Identification and characterization of capsule depolymerase Dpo48 from Acinetobacter baumannii phage IME200

Bacterial strains and culture conditions

A total of 41 A. baumannii strains were isolated from sputum samples (AB1, AB2, AB3, AB5, AB9, AB10, AB11, AB12, AB15, AB16, AB17, AB18, AB19, AB007, AB043, AB139, AB178, AB220, AB237, AB295, AB358, AB1610, AB1611, AB1612, AB1613, AB1614, AB1615, AB-E85, and LAB-9), blood samples (AB4, AB6, AB8, AB13, AB20, AB387, and AB5-2), throat swab samples (AB392 and AB406), a bronchoalveolar lavage fluid sample (AB363), a central venous catheter sample (AB7), and a wound secretion sample (AB14) from patients with severe pneumonia in the intensive care unit at PLA Hospital 307, Beijing, China. These samples were collected from different patients on different dates from 2015 to 2017, and the patients were orally informed that the samples would be used for the screening of bacteria. The supernatants of different samples were cultured on Columbia blood agar plates (Oxoid, Hampshire, UK), and each isolate was selected from a single clone for further cultivation. Identification of the above A. baumannii strains and determination of their susceptibility rates toward antibiotics were carried out on a Vitek 2.0 system (BioMerieux Clinical Diagnostics, Paris, France), and the minimum inhibitory concentrations (MICs) were determined in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Pseudomonas aeruginosa ATCC 27853 served as a control strain. These A. baumannii strains were next verified by 16S ribosomal rRNA gene sequencing. The susceptibility rates of all A. baumannii strains toward different classes of antibiotics are given in Table 1, and all strains manifested resistance to at least three classes of antimicrobial agents, except for five A. baumannii strains (AB19, AB220, AB237, AB295, and LAB-9). All the bacterial strains were stored in Luria-Bertani (LB) broth (Oxoid) with 50% glycerol (v/v) at −70 °C, and single colonies were cultured in LB broth at 37 °C, with shaking at 220 rpm. The protocols for collecting clinical samples and screening of bacteria were approved by the Ethics Committees of PLA Hospital 307 and the Beijing Institute of Microbiology and Epidemiology (Ethical Application Ref: ky-2015-3-17).

Bacteriophage isolation, purification, and plaque characterization

Among all the clinical strains, A. baumannii AB1610 was resistant to all classes of antibiotics, including polymyxins and tetracyclines (Table S1); thus, it could be designated as extensively drug-resistant A. baumannii (Magiorakos et al., 2012; Sweeney et al., 2018). Therefore, bacteriophage IME200 was isolated from raw sewage collected at PLA Hospital 307, using A. baumannii AB1610 as the host bacterium. Briefly, sewage samples were centrifuged at 8,000 × g for 10 min, and the supernatants were collected and passed through a membrane filter with 0.20 µm pore size to remove remaining bacterial debris. To obtain the target phage, 4 mL of filtered sewage and 200 µL of bacterial culture in the exponential growth phase (optical density at 600 nm [OD₆₀₀] = 0.6) were added to 2 mL of a 3 × LB medium at 37 °C for overnight incubation with shaking at 220 rpm. The cultures containing the phage were rid of remaining bacterial cells and stored at 4 °C. The phage was isolated and purified with successive single-plaque assays using double-layer agar plates (Kropinski et al., 2009). To further characterize phage plaques, the double-layer agar plate assay was conducted. Briefly, 100 µL of the purified phage was diluted and combined with 200 µL of overnight-cultured bacteria. The mixture was then added to 4 mL of molten soft nutrient agar (0.75% agar) and rapidly overlaid onto 1.5% agar plates. The plates were incubated at 37 °C after the top layer of soft agar solidified, and phage plaques were examined at 12, 24, 48, and 72 h.

Multilocus sequence typing (MLST) of A. baumannii strains and determination of the lytic spectrum of phage IME200

All strains of A. baumannii isolated from clinical samples were typed by the MLST method according to the protocol described for A. baumannii strains on the MLST website (https://pubmlst.org/abaumannii/) (Bartual et al., 2005). Sequences of seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were aligned using the online National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), and the aligned sequences were then retrieved by comparing them to allele profiles in the A. baumannii MLST (Oxford) database.

