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

Abstract

BackgroundThe emergence of multidrug- or extensively drug-resistant Acinetobacter baumannii has made it difficult to treat and control infections caused by this bacterium. It is urgently necessary to search for alternatives to conventional antibiotics for control of severe A. baumannii infections. In recent years, bacteriophages and their derivatives, such as depolymerases, showed great potential as antibacterial or antivirulence agents against bacterial infections. Nonetheless, unlike broad-spectrum bactericidal antibiotics, phage-encoded depolymerase targets only a limited number of bacterial strains. Therefore, identification of novel depolymerases and evaluation of their ability to control A. baumannii infections is important.MethodsA bacteriophage was isolated from hospital sewage using an extensively drug-resistant A. baumannii strain as the host bacterium, and the phage’s plaque morphology and genomic composition were studied. A polysaccharide depolymerase (Dpo48) was expressed and identified, and the effects of pH and temperature on its activity were determined. Besides, a serum killing assay was conducted, and amino acid sequences homologous to those of putative polysaccharide depolymerases were compared.ResultsPhage IME200 yielded clear plaques surrounded by enlarged halos, with polysaccharide depolymerase activity against the host bacterium. A tail fiber protein with a Pectate_lyase_3 domain was identified as Dpo48 and characterized . Dpo48 was found to degrade the capsule polysaccharide of the bacterial surface, as revealed by Alcian blue staining. Dpo48 manifested stable activity over a broad range of pH (5.0–9.0) and temperatures (20–70 °C). Results from in vitro serum killing assays indicated that 50% serum was sufficient to cause a five log reduction of overnight enzyme-treated bacteria, with serum complement playing an important role in these killing assays. Moreover, Dpo48 had a spectrum of activity exactly the same as its parental phage IME200, which was active against 10 out of 41 A. baumannii strains. Amino acid sequence alignment showed that the putative tail fiber proteins had a relatively short, highly conserved domain in their N-terminal sequences, but their amino acid sequences containing pectate lyase domains, found in the C-terminal regions, were highly diverse.ConclusionsPhage-encoded capsule depolymerases may become promising antivirulence agents for preventing and controlling A. baumannii infections.

Identification of Two Depolymerases From Phage IME205 and Their Antivirulent Functions on K47 Capsule of Klebsiella pneumoniae

“…It was noted that the enzyme mixed with serum could not completely eradicate all bacteria. Similar observation was noted in our previous study (Liu et al, 2019b). We found that Dpo48 depolymerase could form translucent halos on the lawn of non-treated bacteria but not on the lawn of enzyme-treated bacteria, suggesting that the enzyme should have removed the capsules of the bacteria.…”

Genomic and Phenotypic Analyses of Acinetobacter baumannii Isolates From Three Tertiary Care Hospitals in Thailand

“…The enzyme also sensitized the C'-resistant isolate to serum (Liu et al, 2019b), which is highly relevant as the large majority of our GC2 isolates (40/45) were C' resistant, in similar proportion to other recent studies (Sanchez-Larrayoz et al, 2017;Skerniškytë et al, 2019). LPS O-side chains prevent assembly of the C5b-9 complex by steric hindrance; A. baumannii however does not decorate its LOS with O-side chains but is able to modify the lipid A moiety of LOS by acylation, resulting in increased survival in blood (Bartholomew et al, 2019), which could prevent C5b-9 intercalation into the bilayer.…”

Diversity and Function of Phage Encoded Depolymerases

“…The authors found that the enzymatic activity is enhanced by divalent cations (Hernandez-Morales et al, 2018). Liu et al (2019) identified the TSP Dpo48 in phage IME200. The enzyme tolerates pH 5 -9 and temperatures of 70 • C. The K-type specificity of Dpo48 is still unknown (Liu et al, 2019).…”

“…Liu et al (2019) identified the TSP Dpo48 in phage IME200. The enzyme tolerates pH 5 -9 and temperatures of 70 • C. The K-type specificity of Dpo48 is still unknown (Liu et al, 2019). Recently, Oliveira et al (2019b) characterized gp38 of bacteriophage B3, a member of the Autographivirinae.…”

“…The interaction of the purified enzyme and the target bacteria was analyzed during infection. It seems that every tested enzyme renders the ACB bacteria sensitive to serum killing by the complement system and that infected animals either survive significantly longer, or can be even cured after infection in laboratory settings (Oliveira et al, 2017;Liu et al, 2019;Oliveira et al, 2019a,b). One TSP, Dpo1 of phage Petty, was also tested for its ability to degrade biofilms produced by A. baumannii.…”