Evolutionary consequences of intra-patient phage predation on microbial populations

1Traditional views of host–pathogen interactions are of a battle between two opposing organisms. This perspective is being challenged with greater appreciation of the influence of the host's microbial ecosystem on these interactions. Bacteriophages influence bacterial populations in many ecosystems and specifically temperate phages play a role in many diseases through lysogenic conversion. In contrast, the impact of lytic phage predation on bacterial pathogens in the context of human disease is under-appreciated. Vibrio cholerae is a water-borne pathogen responsible for the diarrheal disease cholera. In order for V. cholerae to colonize the human small intestine and elicit diarrhea, it produces a set of virulence determinants including cholera toxin. The ToxR signaling cascade activates expression of these virulence factors in response to host environmental stimuli. Cholera epidemics appear with regular seasonality in regions like Bangladesh and can arise unpredictably in vulnerable regions as exemplified by the epidemic that recently began in Haiti following the single-source introduction of a pandemic V. cholerae O1 strain from another continent. Lytic phages are hypothesized to impact cholera disease burden in Bangladesh. The predation of V. cholerae by phages following their co-ingestion from the environment is central to this hypothesis; however, molecular evidence in support of this hypothesis is lacking.

2We recently described the ICP2 species of V. cholerae-specific, virulent podoviruses that are found sporadically in cholera patient stool samples collected since 2001 in Bangladesh ( and present study). To begin to address the geographic diversity of cholera phages in patient stools, we tested by plaque assay for the presence of phages within nine Haitian cholera patient stool samples collected in 2013. We identified one sample that had a high titer of phage (108 PFU/ml) relative to V. cholerae (105 CFU/ml). Whole genome sequencing and comparative analysis of this phage, named ICP2_2013_A_Haiti, revealed 84% identity over 93% of its genome to ICP2_2011_A, a phage isolated from Bangladesh in 2011. An alignment of the annotated ICP2 genome with the three available ICP2-related isolates shows that synteny is conserved between these geographically distinct phages, with no significant genome rearrangements observed (Figure 1—figure supplement 1). The Haitian ICP2 isolate is strikingly similar, although clearly distinct, from Bangladeshi ICP2 isolates, and is the first lytic phage reported to be associated with epidemic V. cholerae in Haiti.

3Remarkably, we observed that most V. cholerae isolates recovered from the same stool sample as ICP2_2013_A_Haiti were resistant to infection by this phage. We investigated phenotypic heterogeneity within this stool sample by testing 269 single colony isolates for sensitivity to ICP2_2013_A_Haiti and found that 267 (>99%) were phage-resistant. Eight phage-resistant isolates and the two phage-sensitive isolates from this sample were subjected to whole genome sequencing. All 10 isolates were isogenic except for mutations within ompU encoding the major outer membrane porin (Supplementary file 1). Furthermore, the ompU mutations in the eight phage-resistant isolates were heterogeneous. We sequenced ompU from an additional 11 phage-resistant isolates from this stool sample and found a total of six unique ompU alleles (Figure 1A). When each of these alleles was used to replace ompU in a clean genetic background, all conferred resistance to ICP2, yet produced normal amounts of OmpU (Figure 2 and data not shown), suggesting that ICP2 uses OmpU as a receptor to initiate infection and that the mutations disrupt this interaction. These data showing that intra-host V. cholerae isolates are isogenic, only differing in ICP2 resistance mutations, indicates that they diverged within the patient. Furthermore, the presence of multiple different ICP2 resistance mutations within this single host suggests that selection of phage resistance occurred multiple, independent times during the course of infection. We also found evidence for ICP2-mediated selection of OmpU mutants in isolates from Bangladesh. Analysis of the ompU sequence from 54 clinical isolates collected between 2001 and 2011 showed that 15% had non-synonomous mutations (Figure 1A, Figure 1—figure supplement 2) and these alleles were sufficient for ICP2 resistance (Figure 2). One of the OmpU mutants, G325D, was observed in clinical isolates from Haiti and Bangladesh (Figure 1A). Interestingly, seven of the eight mutant OmpU proteins detected in the Haitian and Bangladeshi clinical isolates had alterations in two predicted extracellular loops, while the eighth had an altered transmembrane segment adjacent to the first of these extracellular loops (Figure 1B). This suggests that ICP2 interacts specifically with these exposed loops to initiate infection.

