Dataset Information

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

ABSTRACT: Background:The 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. Methods:A 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. Results:Phage 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. Conclusions:Phage-encoded capsule depolymerases may become promising antivirulence agents for preventing and controlling A. baumannii infections.

SUBMITTER: Liu Y

PROVIDER: S-EPMC6336015 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Identification and characterization of capsule depolymerase Dpo48 from <i>Acinetobacter baumannii</i> phage IME200.

Liu Yannan Y Mi Zhiqiang Z Mi Liyuan L Huang Yong Y Li Puyuan P Liu Huiying H Yuan Xin X Niu Wenkai W Jiang Ning N Bai Changqing C Gao Zhancheng Z

PeerJ 20190114

<h4>Background</h4>The emergence of multidrug- or extensively drug-resistant <i>Acinetobacter baumannii</i> 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 <i>A. baumannii</i> infections. In recent years, bacteriophages and their derivatives, such as depolymerases, showed great potential as antibacterial or antivirulence agents against bacterial infections. Noneth ...[more]

PMID: 30656071

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Project description:Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of how A. baumannii defends against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we identified genome-wide determinants of phage susceptibility in A. baumannii using Tn-seq. These studies focused on the lytic phage Loki, which targets Acinetobacter by unknown mechanisms. We identified 41 candidate loci that increase susceptibility to Loki when disrupted, and 10 that decrease susceptibility. Combined with spontaneous resistance mapping, our results support the model that Loki uses the K3 capsule as an essential receptor, and that capsule modulation provides A. baumannii with strategies to control vulnerability to phage. A key center of this control is transcriptional regulation of capsule synthesis and phage virulence by the global regulator BfmRS. Mutations hyperactivating BfmRS simultaneously increase capsule levels, Loki adsorption, Loki replication, and host killing, while BfmRS-inactivating mutations have the opposite effect, reducing capsule and blocking Loki infection. We identified novel BfmRS-activating mutations, including knockouts of a T2 RNase protein and the disulfide formation enzyme DsbA, that hypersensitize bacteria to phage challenge. We further found that mutation of a glycosyltransferase known to alter capsule structure and bacterial virulence can also cause complete phage resistance. Finally, additional factors including lipooligosaccharide and Lon protease act independently of capsule modulation to interfere with Loki infection. This work demonstrates that regulatory and structural modulation of capsule, known to alter A. baumannii virulence, is also a major determinant of susceptibility to phage. Author summary Antibiotic-resistant infections with Acinetobacter baumannii are a major problem in critical care units and have increased in frequency during the COVID-19 pandemic. The virulence of these infections depends on a polysaccharide capsule surrounding the bacterium. Phage, or viruses that kill bacteria, represent a promising alternative therapy against highly antibiotic-resistant A. baumannii infections, and A. baumannii-specific phage often target the capsule. Here, we use high-throughput genetics to analyze how A. baumannii defends against phage and identify ways to potentiate their killing activity. We found that stressing the bacteria in ways that cause augmented production of capsule also causes hyper-susceptibility to phage. By contrast, turning off the stress response, or mutating the capsule structure, causes complete phage resistance. Altering another surface structure, lipooligosaccharide, or an intracellular protease also enhances phage attack. Modulating the amounts or makeup of capsular polysaccharide is known to influence virulence in A. baumannii. This work thus uncovers a connection between phage pressure and the evolution of virulence in A. baumannii, and it identifies control mechanisms that may be leveraged for improving future phage-based antimicrobial therapies.

Project description:Acinetobacter baumannii is an important pathogen causative of health care-associated infections and is able to rapidly develop resistance to all known antibiotics, including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbaM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule types, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80°C). B9gp69 was demonstrated to be nontoxic in a cell line model of the human lung and to make the K45 strain fully susceptible to serum killing in vitro Contrary to the case with phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.IMPORTANCE Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Here we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterized the phage-encoded depolymerase, which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that the K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection.

Project description:Acinetobacter baumannii is emerging as a major nosocomial pathogen in intensive care units. The bacterial capsules are considered major virulence factors, and the particular A. baumannii capsular type K2 has been associated with high antibiotic resistance. In this study, we identified a K2 capsule-specific depolymerase in a bacteriophage tail spike C terminus, a fragment that was heterologously expressed, and its antivirulence properties were assessed by in vivo experiments. The K2 depolymerase is active under a broad range of environmental conditions and is highly thermostable, with a melting point (Tm ) at 67°C. In the caterpillar larva model, the K2 depolymerase protects larvae from bacterial infections, using either pretreatments or with single-enzyme injection after bacterial challenge, in a dose-dependent manner. In a mouse sepsis model, a single K2 depolymerase intraperitoneal injection of 50 μg is able to protect 60% of mice from an otherwise deadly infection, with a significant reduction in the proinflammatory cytokine profile. We showed that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. Moreover, the K2 depolymerase is highly refractory to resistance development, which makes these bacteriophage-derived capsular depolymerases useful antivirulence agents against multidrug-resistant A. baumannii infections.IMPORTANCEAcinetobacter baumannii is an important nosocomial pathogen resistant to many, and sometimes all, antibiotics. The A. baumannii K2 capsular type has been associated with elevated antibiotic resistance. The capsular depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug-resistant pathogens. They are highly specific, stable, and refractory to resistance, as they do not kill bacteria per se; instead, they remove bacterial surface polysaccharides, which diminish the bacterial virulence and expose them to the host immune system.

Dataset Information

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

Publications

Identification and characterization of capsule depolymerase Dpo48 from <i>Acinetobacter baumannii</i> phage IME200.

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