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

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Microbiology

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Introduction

Materials and Methods

Bacterial strains and culture conditions

Bacteriophage isolation, purification, and plaque characterization

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

DNA extraction, whole-genome sequencing, and bioinformatic analysis

Expression, purification, and identification of Dpo48

Extraction of bacterial surface polysaccharides

Determination of Dpo48 activity and Alcian blue staining

Effects of pH and temperature on Dpo48 activity

Serum sensitivity assay

Statistical analysis

Results

Bacteriophage isolation and plaque formation

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

The putative tail fiber protein (ORF48) has depolymerase activity

The influence of pH and temperature on the activity of Dpo48

Serum killing of the Dpo48-treated bacteria

Homology analysis of phage tail fiber proteins with polysaccharide depolymerase activity

Discussion

Conclusions

Supplemental Information

The antibiotic resistance profile of the clinical strain A. baumannii AB1610

DOI: 10.7717/peerj.6173/supp-1

The list of ORFs in the genome of phage IME200 and their putative functions

DOI: 10.7717/peerj.6173/supp-2

Dpo48 enhanced serum sensitivity of A. baumannii

A total of 104 CFUs of enzyme-treated AB1610 were incubated with human serum at a volume ratio of 1:3. After 3 h at 37 °C, the data were recorded as the survival ratio of bacteria (CFUs of viable bacteria /CFUs of initial inoculum). Data are expressed as the survival ratio of bacteria (means ± SD; n = 3), and Student’s t test was carried out to compare the groups (****P < 0.0001).

DOI: 10.7717/peerj.6173/supp-4

Enzyme-treated A. baumannii was not sensitive to Dpo48

The sensitivity of the host (A) or enzyme-treated bacterium AB1610 (B) to Dpo48 (2 μg) was determined by modified single-spot assays. Formation of a transparent halo as a measure of bacterial sensitivity. PBS served as a negative control.

DOI: 10.7717/peerj.6173/supp-5

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Yannan Liu conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Zhiqiang Mi conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper.

Liyuan Mi performed the experiments.

Yong Huang assembled and analyzed the sequence of the phage genome.

Puyuan Li, Huiying Liu, Xin Yuan, Wenkai Niu and Ning Jiang collected the clinical strains of bacteria.

Changqing Bai and Zhancheng Gao contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.

Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The protocols for collecting clinical samples and screening 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).

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

The sequence of bacteriophage IME200 described here are accessible via GenBank accession number KT804908.2.

Data Availability

The following information was supplied regarding data availability:

The raw data has been supplied as a Supplemental File.

Funding

This project was supported by the National Natural Science Foundation of China (grant no. 81572045), the Capital Health Research and Development of Special (grant no. 2016-2-5062), the Capital Characteristic Clinic Project of Beijing (grant no. Z161100000516181), and the National Key Research and Development Program of China (2016YFC0903800). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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