In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli

Abstract

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-M^EHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT₃EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-M^EHEC(ΦStx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality.

Keywords: Biological Sciences; EHEC; Immunology and Inflammation; Microbiology; T3SS; smart microbe; therapeutic E. coli.

Figure 1:. Mice are equally susceptible to infection with Cr(ΦStx2dact) or Cr(ΦStx2a).

(A) Schematic of seamless cloning approach. (B, C) Seven-week-old female mice were orally inoculated with 1 X 10⁸ CFU of Cr(ΦStx2dact) or Cr(ΦStx2a) by feeding. Five mice were included in each cohort. (B) Viable counts of bacteria in feces were determined by plating. Each point shown represents an individual mouse, and each line represents the geometric mean of log¹⁰ CFU/g of feces. Samples plotted on the x-axis indicate no data available due to a lack of collected feces. All differences in bacterial titers were ns (non-significant) as determined by two-way ANOVA. (C) Kaplan–Meier survival curves of mice infected with Cr(ΦStx2dact) and Cr(ΦStx2a). No evidence of statistical significance between the two cohorts was found using the log-rank (Mantel-Cox) test.

Figure 2:. The extracellular domains of EHEC, EPEC, and Cr Tir are functionally interchangeable.

(A) Schematic of WT and chimeric Tir variants. (B) Secretion assay of Tir variants from designated strains. Supernatants (S) and whole-cell pellet lysates (P) fractions were obtained 6 hours post-transfer to fresh media. Images of immunoblots probed with an anti-Tir or an anti-GroEL antibody are shown. GroEL serves as a lysis control for (S) and loading control for (P). (C-E). Seven-week-old female mice were orally inoculated with 1 X 10⁸ CFU designated Cr strains. Five mice were included in each cohort. (C) Viable counts of bacteria in feces were determined by plating. Each point shown represents an individual mouse, and each line represents the geometric mean of log¹⁰ CFU/g of feces. Samples plotted on the x-axis indicate no data available due to a lack of collected feces. (D) Time course of body weight changes (%) over time. Mean +/-SEM plotted. Data in C and D were analyzed using two-way ANOVA with Bonferroni's post hoc multiple comparison test at a 95% confidence interval. For C, DPI (1–4, 6, 7) = ns; DPI 5, Cr(ΦStx2dact) vs Cr_Tir-M^EHEC(ΦStx2a) **p =0.0036. For D, DPI (1–7) = ns, DPI 8, Cr(ΦStx2a) vs Cr_Tir-M^EHEC(ΦStx2a) *p = 0.0135. (E) Kaplan–Meier survival curves of mice infected with Cr(ΦStx2dact), Cr(ΦStx2a), and Cr_Tir-M^EHEC(ΦStx2a). No evidence of statistical significance was found between the four cohorts using the log-rank (Mantel-Cox) test.

Figure 3:. EcN- but not HS-PROT₃EcT significantly delays infection with Cr(ΦStx2).

Six-seven-week-old female mice pre-treated with (A-C) EcN-PROT₃EcT or (D-F) HS-PROT₃EcT or (A-F) mock media were infected with 1x10⁸ CFU of Cr(ΦStx2dact) (A-C) or Cr(ΦStx2a) (D-F). Five mice were included in each cohort. (A, D) Viable counts of bacteria in feces were determined by plating. Each point shown represents an individual mouse, and each line represents the geometric mean. Samples plotted on the x-axis indicate no data available. Open symbols indicate CFU at the limit of detection (LOD). This value was used when evaluating statistical significance at each time point using two-way ANOVA with Bonferroni's post hoc multiple comparison test (95% CI). In (A), DPI 1: p = 0.0414; DPI 2: p = 0.0007, DPI 4: p = 0.0005; DPI 5: p = 0.0138; DPI 6–7: ns. In (D), all time points were found to be ns. (B, E) Time course of body weight changes (%) over time. Mean +/-SEM plotted. A two-tailed unpaired Studenťs t-test was used to determine statistical significance (95% CI). In (B), DPI 1–5: ns; DPI 6: p = 0.0492, DPI 7: p = 0.0003, DPI 8: 0.0047. In (E), all differences were ns. (C, F) Kaplan–Meier survival curves of mice group pre-treated with designated strains infected with Cr(ΦStx2). Statistical significance was determined by the log-rank (Mantel-Cox) test. Differences in survival in (C) (P = 0.0062) but not (F) were found to be statistically significant.

Figure 4:. HS-PROT₃EcT can be engineered to constitutively secrete SS^OspC2-Nb^TD4.

(A) Schematic of HS-PROT3EcT. (B-D, F-G) Secretion assays of designated strains engineered to secrete noted FLAG-tagged nanobodies, each fused to an OspC2 secretion sequence. Supernatant (TCA precipitated) (S) and whole-cell pellet lysates (P) were obtained at 30 min (B), 1 hr (C), 3 hr (D, F), or at designated time points (G) post transfer of the bacteria to PBS (B) or LB (C-D, F-G). In the case of (B) and (C), IPTG was added to induce expression of the nanobodies. Immunoblots probed with anti-FLAG or anti-DnaK are shown. Blots are representative of three independent experiments. (E) Growth curves of HS E. coli, HS-PROT T₃EcT, and HS-PROT₃EcT-Nb^2xTD4 grown in parallel. Data are representative of mean ± SEM of four technical repeats.

Figure 5:. Pre-treatment with HS-PROT₃EcT-TD4 delays Cr_Tir-M^EHEC(ΦStx2a) colonization and prolongs survival of infected mice.

(A) Study design schematic. (B-D) Seven-week-old female mice pre-treated with PBS (n=5), HS-PROT₃EcT (n=10) or HS-PROT₃EcT-Nb^2xTD4 (N=9) were infected with 1x10⁸ CFU of Cr_Tir-M^EHEC(ΦStx2a). (B) Viable counts of bacteria in feces were determined by plating. Each point shown represents an individual mouse, and each line represents the geometric mean. Shapes plotted on the x-axis indicate no data is available. Open symbols indicate CFU at the limit of detection (LOD). This value was used when calculating statistical significance. (C) Time course of body weight changes (%) over time. Mean +/-SEM plotted. Data in (B) and (C) were analyzed using two-way ANOVA with Bonferroni's post hoc multiple comparison test at a 95% confidence interval. *Denotes comparison to PBS and ^# denotes comparison to HS-PROT₃EcT. For (B), DPI 2 **p = 0.0035, ^##p = 0.0040; DPI 4: *p = 0.0104, ^##p = 0.0029; DPI 6: *p = 0.0114, ^##p = 0.0034. For (C), DPI 6 ****p < 0.0001, ^####p = 0.0001; DPI 7: ***p = 0.0007, ^####p < 0.0001. (D) Kaplan–Meier survival curves of mice pretreated with HS-PROT₃EcT-Nb^2xTD4, HS-PROT₃EcT, and Mock; infected with Cr_Tir-M^EHEC(Stx2a). Statistical significance was determined by the log-rank (Mantel-Cox) test. All possible pairs of survival curves were compared independently. ***p = 0.0003, ****p < 0.0001, ns=non-significant.