Phase-variable capsular polysaccharides and lipoproteins modify bacteriophage susceptibility in Bacteroides thetaiotaomicron

Abstract

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides–phage interactions, we isolated 71 Bacteroides thetaiotaomicron-infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts.

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Fig. 1: Host range of B. thetaiotaomicron phages on strains expressing different CPS.
Fig. 2: Infection of various cps mutant strains by branch 2 and 3 phages.
Fig. 3: Effects of ARB25 phage infection on growth of bacteria expressing different CPSs.
Fig. 4: ARB25 infection of wild-type B. thetaiotaomicron causes altered cps gene expression.
Fig. 5: Infection of acapsular B. thetaiotaomicron selects for increased expression of multiple phase-variable loci, whereas wild-type mostly alters CPS expression.
Fig. 6: Expression of the BT1927 S-layer increases B. thetaiotaomicron resistance to four different phages.

Data availability

Source data for all the experiments, along with corresponding statistical test values, where appropriate, are provided. RNA-seq data for whole-genome transcriptional profiling is deposited in NCBI Gene Expression Omnibus database as accession no. GSE147071.

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Acknowledgements

We thank R. Honrada at the San Jose-Santa Clara Wastewater Treatment Plant and the staff at the Ann Arbor Wastewater treatment plant for assistance in collecting primary sewage effluent and D. Maghini for assistance in identifying shared cps loci between B. thetaiotaomicron strains VPI-5482 and -7330. This work was funded by NIH grants (nos. GM099513 and DK096023 to E.C.M), an NIH postdoctoral NRSA (no. 5T32AI007328 to A.J.H.), a Stanford University School of Medicine Dean’s Postdoctoral Fellowship (to A.J.H.), the NIH Cellular Biotechnology Training Program (no. T32GM008353 to N.T.P.) and NIH Bioinformatics Training grant (no. T32GM070449 to R.C.).

Author information

Affiliations

Authors

Contributions

N.T.P. and A.J.H. performed most of the experiments, including initial phage isolation, host range measurements, construction of additional mutants and subsequent testing. B.D.M. and J.O.G. assisted A.J.H. with the experiments listed. J.J.F. and S.S. performed and analysed RNA-seq experiments, except those shown in Fig. 5. N.T.P. and E.C.M. performed these experiments for Fig. 5. R.W.P.G. and A.J.H. constructed additional capsule mutants for ED4. N.T.P., A.J.H., J.J.F. and E.C.M. designed the experiments, and analysed and interpreted most of the data. R.D.C. and E.S.S. performed whole-genome phylogenetic analysis. J.J.F. conducted the corresponding cps locus search. J.L.S. and E.C.M provided tools and reagents. N.T.P., A.J.H., J.J.F., S.S. and E.C.M. prepared the display items and compiled the Source data. N.T.P., A.J.H., J.J.F. and E.C.M. wrote the paper. All authors edited and approved the manuscript before submission.

Corresponding authors

Correspondence to Andrew J. Hryckowian or Eric C. Martens.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Diversification and structure of cps gene clusters in human gut Bacteroidetes.

Diversification and structure of cps gene clusters in human gut Bacteroidetes. a, The genomes of 53 different human gut Bacteroidetes (predominantly named type strains) were searched for gene clusters that contain two or more different protein families indicative of cps loci (see Methods). The number of cps loci detected in each genome is shown in the context of phylogenetic tree derived from the core genome of the 53 species used for this analysis; species for which cps loci were not detected using our search criteria are marked with a red “X”. Due to gaps in several genomes, which often occur at cps loci, the numbers shown are likely to be an underestimate. b, Schematics of the 8 annotated cps loci in B. thetaiotaomicron VPI-5482, which are singly present in the cps1-cps8 strains used in this study, or completely eliminated in the acapsular strain. Genes are color coded according to the key at the bottom and additional Pfam family designations are provided under most genes. The four main protein families used for informatics analysis are marked with asterisks and highlighted in bold in the key. Source data

Extended Data Fig. 2 Representative pictures of phage plaques for all phages in this study: (a) phages from Ann Arbor (ARB); (b) phages from San Jose (SJC).

Representative pictures of phage plaques for all phages in this study: (a) phages from Ann Arbor (ARB); (b) phages from San Jose (SJC). The top row of images for each phage are unaltered; background and saturated pixels were removed from images in the bottom row to facilitate viewing of the plaques. Experiment was performed only once for photographing plaques, no major variations were observed in additional experiments with the same phage on the same host strain. Scale bar = 2 mm.