The lytic spectrum of phage IME200 was determined in double-layer agar plate assays, with each of the 41 A. baumannii clinical isolates. Plates containing mixtures of the phage and an A. baumannii strain were incubated for 6 h at 37 °C, and plaque-forming units (PFU) were counted for each combination. Relative efficiency of plating (EOP) was calculated as the average PFU number of the phage on target bacteria divided by the average PFU number on host bacteria (Khan Mirzaei & Nilsson, 2015). The experiment was repeated three times.

DNA extraction, whole-genome sequencing, and bioinformatic analysis

DNase I (Thermo Scientific, Waltham, MA, USA) and RNase A (Thermo Scientific) were added to the phage IME200 to a final concentration of 1 µg/mL each and incubated overnight at 37 °C to remove host nucleic acids. The mixture was then inactivated for 15 min at 80 °C. Ethylenediamine tetraacetic acid (EDTA; 200 µg/mL; Thermo Scientific), proteinase K (50 µg/mL; Invitrogen, Carlsbad, CA, USA), and sodium dodecyl sulfate (SDS; 0.5 mg/mL; Invitrogen) were added to the mixture, which was then incubated for 1 h at 56 °C. After that, an equal volume of saturated phenol (pH 7.9; Thermo Scientific) was added, followed by gentle vortexing and centrifugation at 10,000× g for 5 min to separate the upper aqueous phase. Equal volumes of phenol: chloroform: isoamyl alcohol (25:24:1; Sigma-Aldrich, St. Louis, MO, USA) and chloroform (Hushi, Shanghai, China) were next added as described above to purify the phage. Finally, an equal volume of isopropanol (Hushi) was added to the DNA, and the mixture was incubated at −20 °C for at least 1 h and centrifuged at 10,000× g for 10 min, and the precipitate was washed with 75% ethanol (Hushi). The genomic DNA of the phage was dissolved in deionized water and stored at −20 °C.

The genomic DNA of phage IME200 was subjected to high-throughput sequencing on a Life Technologies Ion Personal Genome System (San Francisco, CA, USA). The complete genome sequence of the phage was assembled in Newbler 2.9.1 software (Roche, Indianapolis, IN, USA), and annotated by the Rapid Annotation in Subsystem Technology (RAST; http://rast.nmpdr.org/). The putative function of the protein was predicted using NCBI BLASTP, and tRNAs were predicted in tRNAScan-SE and ARAGORN (Laslett & Canback, 2004; Schattner, Brooks & Lowe, 2005). Homology analysis of the phage genome was performed via NCBI BLAST, and the whole-genome comparison was performed by means of the BLASTN program in local BLAST-2.2.31+. After the highly homologous sequences of phage genomes were analyzed and compared, a comparison of their amino acid sequences that are encoded by putative tail fiber genes was performed in CLC Genomics Workbench 3.6.1 (CLC bio, Aarhus, Denmark). The whole genome sequence of phage IME200 is available from GenBank/EMBL/DDBJ under accession number KT804908.2.

Expression, purification, and identification of Dpo48

Based on the annotation results of the complete genome sequences, the phage tail fiber protein gene (open reading frame 48 [ORF48]; GenBank/EMBL/DDBJ accession number ALJ97635) with a Pectate_lyase_3 domain in the C-terminal sequence was predicted to encode a putative polysaccharide depolymerase (Dpo48). The sequence of ORF48 was PCR-amplified with forward and reverse primers (5′-ATGAATATACTACGCTCATTTACAG-3′ and 3′-TTAAAATCCAGATACCACAGTAAA CATAGC-5′) carrying restriction endonuclease sites Bam HI (New England Biolabs [NEB], MA, USA) and Xho I (NEB), respectively. The amplified target product was cloned into the prokaryotic expression vector pET28a (Novagen, Madison, WI, USA) with a C-terminal His ×6 tag. The recombinant plasmid was verified by DNA sequencing on a 3730XL system (Thermo Scientific) and transfected into E. coli BL21 (DE3; TransGen Biotech, Beijing, China). The cells carrying recombinant plasmids were selected on LB agar plates containing 1 µg/mL kanamycin (Sigma-Aldrich). The recombinant isolates were cultured to the exponential growth phase, and then induced with 23.83 mg/mL isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich). After overnight culturing, the cells were centrifuged at 13,000× g for 10 min at 4 °C, resuspended in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0), and sonicated on ice (8–10 cycles with 30 s pulse and 30 s pause). The bacterial lysate was then centrifuged at 13,000× g for 10 min at 4 °C, and the supernatant was passed through a 0.22 µm filter. The filtrate was loaded onto a Ni-NTA column (Sangon Biotech, Shanghai, China), and proteins were eluted with 5 volumes of imidazole-containing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0) via a step gradient to recover the purified Dpo48. The latter was then dialyzed overnight in a bag of 8–14 kDa molecular-mass-cutoff membrane (Viskase, Willowbrook, IL, USA), against lysis buffer at 4 °C. The molecular weight of Dpo48 was determined by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE; Thermo Scientific) followed by staining with Coomassie blue (Sigma-Aldrich). Dpo48 was then quantified with the Bradford Protein Assay Kit (Thermo Fisher Scientific).

