RNA and sugars, unique properties of bacteriophages infecting multidrug resistant Acinetobacter radioresistens strain LH6

Authors: CRIPPEN, CLAY - University Of Georgia; ZHOU, BIBI - University Of Georgia; ANDRESEN, S - University Of Georgia; PATRY, ROBERT - University Of Georgia; MUSZYNSKI, ARTUR - University Of Georgia; Parker, Craig; COOPER, KERRY - University Of Arizona; SZYMANSKI, CHRISTINE - University Of Georgia

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2021
Publication Date: 8/20/2021
Citation: Crippen, C.S., Zhou, B., Andersen, S., Patry, R.T., Muszynski, A., Parker, C.T., Cooper, K.K., Szymanski, C.M. 2021. RNA and sugars, unique properties of bacteriophages infecting multidrug resistant Acinetobacter radioresistens strain LH6. Viruses. 13(8). Article 1652. https://doi.org/10.3390/v13081652.
DOI: https://doi.org/10.3390/v13081652

Interpretive Summary: Bacteriophages (phages) are viruses that replicate in bacterial hosts and have been gaining attention for their potential use in biocontrol of multiantibiotic resistant pathogenic bacteria. Acinetobacter are a genus of bacteria that include opportunistic human pathogens that are listed by the World Health Organization and the Centers for Disease Control and Prevention as pathogens for which new antimicrobials (including phages) are most desperately needed. Phages are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage-host interactions. The accumulation of knowledge about bacteriophage diversity, life cycles and binding targets are important foundations to better understand these viruses, which can be used by researchers and physicians combatting drug-resistant infections with phage therapy. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1-2), five dsRNA lytic cystoviruses (CAP3-7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the 3 dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane of the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Although further characterization is necessary to obtain structural validation, this is the first evidence of surface glycosylation in any bacteriophage, and the first isolation of cystoviruses that infect non-Pseudomonas species.

Technical Abstract: Bacteriophages (phages) are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage-host interactions. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1-2), five unique dsRNA lytic cystoviruses (CAP3-7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the 3 dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane of the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Although further characterization is necessary to obtain structural validation, this is the first evidence of surface glycosylation in any bacteriophage, and the first isolation of cystoviruses that infect non-Pseudomonas species.