Extended Data Fig. 3 Replication of a subset of host range assays of B. thetaiotaomicron-targeting phages on strains expressing different CPS types.

Replication of a subset of host range assays of B. thetaiotaomicron-targeting phages on strains expressing different CPS types. Ten bacteriophages isolated and purified on the wild-type, acapsular, or the 8 single CPS-expressing strains were re-tested in a spot titer assay to determine phage host range. 10-fold serial dilutions of each phage ranging from approximately 106 to 103 plaque-forming units (PFU) / ml were spotted onto top agar plates containing the 10 bacterial strains. Plates were then grown overnight, and phage titers were calculated. Titers are normalized to the titer on the preferred host strain for each replicate. Each row in the heatmap corresponds to a replicate for an individual phage, whereas each column corresponds to one of the 10 host strains. One to three replicates of the assay were conducted for each phage by the two lead authors (AJH and NTP). Assays were carried out at the same time, and each author used the same set of cultures and phage stocks. For comparison, individual replicates from Fig. 1 are included (marked with *). Experiment was conducted once during a visit of AJH to the University of Michigan research site to compare reproducibility between experimenters with the replication described above. Source data

Extended Data Fig. 4 Effects of eliminating permissive CPS from another B. thetaiotaomicron strain.

Effects of eliminating permissive CPS from another B. thetaiotaomicron strain. a, We identified B. thetaiotaomicron 733056 as the only sequenced and genetically tractable strain that contains VPI-5482-like cps loci (cps2, cps5, and cps6). Gene colors illustrate syntenic genes with >98% amino acid identity but do not indicate function. Please see Extended Data Fig 1 for functional annotation of B. thetaiotaomicron VPI-5482 cps loci. We also observed that the Branch 2 phage SJC01 did not yield productive infection in B. thetaiotaomicron 7330, but could partially clear lawns of B. thetaiotaomicron 7330 at high titers. This ability to clear lawns is a previously described phenomenon known as “lysis from without57”. b, Deletion of permissive capsules (cps2, cps5, and cps6) either alone or in combination affects VPI-5482 infection by SJC01 (n = 7 biological replicates per strain; bars represent the geometric mean). c, While SJC01 plaques on WT B. thetaiotaomicron VPI-5482, it does not form plaques on wild-type B. thetaiotaomicron 7330. However, SJC01 does exhibit a “lysis from without” clearing phenotype at high densities of phage (top two spots, made with 1 microliter of 1e8 and 1e7 PFU per mL, according to titers observed on wild-type VPI-5482). Experiment was independently performed 7 times with VPI-5482 and 8 times with 7330 with similar results. d, B. thetaiotaomicron 7330 strains lacking cps5 (with the exception of 7330 ∆cps5cps6) show the lysis from without phenomenon less frequently than strains that have intact cps5. Experiments were performed with the following number of replicates per strain with similar results: wild-type 7330 (n = 8), Δ2 (n = 8), Δ5 (n = 8), Δ6 (n = 8), Δ2,5 (n = 17), Δ2,6 (n = 5), Δ5,6 (n = 3), Δ2,5,6 (n = 19). For panel b, significant differences in phage titers on each mutant strain were compared to wild type via two-tailed Mann-Whitney test with actual P values shown. For panel c, scale bars = 0.5 cm. Source data

Extended Data Fig. 5 Free CPS does not inhibit ARB25 infection when provided in trans and effect of CPS to phage infection on bacterial growth.

Free CPS does not inhibit ARB25 infection when provided in trans and effect of CPS to phage infection on bacterial growth. a, ARB25 was incubated with purified CPS1 or CPS2 (1 mg/ml, an estimated 109 molar excess of CPS molecules to phage, see Methods) before plating on the acapsular strain, and plaques were counted after overnight incubation. Titers are normalized to mock (H2O) treatment. No significant differences in titers were found compared to mock treatment, as determined by Welch’s t test, 2-tailed (n = 3 biological replicates, bars represent mean ± SEM). b, Post ARB25-infected, surviving cultures still contain infectious phages. Wild-type B. thetaiotaomicron was infected with live or heat-killed ARB25, and bacterial growth was monitored via optical density at 600 nm (OD600). At 0, 6.02, 8.36, and 11.7 h post inoculation, replicate cultures were removed and phage levels were titered (n = 3 and individual replicate curves are shown). No phages were detected in heat-killed controls. Note that the PFU/mL do not increase substantially after the initial “burst” corresponding to decreased bacterial culture density prior to re-growth. c, Ten strains: the wild-type (WT), the acapsular strain (∆cps), or the eight single CPS-expressing strains were infected with either live or heat-killed SJC01. d, 20 different colonies of cps4 or cps5 strains were infected with ARB25. Growth was monitored via optical density at 600 nm (OD600) on an automated plate reading instrument as described in Methods and individual growth curves for live and heat-killed phage exposure are shown separately. Source data

Extended Data Fig. 6 Infection of wild-type B. thetaiotaomicron at a low multiplicity of infection and subsequent effects on cps gene expression.

