A prophage-encoded sRNA limits phage infection of adherent-invasive E. coli

Abstract

Prophages are prevalent features of bacterial genomes that can reduce susceptibility to infection by competing phages, yet the mechanisms involved are often elusive. Here, we identify a small RNA (svsR) encoded by the lambdoid prophage NC-SV in adherent-invasive Escherichia coli strain NC101 that limits infection by virulent coliphages. Comparative genomics revealed that NC-SV-like prophages and svsR homologs are broadly conserved across Enterobacteriaceae. Transcriptomic analyses show that svsR represses maltodextrin transport genes, including lamB, which encodes the outer membrane maltoporin LamB, a known receptor for numerous coliphages. Deletion of the lamB gene reveals that while LamB is not required for replication of the virulent phages tested, it contributes to plaque expansion, indicating a role in phage spread but not as an essential receptor. Nutrient supplementation experiments further linked maltodextrin and glucose availability to changes in plaque expansion and phage adsorption. In vivo, we compared wild-type NC101 and a prophage-deletion strain (NC101∆NC-SV) in mice to assess the impact of NC-SV on lytic phage susceptibility. Although intestinal E. coli densities remained stable across groups, animals colonized with NC101 exhibited markedly reduced phage burdens in both the intestinal lumen and mucosa compared to mice colonized with NC101∆NC-SV. This reduced phage pressure was associated with increased dissemination of E. coli to extraintestinal tissues, including the spleen and liver. Together, these findings highlight a nutrient-responsive, prophage-encoded mechanism that protects E. coli from phage predation and may promote bacterial persistence in and dissemination from the mammalian gut.

Fig 1. The NC-SV prophage protects E. coli NC101 from phage infection by reducing virion adsorption.

(A) Plaques formed by wastewater phage isolate RSB03 on E. coli NC101 or the indicated prophage mutants. Representative images are shown. Scale bars represent 10 mm. (B) The surface area of RSB03 plaques formed on lawns of the indicated strains was measured, N = 50 plaques per condition, from four replicate experiments, ****P < 0.0001 compared to NC101; ns, not significant. (C) Growth (OD₆₀₀) of NC101 and NC101^ΔNC-SV was measured after infection with RSB03 (MOI = 1). Data are the mean of four experiments. (D) The percentage of adsorbed RSB03 virions was measured in NC101 or NC101^ΔNC-SV cells at the indicated times post infection, MOI = 1. Data are the mean ± SEM of three experiments, **P < 0.01, ***P < 0.001. (E) Representative transmission electron microscopy images of negatively stained NC101 and NC101^ΔNC-SV cells 60 minutes post-infection with phage RSB03 (MOI 1). Scale bars represent 500 nm (50 nm in the inset). The final panel depicts an NC101^ΔNC-SV cell undergoing phage-induced lysis, with loss of cell envelope integrity.

Fig 2. The NC-SV prophage restricts RSB03 phage replication and persistence in the mouse gut.

Germ-free C57BL/6J mice were stably monoassociated with either NC101 or NC101^∆NC-SV E. coli, then administered 1x10⁸ PFU RSB03 daily by gavage for five consecutive days. (A) Serial quantification of E. coli log₁₀ CFU from fecal pellets plated on selective LB-chloramphenicol agar, normalized to fecal sample weight (g, mean + /- 95% confidence interval). Vertical dotted lines indicate days of RSB03 administration; P > 0.05, Mixed-effects model with Geisser-Greenhouse correction. (B-C) Log₁₀ difference in E. coli density in fecal pellets from baseline to (B) day 4 (last day of RSB03 treatment) and (C) day 15; ns, no statistically significant difference between groups. P > 0.05, unpaired nonparametric Mann-Whittney test. (D) Serial quantification of plaque forming units (log₁₀ PFU) from fecal pellets, normalized to fecal sample weight (grams, mean ± - 95% confidence interval); P < 0.0001, Mixed-effects model with Geisser-Greenhouse correction. (E-F) Log₁₀ difference in PFU in fecal pellets from baseline to (E) day 4 and (F) day 15; ****P < 0.0001, **P < 0.01, unpaired nonparametric Mann-Whitney test. (G-I) Detectable (G) E. coli log₁₀ CFU, (H) RSB03 log₁₀ PFU, and (I) Phage:Host index (min-max normalized log₁₀ difference of PFU-CFU, with 1 reflecting equal density) for the indicated samples at endpoint (day 17). For bar plots, dots represent individual subjects, bars represent the mean ± 95% confidence interval. Significantly different groups are indicated by compact letter display, where uppercase letters above each bar denote significantly different (P < 0.05) groups by two-way ANOVA and post hoc means testing (Tukey) comparing all groups. Groups that share a letter designation are not statistically different (e.g., A is significantly different from B, but not AB).

