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Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii.

ABSTRACT: Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical β₁α₁β₂β₃α₂β₄ and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.

SUBMITTER: Ciani C

PROVIDER: S-EPMC9313427 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii.

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