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Unpuzzling Friunavirus-Host Interactions One Piece at a Time: Phage Recognizes Acinetobacter pittii via a New K38 Capsule Depolymerase.

ABSTRACT: Acinetobacter pittii is a species that belong to the Acinetobacter calcoaceticus-baumannii complex, increasingly recognized as major nosocomial bacterial pathogens, often associated with multiple drug-resistances. The capsule surrounding the bacteria represents a main virulence factor, helping cells avoid phage predation and host immunity. Accordingly, a better understanding of the phage infection mechanisms is required to efficiently develop phage therapy against Acinetobacter of different capsular types. Here, we report the isolation of the novel A. pittii-infecting Fri1-like phage vB_Api_3043-K38 (3043-K38) of the Podoviridae morphotype, from sewage samples. Its 41,580 bp linear double-stranded DNA genome harbours 53 open reading frames and 302 bp of terminal repeats. We show that all studied Acinetobacter Fri1-like viruses have highly similar genomes, which differentiate only at the genes coding for tailspike, likely to adapt to different host receptors. The isolated phage 3043-K38 specifically recognizes an untapped Acinetobacter K38 capsule type via a novel tailspike with K38 depolymerase activity. The recombinant K38 depolymerase region of the tailspike (center-end region) forms a thermostable trimer, and quickly degrades capsules. When the K38 depolymerase is applied to the cells, it makes them resistant to phage predation. Interestingly, while K38 depolymerase treatments do not synergize with antibiotics, it makes bacterial cells highly susceptible to the host serum complement. In summary, we characterized a novel phage-encoded K38 depolymerase, which not only advances our understanding of phage-host interactions, but could also be further explored as a new antibacterial agent against drug-resistant Acinetobacter.

SUBMITTER: Domingues R

PROVIDER: S-EPMC8614642 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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.

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Unpuzzling Friunavirus-Host Interactions One Piece at a Time: Phage Recognizes Acinetobacter pittii via a New K38 Capsule Depolymerase.

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