Infection of wild-type B. thetaiotaomicron at a low multiplicity of infection and subsequent effects on cps gene expression. a, The wild-type (WT) strain was infected at a low multiplicity of infection (MOI = 1 ×10-4) of live or heat-killed ARB25, and bacterial growth was monitored via OD600 (n = 3 biological replicates and separate curves are shown). b, RNA was harvested from cultures after reaching an OD600 of 0.6-0.7, cDNA was generated, and relative expression of the 8 cps loci was determined by qPCR (histogram bars are mean ± SEM of 3 biological replicates). Individual replicates of high MOI (c) and low MOI (d). In the high MOI experiment, replicate 2 showed higher starting expression of the non-permissive CPS3 compared to others. In the low MOI experiment, replicate 3 showed higher starting expression of the non-permissive CPS3. In both experiments, post phage-exposed replicates displayed nearly identical CPS expression profiles characterized by high expression of CPS3. The experiments in a-c were repeated one time with three parallel biological replicates started from single B. thetaiotaomicron wild-type colonies picked from the same plate. Source data

Extended Data Fig. 7 Determination of phase-variable promoter switching for six loci encoding putative S-layer proteins.

Determination of phase-variable promoter switching for six loci encoding putative S-layer proteins. The hypothesis that the promoters associated with seven additional B. thetaiotaomicron S-layer like lipoproteins are phase variable was validated using a PCR amplicon sequencing strategy. Because of high nucleotide identity in both the regions flanking the 7 additional loci, a nested PCR approach was required to specifically amplify and sequence each site. In the first step, a primer lying in each S-layer gene (Supplementary Table 5 “S-layer gene” primers) was oriented towards the promoter and used in a PCR extension to a primer in the upstream recombinase gene (Supplementary Table 5 “recombinase gene 3” primer). The products of this PCR were purified without gel extraction and used in a second reaction with a nested primer that lies internal to the previous recombinase gene primer (Supplementary Table 5 “recombinase 2” primer). The expected PCR products from this reaction, which are ~1 kb and span promoter sequences in both the ON and OFF orientations, were excised and used for an orientation-specific PCR using the original S-layer gene primer for each site and a universal primer (green schematic) that was designed for each promoter and is oriented to extend upstream of the S-layer gene (e.g., OFF orientation). Resulting products from this third reaction, which should correspond to the ON orientation if a promoter inversion has occurred in some cells, were obtained for 5/7 of the additional identified loci and the BT1927 S-layer locus as a control. In all cases in which an amplicon and sequence were obtained, the expected recombination occurred between the inverted repeat site proximal to the S-layer gene start (new DNA junction), which would orient the promoter to enable expression of the downstream S-layer gene. The sequences shown are the consensus between forward and reverse reads for each amplicon. The putative core promoter -7 sequence is shown in bold/red text, the coding region of each S-layer gene is shown in bold/blue text and the S-layer gene proximal recombination site is noted and highlighted in bold/gold text. Note that the 5′-end of the sequenced amplicon was not resolved for the BT2486 locus.

Extended Data Fig. 8 Recombination between the genes BT1040, BT1042, and BT1046 and effect of BT1033-52 locus.

Recombination between the genes BT1040, BT1042, and BT1046 and effect of BT1033-52 locus. a, Pfam domain schematics of the amino acid sequences of these three genes highlighting that BT1040 and BT1046, as originally assembled in the B. thetaiotaomicron genome sequence, lack additional N-terminal sequences that are present on BT1042. b, Sequencing of the 8 PCR amplicons schematized in Fig. 5d. Amplicons 1, 5 and 8 represent the original genome architecture, while the others represent inferred recombination events that are validated here by sequencing. The 5′ and 3′ ends of the BT1042, BT1040 and BT1046 genes are color-coded to assist in following their connectivity changes after recombination. A series of single-nucleotide polymorphisms (SNPs) present in BT1042, downstream of the proposed recombination site, are highlighted in yellow. The transfer of these SNPs to a fragment containing the 5′ end of BT1040 (Amplicon 4) was used to narrow the recombination region to the 7 nucleotide sequence highlighted in red. Additional SNPs that are specific to the regions upstream of this recombination site are shown in white text for each sequence. Susceptibility of acapsular B. thetaiotaomicron to ARB25 without the BT1022-52 locus is not affected. c, Ten-fold serial dilutions of ARB25 were spotted onto lawns of B. thetaiotaomicroncps (n = 5) and B. thetaiotaomicroncps ∆BT1033-52 (n = 5). Each of the 5 biological replicates contained 3 technical replicates from independent clones and all 15 replicates are shown individually. Plaquing efficiency was determined by normalizing plaque counts on B. thetaiotaomicroncps ∆BT1033-52 relative to plaque counts on B. thetaiotaomicroncps for each replicate. Statistical significance was determined using the 2-tailed Mann-Whitney test and bars represent the geometric mean. Source data

