Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr 14;11(4):e0153361.
doi: 10.1371/journal.pone.0153361. eCollection 2016.

The Tail Associated Protein of Acinetobacter baumannii Phage ΦAB6 Is the Host Specificity Determinant Possessing Exopolysaccharide Depolymerase Activity

Meng-Jiun Lai  1 , Kai-Chih Chang  1 , Shiuan-Wen Huang  2 , Cheng-Hung Luo  3 , Pei-Yu Chiou  3 , Chao-Chuan Wu  4 , Nien-Tsung Lin  2   3
Affiliations

The Tail Associated Protein of Acinetobacter baumannii Phage ΦAB6 Is the Host Specificity Determinant Possessing Exopolysaccharide Depolymerase Activity

Meng-Jiun Lai et al. PLoS One. .

Abstract

Acinetobacter baumannii is a non-fermenting, gram-negative bacterium. In recent years, the frequency of A. baumannii infections has continued to increase, and multidrug-resistant strains are emerging in hospitalized patients. Therefore, as therapeutic options become limited, the potential of phages as natural antimicrobial agents to control infections is worth reconsidering. In our previous study, we isolated ten virulent double-stranded DNA A. baumannii phages, ϕAB1-9 and ϕAB11, and found that each has a narrow host range. Many reports indicate that receptor-binding protein of phage mediates host recognition; however, understanding of the specific interactions between A. baumannii and phages remains very limited. In this study, host determinants of A. baumannii phages were investigated. Sequence comparison of ϕAB6 and ϕAB1 revealed high degrees of conservation among their genes except the tail fiber protein (ORF41 in ϕAB1 and ORF40 in ϕAB6). Furthermore, we found that ORF40ϕAB6 has polysaccharide depolymerase activity capable of hydrolyzing the A. baumannii exopolysaccharide and is a component of the phage tail apparatus determining host specificity. Thus, the lytic phages and their associated depolymerase not only have potential as alternative therapeutic agents for treating A. baumannii infections but also provide useful and highly specific tools for studying host strain exopolysaccharides and producing glycoconjugate vaccines.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Sequence alignment of tail fibers of A. baumannii phages.
(A) Amino acid sequence alignment of the phage tail fibers encoded by ϕAB1 ORF41 and ϕAB6 ORF40, respectively. (B) Predicted domain structure of ϕAB6 ORF40. The numbers on the top indicate size in amino acid residues. The domains are identified as follow: T7 tail (aa 10–112), T7 phage tail domain; PLN03010 (aa 218–274), the PLN03010 domain of polygalacturonase; and PL 3 (aa 226–419) pectate lyase 3 superfamily.
Fig 2
Fig 2. Immunogold-labeling electron microscopy of ϕAB6 phage particles.
The primary antibody targeting ϕAB6 ORF40 is described in Materials and Methods. The secondary antibody was conjugated to 6-nm diameter gold particles (black dots). Scale bar, 100 nm.
Fig 3
Fig 3. Translucent halo formation by phage ϕAB6.
Clear plaques surrounded by translucent halos were observed in the plaque assay of phage ϕAB6 with A. baumannii strain 54149 as the indicator host.
Fig 4
Fig 4. Overexpression and depolymerization activity test of ORF40ϕAB6 and ORF41ϕAB1 proteins.
(A) SDS-PAGE of the recombinant proteins overexpressed. Lanes: M, protein markers; 1 and 4, BL21(DE3) harboring an empty plasmid; 2, uninduced BL21(DE3)/pET-orf40ϕAB6; 3, BL21(DE3)/pET-orf40ϕAB6 induced with 0.1 mM IPTG; 5, uninduced BL21(DE3)/pET-orf41ϕAB1; 6, BL21(DE3)/pET-orf40ϕAB6 induced with 0.1 mM IPTG. The arrowheads indicate the overexpressed recombinant proteins. (B) Enzyme activity of purified proteins was tested by spot test. Halo surrounding stands for positive activity.
Fig 5
Fig 5. Confirmation of chimeric phage ϕAB1tf6 by PCR.
(A) Maps of orf41ϕAB1/orf40ϕAB6 and the surrounding genes. (B) The PCR reactions were performed on the genomes of ϕAB1, ϕAB6, and ϕAB1tf6 as the templates with pf1 and pr3 primers and the amplicons were subjected to separation in 0.8% agarose gel.
Fig 6
Fig 6. Confirmation of chimeric phage ϕAB1tf6 by RFLP.
(A) The SphI-restriction map shows orf41ϕAB1/orf40ϕAB6 and surrounding genes. (B) RFLP was performed on genomic DNA of ϕAB1, ϕAB6, and ϕAB1tf6 with SphI digestion. The digests were subjected to separation in 0.8% agarose gel.
Fig 7
Fig 7. Spot assay for testing the infection ability of ϕAB1, ϕAB6, and ϕAB1tf6 for A. baumannii strains 54149 and M68316.
Fig 8
Fig 8. Adsorption assay for testing the specificity of ϕAB1, ϕAB6, and chimeric phage ϕAB1tf6, as described in Materials and Methods, in binding to A. baumannii strains M68316 and 54149.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21: 538–582. 10.1128/CMR.00058-07 - DOI - PMC - PubMed
    1. Duplessis M, Moineau S. Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages. Mol Microbiol. 2001; 41: 325–336. - PubMed
    1. Dupont K, Vogensen FK, Neve H, Bresciani J, Josephsen J. Identification of the receptor-binding protein in 936-species lactococcal bacteriophages. Appl Environ Microbiol. 2004; 70: 5818–5824. - PMC - PubMed
    1. Le S, He X, Tan Y, Huang G, Zhang L, Lux R, et al. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004. PLoS One. 2013; 8: e68562 10.1371/journal.pone.0068562 - DOI - PMC - PubMed
    1. Rakhuba DV, Kolomiets EI, Dey ES, Novik GI. Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell. Pol J Microbiol. 2010; 59: 145–155. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This work was supported by grants TCSP98-03-01 from Buddhist Tzu Chi General Hospital and TCIRP98003-02 from Tzu Chi University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.