NOVEL KLEBSIELLA PNEUMONIAE VIRULENT BACTERIOPHAGE KPPK108.1 CAPABLE OF INFECTING THE K108 SEROTYPE STRAINS

Evseev P.V.; Shneider M.M.; Mikhailova Y.V.; Shelenkov A.A.; Yanushevich Y.G.; Shagin D.A.; Karlova M.G.; Moiseenko A.V.; Sokolova O.S.

Evseev PV1, Shneider MM1 Mikhailova YuV2, Shelenkov AA2, Yanushevich YuG3, Karlova MG4, Moiseenko AV4, Sokolova OS4, Shagin DA3

1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

2 Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia

3 Pirogov Russian National Research Medical University, Moscow, Russia

4 Lomonosov Moscow State University, Moscow, Russia

Multidrug-resistant Klebsiella pneumoniae strains are one of the major causes of nosocomial infections caused by the antibiotic-resistant bacteria. There are different options for dealing with this threat, among which is the clinical application of bacteriophages. The study was aimed to isolate and describe a virulent bactriophage, having the potential for therapeutic use. The standard phage biology and bioinformatic methods were used, which included the advanced techniques for protein structure prediction (AlphaFold software), and electron microscopy. The virulent podovirus KPPK108.1, being the member of genus Drulisvirus, which is able to specifically infect the K. pneumoniae strains with the KL108 type capsular polysaccharide, has been isolated from the wastewater. The sequence of the bactriophage genome has been defined, the biological properties have been investigated, and the genetic features have been described. Keywords: bacteriophage, Klebsiella pneumoniae, capsular polysaccharide, depolymerase

Funding: the study was funded by the Ministry of Health of the Russian Federation (EGISU R&D № 121052800048-3).

Acknowledgements: the authors wish to thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, for advice on research methods.

Author contribution: Shagin DA — research conceptualization, study management, manuscript writing; Evseev PV, Shelenkov AA — formal analysis of sequencing data, manuscript editing; Shneider MM — methodology, study management; Mikhailova YuV — sequencing, data validation; Yanushevich YuG, Moiseenko AV, Karlova MG — methodology; Sokolova OS — electron microscopy, methodology.

[><1 Correspondence should be addressed: Mikhail M. Shneider

Miklukho-Maklaya, 16/10, Moscow, 117997, Russia; mm_shn@mail.ru

Received: 08.12.2021 Accepted: 22.12.2021 Published online: 30.12.2021

DOI: 10.24075/brsmu.2021.068

НОВЫЙ ВИРУЛЕНТНЫЙ БАКТЕРИОФАГ KLEBSIELLA PNEUMONIAEKPPK108.1, ИНФИЦИРУЮЩИЙ ШТАММЫ СЕРОТИПА K108

П. В. Евсеев1, М. М. Шнейдер1 Ю. В. Михайлова2, А. А. Шеленков2, Ю. Г. Янушевич3, М. Г. Карлова4, А. В. Моисеенко4, О. С. Соколова4, Д. А. Шагин3

1 Институт биохимии имени М. М. Шемякина и Ю. А. Овчинникова, Москва, Россия

2 Центральный научно-исследовательский институт эпидемиологии Роспотребнадзора, Москва, Россия

3 Российский национальный исследовательский медицинский университет имени Н. И. Пирогова, Москва, Россия

4 Московский государственный университет имени М. В. Ломоносова, Москва, Россия

Мультирезистентные штаммы Klebsiella pneumoniae являются одной из самых серьезных причин внутрибольничных инфекций, вызванных бактериями, устойчивыми к антибиотикам. Существуют различные варианты борьбы с этой угрозой, один из них — клиническое использование бактериофагов. Целью работы было выделить и детально охарактеризовать вирулентный бактриофаг, имеющий потенциал для терапевтического применения. Использовали стандартные методы фаговой биологии, биоинформатики, включая современные способы предсказания белковых структур (программа AlphaFold), электронную микроскопию. Из образцов сточных вод был выделен вирулентный подовирус KPPK108.1, относящийся к роду Drulisvirus, специфично инфицирующий штаммы K. pneumoniae, имеющие капсулярный полисахарид типа KL108, определена последовательность его генома, исследованы его биологические свойства и дана генетическая характеристика.

Ключевые слова: бактериофаг Klebsiella pneumoniae, капсулярный полисахарид, деполимераза

Финансирование: работа выполнена при финансовой поддержке Министерства здравоохранения Российской Федерации (ЕГИСУ НИОКТР № 121052800048-3).