Extended Data Fig. 9 Whole genome transcriptional analyses of several additional B. thetaiotaomicron strain and phage combinations.

Whole genome transcriptional analyses of several additional B. thetaiotaomicron strain and phage combinations. a, Infection of the cps1 strain with ARB25, revealing a post-infection response that is largely characterized by increased expression of S-layer/OmpA proteins. b, Infection of wild-type B. thetaiotaomicron with SJC01, revealing that, as with ARB25/wild-type, the bacteria survive phage infection by mostly altering CPS expression. Expression of the non-permissive CPS3 is prominently increased. c, Infection of acapsular B. thetaiotaomicron with SJC01, revealing that in the absence of CPS survival is mostly promoted by increased S-layer/OmpA expression and expression of a phase-variable restriction enzyme system. Transcript abundance values in panels a-c were compared between live and HK treatments to generate fold change (x axis), which is plotted against the adjusted P value (EdgeR) for each gene that was generated using an exact test adapted for overdispersed data55 (n = 3 biological replicates for each panel, peformed with similar results). Please see Supplementary Tables 5-7 for actual adjusted P values for panels (a-c). d, Gene schematic of the phase-variable restriction enzyme system (top) and a lipoprotein contain locus (bottom) that is different from the 8 S-layer loci also revealed in this study. The inverted repeat sequence that was determined to mediate recombination in each locus is shown. e, PCR analysis of the restriction enzyme system and additional lipoprotein promoter orientations with primers designed to detect phase variation from off to on states. Amplicons were sequenced to confirm the re-orientation to the on orientation (not shown). Experiment was performed once. f, Global transcriptional responses of wild-type B. thetaiotaomicron in the ceca of mice after 72 d of co-existence with ARB25. Note that shifts in CPS expression are mostly characterized by increases in permissive CPS, which may be dictated by growth in vivo selecting for these capsules or against the non-permissive CPS3. Correspondingly, wild-type shows increased expression of some but not all S-layer/OmpA systems and the phase-variable restriction enzyme. g, Global transcriptional responses of acapsular B. thetaiotaomicron in the ceca of mice after 72 d of co-existence with ARB25. In the absence of CPS, surviving bacteria show increased expression of only a subset of the identified S-layer/OmpA proteins, with BT1826 expressed most dominantly, along with the BT0291-94 locus and expression of the restriction enzyme system. Experiments in panels f-g are the results of 3 separate biological replicates, statistical tests are identical to those described in panels (a-c). The dashed lines in panels a,b,c,f,g represent the adjusted P value cutoff (≥0.01) that was used to establish significance, which was generated using an exact test adapted for overdispersed data55. Source data

Extended Data Fig. 10 ARB25 (a) or SJC01(b) infection of the acapsular BT1927 locked on and off strains after 1, 2 or 3 days of growth on BPRM.

ARB25 (a) or SJC01(b) infection of the acapsular BT1927 locked on and off strains after 1, 2 or 3 days of growth on BPRM (n = 6 biological replicates per treatment, performed with similar results). Three separate colonies were picked each day as a biological replicate, grown overnight and used to setup infection cultures that were monitored for 24 h in an automated plate reader. Colonies picked after only 1 day show the least resistance to either phage when BT1927 is locked on. After 2 days, resistance is increased and this continues to increase after 3 days, becoming almost complete (compared to HK controls for ARB25). Growth curves represent the mean of 6 biological replicates ± standard error. Source data

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Porter, N.T., Hryckowian, A.J., Merrill, B.D. et al. Phase-variable capsular polysaccharides and lipoproteins modify bacteriophage susceptibility in Bacteroides thetaiotaomicron. Nat Microbiol (2020). https://doi.org/10.1038/s41564-020-0746-5

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