The depolymerase activity of Dpo48 was qualitatively assayed by modified single-spot assays. In brief, 200 µL of overnight bacterial culture was added to 4 mL of molten soft nutrient agar and incubated at 37 °C for 3 h to form a bacterial lawn in plates. The purified enzyme (0.5 mg/mL) was serially diluted, then 4 µL of each dilution was dropped onto an AB1610 bacterial lawn for incubation at 37 °C overnight. The plates were monitored for the formation of semiclear spots as a measure of enzymatic activity. In addition, the sensitivity of other A. baumannii strains to Dpo48 (0.25 µg/mL) was determined in modified single-spot assays.

Extraction of bacterial surface polysaccharides

The extraction and purification of bacterial EPS were performed via previously described methods (Hsieh et al., 2017) with some minor modifications. Briefly, clones of AB1610 were cultured overnight in the LB liquid medium with 0.25% glucose; then, 1 mL of the culture was centrifuged at 10,000× g for 5 min, and the pellet was resuspended in 200 µL of water. An equal volume of water-saturated phenol (pH 6.6; Thermo Scientific) was added, and the mixture was vortexed vigorously. The mixture was then incubated at 65 °C for 20 min, followed by chloroform extraction and centrifugation to remove bacterial debris. The extracted EPS was lyophilized overnight and stored at −20 °C.

Determination of Dpo48 activity and Alcian blue staining

Dpo48 activity was determined by the quantification of reducing sugars released from bacterial surface polysaccharides during a reaction with 3, 5-dinitrosalicylic acid (DNS) (Oliveira et al., 2017). Briefly, the lyophilized EPS powder was resuspended in PBS to a final concentration of 2 mg/mL, and incubated with 10 µg/mL of active or inactivated (by heating at 100 °C for 15 min) Dpo48 to a final reaction volume of 1.0 mL at 37 °C for 1 h. EPS or Dpo48 alone served as controls. Two volumes of the DNS reagent (Solarbio, Beijing, China) was immediately added to each reaction mixture, which was then boiled for 5 min. Absorbance was measured at 540 nm, and the experiment was repeated three times. Data are presented as means ± standard deviation (SD).

Each sample was separated by SDS-PAGE in a 10% gel, followed by staining with Alcian blue as described elsewhere (Karlyshev & Wren, 2001; Pan et al., 2013). In brief, the gel was washed three times (5, 10, and 15 min) with the fix/wash solution (25% ethanol, 10% acetic acid in water), and then soaked in 0.1% Alcian blue (Sigma-Aldrich) dissolved in the fix/wash solution for 15 min in the dark. The gel was finally destained overnight at room temperature in the fix/wash solution. Because the EPS was covalently bound to the cell surface as CPS (Roach & Donovan, 2015), the CPS stained blue.

Effects of pH and temperature on Dpo48 activity

The enzymatic activity at different pH levels or temperatures was assayed by methods from other studies (Majkowska-Skrobek et al., 2016), with some minor modifications. Briefly, the lyophilized extract powder was resuspended in 50 mM sodium acetate buffer (pH 3.0–5.0), 50 mM Na₂HPO₄ buffer (pH 6.0–7.0), or 50 mM Tris-HCl buffer (pH 8.0–9.0) to a final concentration of 2 mg/mL and incubated with 10 µg/mL active Dpo48 for 1 h at 37 °C. The final reaction volume was 1.0 mL. In addition, the polysaccharide was dissolved in a buffer with optimal pH, and incubated with Dpo48 at 20, 37, 50, 60, 70, or 80 °C for 1 h, followed by cooling to room temperature. EPS or Dpo48 alone in optimal-pH buffer incubated at 37 °C served as a control. The Dpo48 activity was then immediately measured as the reducing sugars produced, as described above. All the experiments were repeated at least three times, and the data were presented as means ± SD.

