RNA and Sugars, Unique Properties of Bacteriophages Infecting Multidrug Resistant Acinetobacter radioresistens Strain LH6

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 three 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 from 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. Together, this study demonstrates the isolation of new Acinetobacter-infecting phages and the determination of their cell receptors. Further, we describe the genomes of a new genus of Cystoviruses and perform an initial characterization of membrane-associated glycoconjugates.

Keywords: Acinetobacter; bacteriophages; capsular polysaccharides; lipooligosaccharides; pilin; segmented RNA viruses.

Figure 1

Proposed schematic life cycle of A. radioresistens infecting cystovirus CAP3. (a) CAP3 binds to the protruding pilus. (b) CAP3 fuses with the host outer membrane, degrading host peptidoglycan with its muramidase. (c) CAP3 capsid enters the cytoplasm. (d) dsRNA segments are polymerized out of the capsids as +ssRNA (prior to entry into a new capsid for dsRNA synthesis) and viral replication takes place. (e) The cell inner membrane, the site of most bacterial glycoconjugate biosynthetic assemblies, is used to envelop virion particles. (f) Mature virion particles are constructed, potentially with intermediates of host glycosylation embedded in the membrane, ready to lyse the cell and be released.

Figure 2

Characterization of LH6-infecting phages. Transmission electron micrographs of (a) CAP1, (b) CAP3 and (c) SLAP1. (d) Plaque morphology of isolated phages. (e) CAP1 and CAP3 genome before and after treatment with DNase I or RNase A. (f) CAP3 genome is not degraded with RNase I_f. (g) PCR analysis of SLAP1 and LH6 gDNA using capsid primers from prophage 1 (lanes 1) and prophage 2 (lanes 2) in the LH6 genome [6].

Figure 3

Determination of host binding receptor for CAP1 and CAP3. (a) Determination of plaquing efficiency for CAP1 (red circles) and CAP3 (blue circles). Significance was calculated using unpaired t-tests. *** p ≤ 0.001; **** p ≤ 0.0001 (b) Transmission electron micrograph of LH6 WT showing CAP3 attachment to pili (indicated by red arrows). Inset: enlargement of single pilus. (c) Schematic of effects on LH6 structures when pglC, pilA, lpsC and clsB genes are deleted. (d) Transmission electron micrograph of LH6 ∆pilA showing a lack of pili and reduced CAP3 association. Inset: enlargement of cell surface showing no observable pili. (e) Syntenic comparisons of M genomic RNA segments from Phi6, Phi13 and CAP3 showing differences and similarities in attachment proteins. Genes are drawn to scale and correspond to Figure S3 (S and L segments are shown in Figures S2 and S4, respectively).

Figure 4

Genomic sequence comparison of CAP3–CAP7 phages. (a) Molecular phylogenetic analysis was determined by the Maximum Likelihood method. Each segment (S, M, L) was compared independently, and the trees with the highest likelihood are shown. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site, and bootstrap values are indicated at the appropriate branch points. (b) Percent similarity matrices compare each RNA segment (S, M, L) to the other CAP phage RNA segments.

Figure 5

Representative CAP genome schematic. CAP7 was used as a representative genome for illustrative purposes. The L-gp1 open reading frame (ORF) is absent in the CAP3-L RNA segment (denoted by the striped arrow). Putative protein functions are listed below each gene when a function could confidently be assigned, based on a combination of synteny with other sequenced Pseudomonas cystovirus genomes, annotation, functional characterization, or protein modeling (RAST, Phyre2 and HHpred were used). The gene segments are drawn to scale.

Figure 6

RNA segment comparison between CAP3, CAP7 and the previously sequenced Pseudomonas-infecting cystoviruses. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site, and bootstrap values are indicated at the appropriate branch points.

Figure 7

Pro-Q™ Emerald 300 staining of CAP3 glycans. (a) Proteinase K digests of CAP3 propagated on LH6 WT, ∆pglC, ∆lpsC and ∆clsB, and CAP1 phages were separated on a 15% SDS-PAGE gel and stained with Pro-Q™ Emerald 300 glycan staining kit (compare with Figure S5). CAP3 was treated with 1% acetic acid to test whether the low molecular weight glycan is a lipid-linked glycoconjugate (and Figure S6). (b) Proteinase K digests of LH6 WT, ∆pglC, ∆lpsC and ∆clsB were separated on a 15% SDS-PAGE gel and stained to show differences in glycosylation. BHI medium was also tested to determine the origin of the high molecular weight glycan in the phage samples. (c) CAP3 propagated on LH6 WT, ∆pglC, ∆lpsC and ∆clsB, and CAP1 phages were loaded with 4x volume (from Figure S6). (d) Proteinase K digests of LH6 WT, ∆pglC, ∆lpsC and ∆clsB were separated on a 12.5% SDS-PAGE gel to better resolve high molecular weight capsular polysaccharides (from Figure S7).

Figure 8

GC-MS chromatograms of CAP3 and LH6 extracts. (a) Chromatogram of CAP3 and (b) LH6 aqueous phase extracts (from Figure S8).