Dataset Information

Capsule Protects Acinetobacter baumannii From Inter-Bacterial Competition Mediated by CdiA Toxin.

ABSTRACT: Currently, Acinetobacter baumannii is considered as one of the most important infectious agents causing hospital acquired infections worldwide. It has been observed that many clinically important pathogens express contact-dependent growth inhibition (CDI) phenomenon, which modulates cell-cell and cell-environment interactions, potentially allowing bacteria to adapt to ever-changing conditions. Mainly, these systems are used for the inhibition of the growth of genetically different individuals within the same species. In this work, by performing cell competition assays with three genotypically different (as determined by pulse-field gel electrophoresis) clinical A. baumannii isolates II-c, II-a, and II-a1, we show that A. baumannii capsule is the main feature protecting from CDI-mediated inhibition. We also observed that for one clinical isolate, the two-component BfmRS system, contributed to the resistance against CDI-mediated inhibition. Moreover, we were able to demonstrate, that the effector protein CdiA is released into the growth media and exhibits its inhibitory activity without the requirement of a cell-cell contact. Lastly, by evaluating the remaining number of the cells pre-mixed with the CdiA and performing live/dead assay, we demonstrate that purified CdiA protein causes a rapid cell growth arrest. Our results indicate, that capsule efficiently protects A. baumannii from a CDI-mediated inhibition by a clinical A. baumannii V15 strain, which is able to secrete CdiA effector into the growth media and cause target cell growth arrest without a cell-cell contact.

SUBMITTER: Krasauskas R

PROVIDER: S-EPMC7396552 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Capsule Protects <i>Acinetobacter baumannii</i> From Inter-Bacterial Competition Mediated by CdiA Toxin.

Krasauskas Renatas R Skerniškytė Jūratė J Martinkus Julius J Armalytė Julija J Sužiedėlienė Edita E

Frontiers in microbiology 20200717

Currently, <i>Acinetobacter baumannii</i> is considered as one of the most important infectious agents causing hospital acquired infections worldwide. It has been observed that many clinically important pathogens express contact-dependent growth inhibition (CDI) phenomenon, which modulates cell-cell and cell-environment interactions, potentially allowing bacteria to adapt to ever-changing conditions. Mainly, these systems are used for the inhibition of the growth of genetically different individ ...[more]

PMID: 32849318

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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.

Project description: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.

Project description:Acinetobacter baumannii is emerging as a leading global multiple-antibiotic-resistant nosocomial pathogen. The identity of genes essential for pathogenesis in a mammalian host remains largely unknown. Using transposon-directed insertion-site sequencing (TraDIS), we identified A. baumannii genes involved in bacterial survival in a leukopenic mouse model of bloodstream infection. Mice were inoculated with a pooled transposon mutant library derived from 109,000 mutants, and TraDIS was used to map transposon insertion sites in the genomes of bacteria in the inoculum and of bacteria recovered from mouse spleens. Unique transposon insertion sites were mapped and used to calculate a fitness factor for every insertion site based on its relative abundance in the inoculum and postinfection libraries. Eighty-nine transposon insertion mutants that were underrepresented after experimental infection in mice compared to their presence in the inocula were delineated as candidates for further evaluation. Genetically defined mutants lacking feoB (ferrous iron import), ddc (d-ala-d-ala-carboxypeptidase), and pntB (pyridine nucleotide transhydrogenase subunit) exhibited a fitness defect during systemic infection resulting from bacteremia. In vitro, these mutants, as well as a fepA (ferric enterobactin receptor) mutant, are defective in survival in human serum and within macrophages and are hypersensitive to killing by antimicrobial peptides compared to the survival of the parental strain under these conditions. Our data demonstrate that FepA is involved in the uptake of exogenous enterobactin in A. baumannii. Genetic complementation rescues the phenotypes of mutants in assays that emulate conditions encountered during infection. In summary, we have determined novel A. baumannii fitness genes involved in the pathogenesis of mammalian infection. IMPORTANCE A. baumannii is a significant cause of bacterial bloodstream infection in humans. Since multiple antibiotic resistance is becoming more common among strains of A. baumannii, there is an urgent need to develop novel tools to treat infections caused by this dangerous pathogen. To develop knowledge-guided treatment approaches for A. baumannii, a thorough understanding of the mechanism by which this pathogen causes bloodstream infection is required. Here, using a mouse model of infection, we report the identification of A. baumannii genes that are critical for the ability of this pathogen to cause bloodstream infections. This study lays the foundation for future research on A. baumannii genes that can be targeted to develop novel therapeutics against this emerging human pathogen.

Dataset Information

Capsule Protects Acinetobacter baumannii From Inter-Bacterial Competition Mediated by CdiA Toxin.

Publications

Capsule Protects <i>Acinetobacter baumannii</i> From Inter-Bacterial Competition Mediated by CdiA Toxin.

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