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

Abstract

Prophages are prevalent features of bacterial genomes that can reduce susceptibility to lytic phage infection, 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 (AIEC) strain NC101 that confers resistance to lytic coliphages. Comparative genomic analyses revealed that NC-SV-like prophages and svsR homologs are conserved across diverse Enterobacteriaceae. Transcriptional analyses reveal that svsR represses maltodextrin transport genes, including lamB, which encodes the outer membrane maltoporin LamB-a known receptor for multiple phages. Nutrient supplementation experiments show that maltodextrin enhances phage adsorption, while glucose suppresses it, consistent with established effects of these sugars on lamB expression. In vivo, we compared wild-type NC101 and a prophage-deletion strain (NC101^ΔNC-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^ΔNC-SV. This reduced phage pressure was associated with increased dissemination of NC101 to extraintestinal tissues, including the spleen and liver. Together, these findings highlight a nutrient-responsive, prophage-encoded mechanism that protects AIEC from phage predation and may promote bacterial persistence and dissemination in the inflamed gut.

Figure 1:. The NC-SV prophage protects E. coli NC101 from lytic 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, ^****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). (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 cells of the indicated strains 60 minutes post-infection with phage RSB03 (MOI 1). Scale bars represent 500 nm (50 nm in the inlay).

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

(A) Serial quantification of E. coli log₁₀ CFU from fecal pellets, normalized to fecal sample weight (g). Vertical dotted lines indicate days of RSB03 administration. (B) Log₁₀ difference in E. coli density in feces from baseline to day 4 (last day of RSB03 treatment). (C) Log₁₀ difference in E. coli density in feces from baseline to day 15. (D) Serial quantification of plaque forming units (log₁₀ PFU) from fecal pellets, normalized to fecal sample weight (g); p<0.0001. (E) Log₁₀ difference in PFU from fecal pellets from baseline to day 4. (F) Log₁₀ difference in PFU from fecal pellets from baseline to day 4. (F) Log₁₀ difference in PFU from fecal pellets from baseline to day 15. (G-I) Detectable E. coli (G) log₁₀ CFU, (H) RSB03 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, individual subjects are represented by dots overlaying the mean +/− 95% confidence interval. Significantly different groups are indicated by symbols or compact letter display. ns, not significant, *P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001.

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

RNAseq was performed on mid-log NC101 or NC101^ΔNC-SV cells growing in LB broth 10 minutes post infection with lytic 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.

Figure 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 40 mM of 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; ^****P<0.0001 compared to the no supplement control; ns, not significant. (B-D) 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.

Figure 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. Each dot represents a genome, colored by species or strain.

Figure 6.. The svsR sRNA downregulates maltodextrin transport genes and protects E. coli from lytic 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 post infection with lytic 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.