Благодарности: авторы благодарят Центр высокоточного редактирования и генетических технологий для биомедицины РНИМУ им. Н. И. Пирогова за консультации по методической части исследования.

Вклад авторов: Д. А. Шагин — концептуализация, руководство исследованием, редактирование рукописи; П. В. Евсеев, А. А. Шеленков — формальный анализ данных секвенирования, редактирование рукописи; М. М. Шнейдер — методология, руководство исследованием; Ю. В. Михайлова — секвенирование, валидация данных; Ю. Г. Янушевич, А. Моисеенко, М. Г. Карлова — методология; О. С. Соколова — электронная микроскопия, методология.

123 Для корреспонденции: Михаил Маркович Шнейдер

ул. Миклухо-Маклая, д. 16/10, г. Москва, 117997, Россия; mm_shn@mail.ru

Статья получена: 08.12.2021 Статья принята к печати: 22.12.2021 Опубликована онлайн: 30.12.2021 DOI: 10.24075/vrgmu.2021.068

The modern personalized approach to phage therapy is based on the detailed assessment of the interaction between phages and bacterial cells. Bacterial carbohydrates exposed on the cell surface, O-antigens and capsular polysaccharides, are one of the most important specificity determinants in the phage-cell

interaction. Capsular polysaccharides of K. pneumoniae, being the virulence factors [1], are highly diverse in their structure. Currently, bioinformatics databases indicate the existence of at least 134 genetic variants [2]. To meet the challenges of clinical practice it is necessary to create the collection of phages with

the defined specificity, inter alia based on their capacity of adsorption through recognition of capsular polysaccharides with certain structure. The study was aimed to isolate the virulent phage, which specifically infected the previously described K. pneumoniae strains with the KL108 capsular polysaccharide, as well as to fully explore the biological and genetic features of the phage. Standard phage biology methods, electron microscopy, and bioinformatics, including the advanced methods for protein structure prediction (AlphaFold software) were used in order to provide the comprehensive description of the phage.

METHODS

The P224 (1732) and P225 (1333) clinical strains of K. pneumoniae with the K1 08 type capsular polysaccharide were obtained from the collection of the Institute of Epidemiology (Moscow, Russia). The wastewater samples, collected from the wastewater treatment facilities in Moscow, were used for bacteriophage isolation. Dry components of the bacterial culture medium (trypton, 10 g/L, yeast extract, 5 g/L, NaCl, 5 g/L) were added to the wastewater samples previously clarified by centrifugation, then the media were inoculated with the bacterial cells culture being in the phase of exponential growth. Cultivation was carried out at 37 °C for 16 h. The bacterial culture was subsequently inactivated with chloroform, and the samples were clarified by centrifugation. Phages were detected by titration using the double-layer agar plate method. The isolated phage was titrated twice in a row in order to obtain single phage plaques. Preparative bacteriophage growth was performed in 1 L of the P224 strain culture at 37 °C. Bacteriophage was precipitated with polyethylene glycol and purified by caesium chloride density gradient ultracentrifugation [3].

Genomic DNA of the phage was extracted from the purified phage preparation by incubation with the solution, containing 100 mM Tris-HCl (pH 7.5), 25 mM EDTA, 1.5 M NaCl, 2% (w/v) CTAB buffer, 0.3% (v/v) p- mercaptoethanol, and 50 mg/mL of proteinase K, at 50 °C for 30 min, with subsequent chloroform DNA extraction, and precipitated by adding 0.6 volume of isopropyl alcohol. Genome sequencing was performed on the MiSeq platform using the Nextera DNA library preparation kit (Illumina; USA). A single contig was assembled from the resulting sequences using v. 3.13 of the SPAdes software [4].

The experiment aimed at assessing the phage particle production was performed in accordance with the previously reported protocol [5].

Negative contrast electron microscopy was utilized to visualize the phage particles. Phage preparation with a volume of 3 pL was applied to a carbon-coated 400 mesh grid. The negatively contrasted preparation was obtained by 1% uranyl acetate staining for 30 s. Imaging was performed with the JEOL JEM-2100 200 kV transmission electron microscope (JEOL USA Inc.; USA) at 30,000x magnification.

