Lytic myophage Abp53 encodes several proteins similar to those encoded by host Acinetobacter baumannii and phage phiKO2

Abstract

Acinetobacter baumannii is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant A. baumannii isolates has increased in recent years. Directed toward phage therapy, a lytic phage of A. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to Myoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the A. baumannii isolates tested, which were all multiple drug resistant, but not other bacteria. Mg(2+) enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by A. baumannii strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of Klebsiella phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of A. baumannii ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.

Fig. 1.

Dendrogram based on PFGE patterns of ApaI-digested chromosomal DNAs from 10 different A. baumannii strains. Electrophoresis was performed with 0.8% agarose at 13°C (6 V/cm; high, 35 s; low, 2.2 s; in a linear manner for 18 h). The dendrogram was constructed with BioNumerics software (Applied Maths, Kortrijk, Belgium) as described in Materials and Methods. Percent similarity is indicated on the scale.

Fig. 2.

Transmission electron microscopy of Abp53 phage particles (A) and an A. baumannii cell with Abp53 phage particles adsorbed to the surface (B).

Fig. 3.

(A) Time course of phage Abp53 adsorption to the host cells of A. baumannii strain Ab53. Symbols: ○, LB broth; •, LB broth containing 10 mM MgSO₄. (B) Lysis of cells of Ab53 by phage Abp53. Curves labeled with only MOI are for assays performed in LB medium with 10 mM MgSO₄, while the upper three curves represent results of experiments performed for comparison. (C) One-step growth curve of Abp53 on A. baumannii strain Ab53. The medium used was LB containing 10 mM MgSO₄.

Fig. 4.

(A) Agarose gel (0.7%) electrophoresis of phage Abp53 DNA digested with different restriction endonucleases (shown above the lanes). Lane M contains a 1-kb DNA ladder (from Protech Biotechnology, Taiwan) as size markers. (B) PFGE of the Abp53 genomic DNA (ca. 30 ng). Electrophoresis was performed with a 1% agarose gel at 14°C and 200 V (with an initial time of 1 s and a final time of 40 s), running for 20 h. Lane M contains a lambda DNA ladder (New England BioLabs) as molecular size markers.

Fig. 5.

Genome organization of the 10-kb region from Abp53 (top), similar genes from A. baumannii strain ACICU, including the predicted tail protein gene ACICU02717 (middle), and predicted tail protein gene gp21 from K. oxytoca phage phiKO2 (bottom). Similar regions in the predicted proteins are linked by shading, with degrees of similarity shared being labeled. Short thick arrows indicate duplicated domains.