Hsieh Pei-Fang PF Lin Yi-Tsung YT Wang Jin-Town JT

Microbial biotechnology 20190131 3

Klebsiella pneumoniae is an important human pathogen causing opportunistic nosocomial and community-acquired infections. A major public health concern regarding K. pneumoniae is the increasing incidence of multidrug-resistant strains. Here, we isolated three novel Klebsiella bacteriophages, KN1-1, KN3-1 and KN4-1, which infect KN1, KN3 and K56, and KN4 types respectively. We determined their genome sequences and conducted a comparative analysis that revealed a variable region containing capsule ...[more]

PMID: 30706654

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Project description:The genome of the multihost bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5, was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional eight putative capsule depolymerases (S2-4 against K1, S1-1 against K11, S1-3 against K21, S2-2 against K25, S2-6 against K30/K69, S2-3 against K35, S1-2 against KN4, and S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutants of S1-2, S2-2, S2-3, or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified nine functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all ten hosts of this phage, providing an example of type-specific host infection mechanisms in a multihost bacteriophage.IMPORTANCE We currently identified eight novel capsule depolymerases in a multihost Klebsiella bacteriophage and correlated the activities of the gene products to all hosts of this phage, providing an example of carriage of multiple depolymerases in a phage with a wide capsular type host spectrum. Moreover, we also established a recombineering system for modification of Klebsiella bacteriophage genomes and demonstrated the importance of capsule depolymerase for infecting specific hosts. Based on the powerful tool for modification of phage genome, further studies can be conducted to improve the understanding of mechanistic details of Klebsiella phage infection. Furthermore, the newly identified capsule depolymerases will be of great value for applications in capsular typing.

Project description:Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority» category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types.

Project description: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.

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Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types.

Publications

Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types.

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