4We also tested for V. cholerae population heterogeneity within an ICP2-positive stool sample isolated in Bangladesh in 2011. Of 46 single colony isolates tested for sensitivity to ICP2_2011_A, 22% were resistant to the phage. Whole genome sequencing of two phage-sensitive and four phage-resistant isolates from this sample revealed that the strains were isogenic, except that phage-resistant isolates had nonsense mutations in toxR, the direct transcriptional activator of ompU (; Supplementary file 2). We sequenced toxR from an additional eight phage-resistant isolates from this stool sample and found that, analogous to what was observed in the Haitian patient sample, there were multiple unique toxR alleles, which included two non-synonomous and two nonsense mutations (Figure 1C). Each of the four mutant toxR alleles conferred ICP2 resistance and abrogated expression of OmpU when used to replace wild-type toxR in a clean genetic background (Figure 2). Both ICP2-sensitivity and OmpU expression were restored to the clinical toxR mutants by expressing ompU in trans or by reverting the toxR allele to wild-type (data not shown), indicating that these mutant toxR alleles are necessary and sufficient for ICP2 resistance and that this resistance is mediated through loss of OmpU expression.

5To address the potential consequences of the phage-resistance mutations on V. cholerae fitness, we tested the four most frequently isolated ompU alleles and all four toxR alleles in survival and growth assays. Recent Tn-seq analyses of V. cholerae showed that OmpU is critical for fitness in an infant rabbit infection model and for dissemination from the host into pond water. OmpU has also been shown to be important for protection against the bactericidal effect of bile salts, cationic peptides, and intestinal organic acids. When we tested for survival in bile and for competitive fitness in pond water we found that the four OmpU mutants are fully fit (Figure 3A,B), consistent with their wild-type levels of OmpU expression (Figure 2). Moreover, the OmpU(G325D) mutant was fully virulent in a single round, competitive infection in infant rabbits (Figure 4, first column). However, the OmpU mutants, particularly the A195T and G325D mutants, showed a mild competitive defect after multiple passaging in growth medium (Figure 3C). The mild growth defect of the OmpU mutants in conjunction with the low prevalence of ICP2 may explain why the ICP2-resistant ompU variants do not become fixed in the population (Figure 1—figure supplement 2). In sharp contrast, in the presence of ICP2 we observed a 10,000-fold enrichment of the OmpU(G325D) mutant over the wild-type after infection of infant rabbits (Figure 4), indicating that strong selective pressure is imposed by phage predation during V. cholerae infection. This emulates what we hypothesize happens in human infections in the presence of ICP2 and is the first demonstration of phage predation of V. cholerae in the context of a diarrheal disease model.

6V. cholerae strains harboring any of the four ToxR mutations were avirulent in the infant mouse model of infection and were indistinguishable from a ΔtoxR mutant (Figure 3D). The clinical isolates containing these mutations were also at least 100-fold attenuated (Figure 3D—figure supplement 1), indicating that these isolates do not harbor compensatory mutations. The cytoplasmic amino terminus (D17-E112) of ToxR where the two non-synonomous mutations mapped has homology to the winged helix-turn-helix family of transcription activators and is involved in DNA binding and transcriptional activation. A number of point mutations in this domain were previously shown to abrogate activation of ompU and virulence genes. The severe virulence defects of the two non-synonomous toxR mutants from the Bangladeshi patient sample (Figure 3D) are consistent with an inability of the encoded mutant ToxR proteins to activate these genes. These toxR mutants would not colonize the human small intestine and be recovered in the secretory diarrhea if ingested, which further supports our conclusion that these mutants arose due to ICP2 predation during cholera infection in this individual. This also indicates that, in this patient, predation and selection of phage-resistant mutants likely occurred late in infection when colonization and virulence gene expression were no longer required for the development of acute symptoms.

7We have shown that lytic phages are an unexpected ‘third party’ that impose significant bactericidal pressure on an environmentally transmitted pathogen during the natural course of infection in humans. We observed that adaptations to phage predation involve tradeoffs in evolutionary fitness and provide a molecular mechanism for phage predation impacting V. cholerae transmission and seeding of environmental reservoirs. Our results highlight that host–pathogen interactions are embedded within and strongly affected by a complex microbial ecosystem. Predator-prey dynamics are notably absent between microbiotal phage and their bacterial hosts in the intestinal ecosystem in healthy humans. In contrast, our findings indicate that diseased states, which are often accompanied by significant bacterial proliferation, likely facilitate these predatory interactions.