The Klebsiella bacteriophage KPPK108.1 was annotated with the Prokka tool [6] using the embedded databases. The functions of the genome protein products were predicted with the BLAST search tool [7] based on the known homologs, and by the HMM-HMM comparison, performed with the Hhpred [8] and Phyre2 [9] web-based tools using the SC0Pe70_2.07, EC0D_EC0D_F70, and UniProt-SwissProt-viral70 databases. E value < 10-5 was used as a criterion of significant similarity in BLAST analysis; the Phyre2 "confidence" and HHpred "probability" values exceeding 95% were used as the criteria of significant similarity for the HMM-HMM comparison. Genetic mapping was carried out with the Geneious Prime software [10].

Genome sequences of bacteriophages to be used for comparison with the KPPK108.1 phage were downloaded from the NCBI Genome database [11]. The average nucleotide identity was calculated using the VIRIDIC online service [12] and the orthoANIu software [13]. Phylogenetic analysis was performed by the maximum likelihood method implemented in the RaxML program [14] with the use of the GAMMA LG amino acid substitution model [15] and the concatenated amino acid sequence alignment of the major capsid protein, terminase large subunit, DNA polymerase, and RNA polymerase. The sequences were aligned with the MAFFT program [16] and concatenated with the Geneious Prime software [10]. The intergenomic comparison diagram was created with the Easyfig application [17] using the TBLASTX tool [7] to find the homologous regions within genomes.

The models of the gene 8 product tertiary structure and the tailspike protein quaternary structure for the Klebsiella bacteriophage KPPK108.1 were constructed with the AlphaFold-Multimer application [18, 19]. The tailspike protein structure of the Enterobacteria phage -92 was dowloaded from the PDB database [20]. Electrostatic surface charge of the tailspike protein was calculated using the APBS program [21]. The UCSF Chimera program was used for structure alignment and visualization [22].

RESULTS

The KPPK108.1 bacteriophage forms clear plaques, 5 mm in diameter, surrounded by the translucent halos, in the bacterial cultures grown on the agar plates (Fig. 1). The presence of a halo typically indicate the presence of phage-derived depolymerase, which has been confirmed by further research. The one-step growth curve showed a latent period of 15 min and burst size of 46 phage particles per one infected cell.

Klebsiella bacteriophage KPPK108.1 has a genome, typical for the Autographiviridae phages, which consists of double-stranded DNA 43,755 bp in length with the direct terminal repeats 244 bp in size. The GC-content is 53.6%, somewhat lower than the value of 57.5%, characteristic of the sequenced K. pneumoniae strain HS11286 (GenBank Accession

Fig. 1. Electron microscopy of Klebsiella bacteriophage KPPK108.1. Negative staining with 1% uranyl acetate, magnification x3QiQQQ

CP003200.1). The search for coding sequences revealed a total of 56 protein-coding genes and no tRNA-encoding genes in the genome (Fig. 2). The search for homologous and similar sequences using the BLAST algorithm and the HMM-HMM comparison in public databases and web servers made it possible to predict the functions of 29 proteins, encoded in the genome. It was not possible to identify the functions of 27 proteins. No genes, encoding integrases or other proteins specific to temperate phages, were found in the genome.

Comparison of the average nucleotide identity (ANI), involving all 14,923 genes of tailed bacteriophages, deposited in the NCBI Genome database, revealed a group of Klebsiella bacteriophages of the genus Drulisvirus, being the most close to phage KPPK108.1 based on this parameter (Fig . 3). The ANI values of phage KPPK108.1 and a typical phage of the genus Drulisvirus, Klebsiella phage KP34, are 73.0%. Phylogenetic analysis, performed with the use of the concatenated amino acid sequences of the major capsid protein, large terminase subunit, DNA polymerase, and RNA polymerase, shows that Drulisvirus bacteriophages and KPPK108.1 phage form a monophyletic group (Fig. 4). The genetic makeup and genomic organization of the phage KPPK108.1 are generally similar to those of the other Autographiviridae phages (Fig. 5), and are almost identical to those of other members of the genus Drulisvirus. An interesting feature of the gene 8 product was found. Protein structural modeling revealed unusual L-shape of the protein with a tubular C-terminal region (Fig. 6.1). This region was characterized by the number of positively charged amino acid residues above the average. Electrostatic field simulation

showed that the C-terminal region of the gene 8 product had a significant negative surface charge (up to -5) (Fig. 6.2).

Bioinformatic analysis of the KPPK108.1 phage genome revealed genes, encoding the head-tail connector and tailspike proteins. Modeling and analysis of the tailspike protein structure was performed (Fig. 6). The search for similar structures revealed a high degree of similarity between the tailspike of the phage KPPK108.1 and the tailspike of the Enterobacteria phage -92 (PDB entry 6E0V) (Fig. 6) exhibiting colanidase activity confirmed by experimental data [23].

