Project description:Bacteriophage genomes exhibit exceptional diversity in nucleobase modifications, which primarily function to counteract host immunity and reshape DNA physicochemical properties. Recent discoveries of aGPT-Pplase2-catalyzed novel thymidine hypermodifications reveal a broader enzymatic and chemical diversity in phage DNA modification systems. However, the diversity of thymidine hypermodification in other bacteriophages remains largely unexplored. Here we discovered a novel thymidine hypermodification, Na-dapT, in the Acinetobacter baumannii phage SH-Ab 15599, and elucidated its biosynthetic pathway, including the key diamine DNA transferase (DADT, formerly aGPT-Pplase2). DADT utilizes the abundant metabolite 1,3-diaminopropane of host to modify 5hmdU-DNA, exhibiting broad in vitro substrate specificity but a strong in vivo preference for 1,3-diaminopropane. Structural and mutagenesis analyses revealed the molecular basis for substrate recognition and catalysis. The Na-dapT modification occurs specifically at TG dinucleotides and confers resistance to multiple host restriction enzymes, enabling phage escape from the host restriction-modification system. Furthermore, RNA-seq analysis showed that phage infection reprograms host metabolism, upregulating genes for 1,3-diaminopropane synthesis to supply the modification precursor. Our study identified SH-Ab 15599 DADT as a versatile enzyme responsible for thymidine hypermodification, and comprehensively describes a viral strategy of exploiting host metabolites for DNA modification to evade bacterial defense.
Project description:The emergence of carbapenem-resistant Acinetobacter baumannii has been increasingly reported, leading to more challenges in treating its infections. With the development of phage therapy and phage-antibiotic combinations, it is possible to improve the treatment of bacterial infections. In the present study, a vB_AbaP_WU2001 (vWU2001 for short) phage-specific CRAB was isolated and the genome size is 40,792 bp in length. The novel phage vWU2001 belongs to the Autographiviridae family and the order Caudovirales. Shotgun proteomics identified 289 proteins. The broad host range phage vWU2001 displayed a high adsorption rate, short latent period, large burst size and good stability. The phage could reduce preformed biofilms and inhibit biofilm formation. The combination of phage vWU2001 and colistin had significantly higher bacterial growth inhibition activity than that of phage, or colistin alone. The efficacy of the combined treatment was also evaluated in Galleria mellonella. The evaluation of its therapeutic potential revealed that the combination of phage and colistin showed a significantly greater increase in G. mellonella survival and clearance of bacterial number compared to that of phage or colistin alone, indicating that the combination was synergistic against CRAB. The results demonstrated that phage vWU2001 has the potential to be developed as an antibacterial agent.