8Strains utilized in this study are listed in Supplementary file 3. Stool specimens and V. cholerae isolates from the International Centre for Diarrheal Disease Research, Bangladesh were collected and stored from previous studies. Phage (ICP2_2011_A and ICP2_2013_A_Haiti) were isolated from cholera rice-water stool samples as described. V. cholerae isolates were isolated from stool samples on Luria–Bertani agar with sulfamethoxazole (24 µg/ml), trimethoprim (32 μg/ml), nalidixic acid (20 µg/ml) and streptomycin (Sm) (100 µg/ml). Mutations were introduced using pCVD442-lac as previously described. Strains containing the pMMB67EH vector were grown in the presence of 50 μg/ml ampicillin and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG).

9V. cholerae and phage genomic libraries were generated and sequenced using an Illumina HiSeq2000 (Tufts University Core Facility). Sequence reads from each V. cholerae isolate were mapped to the reference genome V. cholerae O1 2010EL-1786 (for Haitian isolates, accession numbers NC_016445.1 and NC_016446.1) or to V. cholerae O1 N16961 (for Bangladeshi isolates, accession numbers NC_002505.1 and NC_002506.1) and mutations were identified using Varscan v2.3.6 and CLC Genomic Workbench software (Version 6.8; CLC Bio, Denmark). Variants were called as being present (different allele than reference), absent (the same allele as reference) or low-coverage (<15×; in which case no call was made).

10Cultures were grown to mid-exponential growth phase in Luria–Bertani (LB) broth at 37°C with aeration. Bacteria were re-suspended in 200 mM Tris–HCl, pH 8.0 and 2 mM EDTA. Sucrose was added to a final concentration of 20% followed by lysozyme treatment for 10 min at 37°C. Using a dry ice/ethanol bath, samples were freeze-thawed twice followed by DNAse I treament for 20 min at room temperature. Samples were spun for 5 min at 16,100×g to pellet the membrane fraction. The pellets were re-suspended in 1% Triton X-100, 10 mM MgCl2, and 50 mM Tris–HCl, pH 8.0 and incubated at 37°C for 20 min. Samples were spun again for 5 min at 16,100×g to separate the inner membrane from the outer membrane. The pellets, which contain the outer membrane proteins, were re-suspended in 200 mM Tris–HCl, pH 8.0.

11Outer membrane fractions were boiled for 10 min in sample buffer containing sodium dodecyl sulfate and β-mercaptoethanol and separated on a NuPAGE 4–12% Bis-Tris polyacrylamide gel (Life Technologies, Carlsbad, CA). Protein gels were transferred to a nitrocellulose membrane for Western blotting. Membranes were probed with rabbit polyclonal antisera against V. cholerae OmpU (gift of James Kaper). A Cy5 goat anti-rabbit antibody was used to develop the blot.

12Strains (grown to OD600 = 0.5) were assessed for their ability to survive 0.2% porcine bile (Sigma, St. Louis, MO) in 0.85% NaCl for 1 hr at room temperature. Pond water survival assays were done as described previously at 30°C for 48 hr. Competition experiments were performed between the strain of interest (lacZ+) and the appropriate control strain (ΔlacZ), and outputs were plated on LB agar plates containing 100 µg/ml Sm and 40 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal).

13All animal experiments were in accordance with the rules of the Department of Laboratory Animal Medicine at Tufts Medical Center. Infant mouse colonization assays and in vitro controls were done as described previously. 3-day old infant rabbits were pre-treated with Cimetidine-HCL (Morton Grove Pharmaceuticals, Morton Grove, IL) 3 hr prior to infection and infected with ∼5 × 108 CFU in 2.5% sodium bicarbonate buffer (pH 9). A 1:1 mixture of wild-type to mutant (for experiments without phage) or 10:1 wild-type to mutant (for experiments including phage) was used. For experiments involving the addition of phage, phage were added immediately before intragastric inoculation to each rabbit inoculum to limit phage adsorption ex vivo (phage were in contact with the bacterial inoculum for ∼30–60 s prior to gavage of each animal). Rabbits were euthanized ∼12 hr post-inoculation and cecal and/or small intestinal fluid was collected by puncture. As with the in vitro assays, the competing strains were enumerated on LB agar plates containing 100 µg/ml Sm and 40 µg/ml X-gal.