DISCUSSION

The genome-wide similarity score of the phages KPPK108.1 and KP34 exceeding 70% of the genus boundary, together with the results of the phylogenetic analysis performed based on the concatenated sequences of conservative genes, show that the Klebsiella phage KPPK108.1 belongs to the genus Drulisvirus, subfamily Slopekvirinae, family Autographiviridae. Intergenomic comparisons support this finding. Minor differences in genome organization can be explained by the recombination events that took place during the Klebsiella bacteriophages' evolution, as confirmed by the presence of NHN endonuclease genes in the genomes of KPPK108.1 and other related bacteriophages. The genome structure of the KPPK108.1 phage is typical for bacteriophages of the Autographivirinae family and is characterized by the presence of the early gene region [24], comprising gene 8, encoding a hypothetical protein with an unusual L-shaped tertiary structure. Regardless of the fact that

replication and transcription

virion

packaging

lysis

NHN homing endonucleases

others

hypothetical

direct terminal repeats

Fig. 2. Genetic map of Klebsiella bacteriophage KPPK108.1. Genes are colored in accordance with the functions of their products (see caption). Arrows indicate gene directions in accordance with their encoding functions

Fig. 3. Average nucleotide identity (ANI) distance matrix of the Klebsiella phage KPPK108.1 generated with the VIRIDIC web-based tool using the genomes of various Autographiviridae family members

acid. Colanic acid, the extracellular polysaccharide, consisting of several types of carbohydrate residues (such as L-fucose, D-glucose, D-galactose, and D-glucuronic acid), which is released into the extracellular environment by bacteria of the Enterobacteriaceae family, is the colanidase substrate [29]. Colanidases have been relatively recently discovered in phage RBPs [30]. Colanidases are present in a number of virulent bacteriophages of the evolutionarily distant groups, such as podoviruses and myoviruses [23, 30], some of which have proven to be effective when used in phage cocktails for phage therapy [30]. It is essential to define the structure of the type K108 K. pneumoniae capsular polysaccharide to clarify the question of the similarity of this polymer to colanic acid.

CONCLUSIONS

Klebsiella bacteriophage KPPK108.1 is a virulent bacteriophage of the genus Drulisvirus, family Autographiviridae. Thorough

the function of this protein has not been defined by searching for homologues using BLAST, or searching for similar proteins by HMM-HMM comparison, the surface charge distribution makes it possible to assume that this protein mimics nucleic acid, like Ocr proteins, which are also located within the early gene regions in the genomes of other Autographiviridae, and are capable of DNA mimicking [25, 26]. It has been shown that Ocr protein effectively inhibits the BREX restriction modification system, facilitating phage infection [27].

Bioinformatic analysis of the KPPK108.1 phage genome makes it possible to predict the organizational structure of the adsorption apparatus comprised of the head-tail connector and the tailspike protein, possessing enzymatic properties. The tailspike protein seems to be the receptor-binding protein (RBP), which determines host specificity and the host spectrum of the phage [28]. The tailspike protein structure analysis indicates the presence of depolymerizing activity against the polysaccharide, presumed to be related to the E. coli colanic

Klebsiella phage KpV47S - Slopekvirinae; Drulisvirus Klebsiella phage KpV71 - Slopekvirinae; Drulisvirus Klebsiella phage KpV41 - Slopekvirinae; Drulisvirus Klebsiella phage Fl9 - Slopekvirinae; Drulisvirus Klebsiella phage vB_KpnP_KpV48 - Slopekvirinae; Drulisvirus Klebsiella phage 1 TK-2018 - Slopekvirinae; Drulisvirus Klebsiella phage VLC5 - Slopekvirinae; Drulisvirus Klebsiella phage phiKpS2 - Slopekvirinae; Drulisvirus Klebsiella phage vB_KpnP_Dlv622 - Slopekvirinae; Drulisvirus Klebsiella virus KpV2883 - Slopekvirinae; Drulisvirus Klebsiella phage vB_KpnP_IME308 - Slopekvirinae; Drulisvirus CKlebsiella phage KPPK1081)