Serum sensitivity assay

To determine the optimal volume ratio of serum to enzyme-treated bacteria in a killing assay, an experiment was conducted as previously described with some minor modifications (Pan et al., 2015). Briefly, a total of ∼10⁹ CFU/mL of overnight culture AB1610 was incubated with or without 10 µg/mL Dpo48 at 37 °C for 1 h. Approximately 10⁷ CFUs of enzyme-treated bacteria were then added to human serum from healthy donors at volume ratios of 3:1, 1:1, or 1:3 to a final reaction volume of 100 µL. After the samples were incubated for 3 h at 37 °C, the bacteria were counted in serial 10-fold dilutions on LB agar plates. The result was expressed as the CFUs of bacterial reduction, which was calculated as the initial inoculum minus viable counts. To evaluate the role of serum complement in the serum killing assay, human serum was heated to 56 °C for 30 min to inactive the serum complement. Approximately 10⁷ CFUs of enzyme-treated bacteria were added to active or inactive serum at a volume ratio of 1:1 and cultured for 3 h at 37 °C. The untreated bacteria were incubated with Dpo48 or serum as a control, and the result was expressed as the counts of bacterial reduction. All the experiments were conducted independently three times, and the results were presented as means ± SD.

Statistical analysis

All experimental data are represented as means ± SD. The independent Student’s t test was utilized to compare two groups, and the one-way analysis of variance (ANOVA) was used to compare multiple groups. All statistical analyses were performed, and the results plotted in Prism 7 (GraphPad Software, La Jolla, CA, USA). Data with P < 0.05 were considered statistically significant.

Bacteriophage isolation and plaque formation

A bacteriophage was isolated from raw sewage collected at PLA Hospital 307 and was designated as IME200. When plated on A. baumannii AB1610, the isolated phage formed small, round, clear plaques on double-layer agar plates. The plaques were surrounded by a translucent halo, which increased in diameter over time (Fig. 1). The clear plaques were ∼2 mm in diameter, while the translucent halos were ∼6, 8, 10, and 12 mm at 12, 24, 48, and 72 h, respectively. The growing halos were assumed to result from the activity of depolymerase, an enzyme that can degrade bacterial surface polysaccharides (Pires et al., 2016).

MLST of A. baumannii strains and the lytic spectrum of phage IME200

Because phages are highly specific to their target bacteria, it is necessary to evaluate the lytic range of a phage before it can be used to combat bacterial infections. In a double-layer agar plate assay, the lytic spectrum of phage IME200 was tested against a panel of 41 A. baumannii strains, including the host bacterium. Results of the MLST analysis revealed that the 41 A. baumannii strains included 14 sequence types, whereas five other strains were untypeable (Table 2). Strains infected by phage IME200 belonged to ST 208 (AB5-2, AB1610, AB1611, AB1614, AB1615, AB-E85), 238 (AB4, AB5), 420 (AB220) and 1106 (LAB-9), and efficiency of plating (EOP) was greater than 0.25 (Table 2). Moreover, those 10 A. baumannii strains were also sensitive to Dpo48, which had the same spectrum of activity as did phage IME200.

The putative tail fiber protein (ORF48) has depolymerase activity

The assembly results indicated that the genome of phage IME200 is 41,243 bp, with G+C content of 39.3%, and no tRNA genes were found. The annotation results from RAST indicated that the complete genome of phage IME200 contains 54 putative ORFs, 20 of which were predicted by NCBI BLASTP to encode functional proteins (Fig. 2A and Table S2). As also depicted in Fig. 2A, ORF48 was predicted to contain a Pectate_lyase_3 domain in the C-terminal region, which is commonly recognized as a polysaccharide depolymerase, with a predicted length and molecular weight of 693 aa and 75.141 kDa, respectively. To verify whether ORF48 has depolymerase activity, the coding sequence of the gene was cloned into a prokaryotic expression vector. As predicted, the purified expressed protein, designated as Dpo48, was found to be 75 kDa as revealed by SDS-PAGE in the 10% gel (Fig. 2B), and the concentration of purified Dpo48 turned out to be 0.5 mg/mL. A single-spot assay using the purified Dpo48 revealed that ORF48 had polysaccharide depolymerase activity and could cause a translucent halo on the lawn of the host bacteria. The size of the semiclear circles correlated with Dpo48 concentration, with the halo disappearing when the enzyme was diluted to 0.1 ng (Fig. 2C).

