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. 2017 Nov 16:8:2257.
doi: 10.3389/fmicb.2017.02257. eCollection 2017.

Therapeutic Application of Phage Capsule Depolymerases against K1, K5, and K30 Capsulated E. coli in Mice

Han Lin  1 , Matthew L Paff  1   2 , Ian J Molineux  2   3 , James J Bull  1   2   4
Affiliations

Therapeutic Application of Phage Capsule Depolymerases against K1, K5, and K30 Capsulated E. coli in Mice

Han Lin et al. Front Microbiol. .

Abstract

Capsule depolymerase enzymes offer a promising class of new antibiotics. In vivo studies are encouraging but it is unclear how well this type of phage product will generalize in therapeutics, or whether different depolymerases against the same capsule function similarly. Here, in vivo efficacy was tested using cloned bacteriophage depolymerases against Escherichia coli strains with three different capsule types: K1, K5, and K30. When treating infections with the cognate capsule type in a mouse thigh model, the previously studied K1E depolymerase rescued poorly, whereas K1F, K1H, K5, and K30 depolymerases rescued well. K30 gp41 was identified as the catalytically active protein. In contrast to the in vivo studies, K1E enzyme actively degraded K1 capsule polysaccharide in vitro and sensitized K1 bacteria to serum killing. The only in vitro correlate of poor K1E performance in vivo was that the purified enzyme did not form the expected trimer. K1E appeared as an 18-mer which might limit its in vivo distribution. Overall, depolymerases were easily identified, cloned from phage genomes, and as purified proteins they proved generally effective.

Keywords: antibiotic; bacterial capsule; capsule depolymerase; infection; phage.

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Figures

FIGURE 1
FIGURE 1
Expression and purification of recombinant K1, K5, and K30 depolymerases. (A) SDS-PAGE showing induced expression and purification of K1E, K1F, and K1H depolymerases. 50 μg of uninduced or induced whole cell lysate obtained after sonication, or 2 μg of enzyme purified was fractionated by SDS-PAGE followed by Coomassie Blue staining. U, uninduced whole cell lysate; I, induced whole cell lysate; P, purified enzyme. (B) SDS-PAGE of K5 and putative K30 depolymerases after a similar induction and protein purification.
FIGURE 2
FIGURE 2
Mouse survival at Day 5 post infection and depolymerase treatment. 1.2–3.5 × 108 CFU of Escherichia coli RS218 (A), 1.7–3.7 × 108 of E. coli ATCC 23506 (B), or 1.0–3.7 × 108 of E. coli E69 (C) were injected to the left thigh, followed by corresponding depolymerase injection in the right thigh at various doses. Three mice each dose were preliminarily tested for dose titration, then more mice were repeated at select doses to validate the treatment efficacy. Mouse survival was monitored for 5 days. The numbers of surviving and dead mice at day 5 were evaluated by Fisher’s Exact Test for treatments with n >= 8: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control or as noted.
FIGURE 3
FIGURE 3
Mouse survival curves of depolymerase treatment. Kaplan–Meier survival curves of the 20 μg-K1 (A), K5 (B), or K30 (C) depolymerase treated mice and control mice in Figure 2 were plotted with the cumulative probability of survival over 5 days. The total mouse number n of each treatment is labeled by each curve. Log Rank test or generalized Wilcoxon test: ∗p < 0.05, ∗∗∗p < 0.001 compared to control.
FIGURE 4
FIGURE 4
Capsule degradation by depolymerases. (A) 10–20 μg of K1 capsule was incubated with serial dilutions of K1E, K1F, or K1H depolymerase at 37°C for 1 h, and then fractionated using 12% TBE-PAGE gel followed by Alcian Blue staining. Protein standards and dyes (XC, xylene cyanol FF; BPB, bromophenol blue; PR, phenol red) were loaded as molecular weight markers. Similar assays were performed using K5 (B) or putative K30 (C) depolymerase and their respective capsules. (D) Quantitative assay of capsule degradation by depolymerases. 30–45 μg of capsule was incubated with ranged doses of depolymerase for 30 min at 37°C. The product of reducing sugar was quantified by dinitrosalicylic acid (DNSA), with glucose as standard. The calculated amount of reducing sugar (nmol glucose equivalents) was plotted against enzyme doses.
FIGURE 5
FIGURE 5
Serum sensitivity assay. 4 × 107 CFU of E. coli RS218 (A), ATCC 23506 (B), or 6 × 107 of E. coli E69 (C) were incubated with or without their respective depolymerases (100 μg/ml) for 1.5–2 h at 37°C. The mixtures were diluted and 4–6 × 104 cells were treated with 75% serum for 1.5–2 h at 37°C before plating to determine CFU. Assays were repeated at least three times. The serum’s bactericidal effect was analyzed by Student’s t-test to compare cell survival in serum to that in PBS within each treatment: #p < 0.05, ###p < 0.001. The enzyme’s effect on serum sensitivity of the bacteria was analyzed by Student’s t-test to compare the ratio of cell survival in serum over survival in PBS between different enzyme treatments or the control (PBS): ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control or as noted.
FIGURE 6
FIGURE 6
Size exclusion chromatography of purified depolymerases. 1 mg K1E (A), 200 μg K1F (B), 500 μg K1H (C), 500 μg K5 (D), 200 μg K30 gp41 (E) or 200 μg K30 gp42 (F) was loaded to a Superose 6 10/300 GL column for chromatographic analysis. mAu, micro absorbance unit. Molecular weight of each peak was estimated using calibration standards (GE Healthcare). The estimated multimeric status and percentage are indicated.
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