Fig 3. The NC-SV prophage transcriptionally regulates maltodextrin transport and carbon metabolism in response to phage infection.

RNAseq was performed on mid-log NC101 or NC101^ΔNC-SV cells growing in LB broth 10 minutes post infection with phage RSB03 (MOI = 1). (A) Volcano plot showing the 235 differentially expressed genes in NC101 compared to NC101^ΔNC-SV with a ± 2-fold change and P ≤ 0.05. Data are representative of quadruplicate experiments. (B) Gene enrichment analysis was performed on significantly differentially regulated genes shown in panel A. (C) Phage titers (PFU/mL) recovered from RSB03 infections of the indicated strains in broth culture. Data are the mean + /-SEM of three independent experiments. (D) Plaque area of RSB03 on lawns of NC101, ΔlamB, NC101^ΔNC-SV, or NC101^ΔNC-SVΔlamB. Plaques area was measured using Image J after 18 h of growth; each point represents one plaque, data are the mean + /-SEM of three independent experiments, ****P < 0.0001, ns = not significant.

Fig 4. Exogenous sugars modulate phage adsorption to E. coli NC101.

E. coli NC101 was grown in diluted (10%vol/vol) LB broth or agar supplemented with the indicated carbon sources at 37°C. (A) RSB03 plaque areas on NC101 lawns were measured after 18 hours. N = at least 30 plaques per condition. Statistical comparisons between glucose and maltodextrin at the same concentrations (0.4, 4, and 40 mM) were performed using unpaired Student’s t tests; *P < 0.05, **P < 0.01. (B and C) The percentage of adsorbed RSB03 virions were measured in cultures supplemented with 40 mM of the indicated sugar at the indicated times post infection with an initial MOI of one. Data are the mean ± SEM of three experiments, *P < 0.05, **P < 0.01 by Student’s t test.

Fig 5. The NC-SV prophage encodes a conserved co293-like small RNA, svsR, found across Enterobacteriaceae.

(A) Genome organization of the NC-SV prophage. The predicted svsR sRNA is encoded on the positive strand at the 3′ end of the cI phage repressor gene (highlighted in purple). (B) Sequence alignment between svsR and the co293 sRNA from E. coli MG1655 reveals 58% identity (45/77 nucleotides). (C) Predicted secondary structure (left) and AlphaFold3 structural model (right) of svsR, reveal a stable hairpin conformation. (D) Conservation of svsR among NC-SV–like prophages in E. coli genomes with ≥30% NC-SV genome coverage. Each point represents a prophage, with % coverage plotted against % identity to the NC-SV reference. Open circles indicate the absence of svsR, while pink symbols indicate its presence; triangle shapes denote svsR embedded within the cI gene. (E) NC-SV–related prophages found in non-E. coli Enterobacteriaceae genomes with ≥10% genome coverage with each dot representing a genome.

Fig 6. The svsR sRNA downregulates maltodextrin transport genes and protects E. coli from phage infection.

(A and B) Growth (OD₆₀₀) of NC101^ΔNC-SV carrying an empty expression construct or a svsR sRNA expression construct was measured at the indicated times in (A) uninfected cells or (B) post infection with phage RSB03 at an MOI of one. Data are the mean ± SEM of three experiments. (C) Representative images of RSB03 plaques on the indicated lawns. (D) RSB03 plaque area was measured after 18 hours on lawns of NC101^ΔNC-SV expressing svsR in trans or empty vector control. (E) The percentage of RSB03 virions adsorbed to NC101^ΔNC-SV cells carrying the empty vector or the svsR expression vector was measured at the indicated times post infection with an initial MOI of one. Data are the mean ± SEM of three experiments; *P < 0.05,***P < 0.001. (F) Volcano plot showing the 83 differentially expressed genes in NC101^ΔNC-SV expressing svsR compared to the empty vector control with ±2-fold change and P ≤ 0.05. Data are representative of quadruplicate experiments. (G) Gene enrichment analysis was performed on the significantly differentially regulated genes shown in panel F.