Klebsiella phage vB_KpnP_Bp5 - Slopekvirinae; Drulisvirus

Klebsiella phage vB_KpnP_SU552A - Slopekvirinae; Drulisvirus

Klebsiella phage vB_KpnP_SU503 - Slopekvirinae; Drulisvirus

Klebsiella phage Pone - Slopekvirinae; Drulisvirus

Klebsiella phage CX1 - Slopekvirinae; Drulisvirus

Klebsiella phage KP34 - Slopekvirinae; Drulisvirus

Klebsiella phage vB_KpnP_cmc20191 - Slopekvirinae; Drulisvirus

Klebsiella phage myPSH1235 - Slopekvirinae; Drulisvirus

Klebsiella phage phi BO 1E - Slopekvirinae; Drulisvirus

Klebsiella phage VLC1 - Slopekvirinae; Drulisvirus

Klebsiella phage Kp2 - Slopekvirinae; Drulisvirus

Escherichia phage Minorna - Slopekvirinae; Drulisvirus

Shigella phage SFN6B - Slopekvirinae; Drulisvirus

Klebsiella phage KPV811 - Slopekvirinae; Drulisvirus

Shigella phage Buco - Slopekvirinae; Bucovirus

Enterobacter phage phiKDA 1 - Slopekvirinae; Koutsourovirus

Pantoea phage LIMElight - Limelightvirus

Proteus phage vB_PmiP_RS8pmA - Slopekvirinae; Novosibovirus

Providencia phage vB PstP PS3 - Kakivirus

Vibrio phage OWB - Maculvirus

Dickeya phage BF25/12 - Corkvirinae; Stompvirus

Pectobacterium phage PPWS1 - Corkvirinae; Kotilavirus

Pectobacterium phage PhiM 1 - Corkvirinae; Phimunavirus

Pseudomonas phage YMC11/06/C171_PPU_BP - Corkvirinae; Kantovirus

Acinetobacter phage Petty - Beijerinckvirinae; Pettyvirus

Vibrio phage vB_VhaP_VH-5 - unclassifiedAutographiviridae

Acinetobacter phage vB_AbaP_Acibel007 - Beijerinckvirinae; Daemvirus

Acinetobacter phage Fri1 - Beijerinckvirinae; Friunavirus

Acinetobacter phage Aristophanes - Beijerinckvirinae; unclassified

Pseudomonas phage phiNV3 - Krylovirinae; Kirikabuvirus

Pseudomonas phage phiKMV- Krylovirinae; Phikmvvirus

Pseudomonas phage phi-2 - Krylovirinae; Tunggulviirus

Pseudomonas phage LKA1 - Krylovirinae; Stubburvirus

Ralstonia phage RSB3 - Jiaoyazivirus

Ralstonia phage phiAp 1 - Ayakvirus

Escherichia phage Lidtsur - Bonnellvirus

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Pantoea phage LIMEzero - Waewaevirus

Cronobacter phage Dev-CD-23823 - Melnykvirinae; Cronosvirus

Fig. 4. Phylogenetic tree of Klebsiella phage KPPK108.1 and other Autographiviridae phages generated with the RaxML program using the concatenated amino acid sequences of the major capsid protein, terminase large subunit, DNA polymerase, and RNA polymerase

bioinformatic analysis has revealed the lytic nature of the phage infection cycle. The analysis makes it possible to predict the structure of the phage adsorption apparatus comprised of the head-tail connector and the tailspike protein, exhibiting

colanidase activity. The predicted characteristics of KPPK108.1 bacteriophage indicate the feasibility of using KPPK108.1 in phage cocktails for phage therapy. As far as we know, KPPK108.1 is the first fully described phage specific for capsular type KL108.

^^RNA polymerase ^^DNA primase/helicase ■"jt-DNA polymerase major capsid protein tubular proteins

. internal core proteins L^^ tail Fibres/spikes lysis proteins terminase

Fig. 5. Intergenomic comparison diagram created with EasyFig and TBLASTX using the genomes of Klebsiella phage KPPK108.1 and other Autographiviridae phages. The vertical lines are colored in accordance with the color scale showing the degree of similarity

Fig. 6. Predicted tertiary structure of the KPPK108.1 phage gene 8 product painted with rainbow colors, where blue indicates N-terminal region, and red indicates C-terminal region of the protein (1). Predicted tertiary structure of the gene 8 product painted in accordance with the charge of the protein surface electrostatic field (2). Predicted tertiary structure of the KPPK108.1 phage tailspike trimer painted with rainbow colors, where blue indicates N-terminal region, and red indicates C-terminal region of the protein, longitudinal view (3). Predicted tertiary structure of the KPPK108.1 phage tailspike trimer with monomers painted with different colors, view along transverse axis (4)

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Литература

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