The Dpo48 activity was quantified as the reducing sugars released from bacterial surface polysaccharides. As presented in Fig. 3A, OD₅₄₀ of EPS only and of Dpo48 in PBS was 0.122 ± 0.006 and 0.126 ± 0.003, respectively. OD₅₄₀ of EPS treated with active Dpo48 was 0.419 ± 0.035. By contrast, when the polysaccharide extracts were incubated with inactive Dpo48, OD₅₄₀ decreased to 0.129 ± 0.005. The increased OD₅₄₀ of the EPS treated with Dpo48, resulting from the production of reducing sugars, meant that the surface polysaccharides extracted from the host bacteria could be degraded by the depolymerase. In addition, the bacterial-capsule–degrading properties of Dpo48 were validated by gel electrophoresis followed by Alcian blue staining (Fig. 3B). Results of the gel electrophoresis also revealed that EPS could apparently be degraded by Dpo48, as compared with EPS alone, or the mixture of EPS and inactivated Dpo48, or Dpo48 alone, which failed to stain with Alcian blue. Dpo48 could therefore degrade the CPS of the bacterial surface.

The influence of pH and temperature on the activity of Dpo48

The activity of Dpo48 at different pH levels was determined by quantifying the production of reducing sugars (Fig. 4A). Results on OD₅₄₀ of EPS treated with Dpo48 in buffers with different pH levels indicated that Dpo48 remained active in buffers ranging from pH 5.0 to 9.0, with the optimal pH being 5.0 (one-way ANOVA, P < 0.0001). The activity of Dpo48 diminished when pH was 4.0 and almost disappeared compared with that of EPS alone or enzyme alone when pH was 3.0 (one-way ANOVA, P < 0.0001). Next, the activity of Dpo48 in sodium acetate buffer (pH 5.0), at temperatures ranging from 20 to 80 °C, was determined (Fig. 4B). OD₅₄₀ indicated that Dpo48 could maintain optimal activity at temperatures ranging from 20 to 70 °C, and that enzymatic activity diminished at 80 °C (one-way ANOVA, P < 0.05).

Serum killing of the Dpo48-treated bacteria

The enzyme-treated host bacteria AB1610 significantly decreased in number when they were mixed with different volumes of serum, as compared with untreated bacteria (Student’s t test, P < 0.001). The optimal volume ratio of enzyme-treated bacteria to serum was 1:1 (Fig. 5A). In other words, approximately five logs of enzyme-treated bacteria were killed by a 50% volume of serum, and the number of enzyme-treated bacteria did not significantly decrease further, despite the volume of serum being increased from 50% to 75%. For evaluating the role of serum complement in the serum killing assays, the viable counts of enzyme-treated bacteria mixed with active or inactive serum were determined (Fig. 5B). The number of surviving enzyme-pretreated bacteria incubated with active serum effectively decreased as compared with the other three groups (one-way ANOVA, P < 0.0001). In contrast, when the enzyme-treated bacteria were incubated with inactive serum, the reduction in the number of bacteria was almost the same as that in two control groups (one-way ANOVA, P = 0.2048).

Homology analysis of phage tail fiber proteins with polysaccharide depolymerase activity

Online BLAST analysis showed that the complete genomic sequence of phage IME200 shares high similarity of 93% to 97% with that of 17 Acinetobacter phages, viz. AS11 (KY268296.1), Abp1 (JX658790.1), B1 (MF033347.1), B3 (MF033348.1), Fri1 (KR149290.1), B5 (MF033349.1), SH-Ab 15519 (KY082667.1), WCHABP5 (KY888680.2), AS12 (KY268295.1), AB3 (KC311669.1), P1 (MF033350.1), P2 (MF033351.1), phiAB1 (HQ186308.1), phiAB6 (KT339321.1), PD-AB9 (KT388103.1), PD-6A3 (KT388102.1) and D2 (MH042230.1) (Chang et al., 2011; Hua et al., 2017; Huang et al., 2013; Lai et al., 2016; Oliveira et al., 2017; Popova et al., 2017; Zhang, Liu & Li, 2015), all of which belong to the genus Fri1virus within the subfamily Autographivirinae (Turner et al., 2017). Whole-genomic comparison indicated that these phage genomes have similar structures, but the regions encoding phage tail fiber proteins are significantly different. Because the phage tail fiber gene is related to host specificity and may encode a polysaccharide depolymerase, the amino acid sequences encoded by putative tail fiber proteins were compared. Results of an alignment by the neighbor-joining method indicated that although these phage tail fiber proteins have a relatively short, highly conserved region (from 1 aa–150 aa) at their N-terminal regions, most of their amino acid sequences were obviously diverse in their C-terminal regions (Fig. 6).