Iron-regulated lysis of recombinant Escherichia coli in host releases protective antigen and confers biological containment

Abstract

The use of a recombinant bacterial vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live bacterial vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) vector and found one, P(viuB), with characteristics suitable for our use. We fused P(viuB) with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible P(T7)-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in bacterial vector vaccine applications.

Fig. 1.

Iron-regulated promoter screening. (A) Plasmid map of the reporter vector pUTG. Gene coding regions are represented on the vector map by arrows. rrnBT1T2, ribosomal terminators T1 and T2; gfp, the green fluorescence protein reporter gene; MCS, multiple-cloning site; Amp^r, ampicillin resistance gene. (B) GFP expression levels in E. coli carrying P_suf, P_fes, P_fhuA, P_viuA, and P_viuB when grown in LB medium containing 40 μM FeSO₄ (Fe 40) as an iron-rich condition or 200 μM 2,2′-dipyridyl (DP200) as an iron-limiting condition. The error bars represent the standard deviations (SD) for three independent experiments performed in triplicate. (C) Fur titration assay for P_viuB. E. coli H1717/pMD19T and H1717/pMD19TB were plated separately on MacConkey medium with 25 μM FeSO₄.

Fig. 2.

GFP expression controlled by P_viuB in vivo in zebrafish. (A) GFP expression levels of BL21(DE3)/pUTG (gray bars) and BL21(DE3)/pUTBG (black bars) incubated in immersion solution. BL21(DE3)/pUTBG (white bars) was induced to synthesize GFP as a positive control. Immersion solution was prepared as phosphate-buffered saline (PBS) with 1% bovine serum albumin. (B) Visualization of GFP synthesis under the control of P_viuB in zebrafish by fluorescence microscopy. Fish were immersed in bacterial suspensions (10⁸ CFU/ml) of BL21(DE3)/pUTG (panel I) and BL21(DE3)/pUTBG (panel II) for 1 h and detected at 9 h postinfection. For further analysis, zebrafish infected by BL21(DE3)/pUTBG were observed by confocal microscopy. Panels III, IV, and V, different parts of the fish from head to tail, respectively (magnification, ×400); panel VI, details of the site marked by a box in panel IV (magnification, ×1,000).

Fig. 3.

Growth and lysis of BL21(DE3)/pUTaBE induced under iron-limiting conditions. (A) Growth and lysis (solid symbols) and viable cell counts (open symbols) of E. coli grown in LB. BL2(DE3)/pUTa with 2,2′-dipyridyl induction (diamonds), BL21(DE3)/pUTaBE with 2,2′-dipyridyl induction (circles), and BL21(DE3)/pUTaBE without 2,2′-dipyridyl induction (triangles) are shown. At 0 min, 2,2′-dipyridyl was added (↓). Standard deviations were calculated from the results from three independent experiments. (B) Scanning electron micrographs of E. coli BL21(DE3)/pUTa (without gene E) with 2,2′-dipyridyl addition (panel I) and of E. coli BL21(DE3)/pUTaBE (with gene E) without 2,2′-dipyridyl addition (panel II) and at 30 min (panel III) and 2 h (panel IV) after 2,2′-dipyridyl addition.

Fig. 4.

Cell lysis and antigen release in vitro of BL21(DE3)/pETGA+pUTaBE. (A) Plasmid maps for pETGA, the antigen expression vector, and pUTaBE, the iron-regulated lysis vector. gapA, gene encoding A. hydrophila GAPDH; E, lysis gene E from bacteriophage φX174; P_T7, T7 promoter; P_viuB, promoter of the viuB gene from V. cholerae. (B) Growth and lysis curves of BL21(DE3)/pETGA+pUTa and BL21(DE3)/pETGA+pUTaBE with 2,2′-dipyridyl induction. At 0 min, 2,2′-dipyridyl was added (↓). (C) The relative amount of GAPDH in the culture supernatant and whole cells were roughly evaluated by ELISA, and the ratio of the released GAPDH in the supernatant to the total GAPDH in the whole-cell lysate was calculated for each strain. Error bars indicate standard deviations from three independent experiments.

Fig. 5.

Cell lysis and antigen release in vivo in zebrafish. After intraperitoneal injection with 5 μl of PBS, E. coli BL21(DE3)/pETGA+pUTa (10⁷ CFU per fish), or E. coli BL21(DE3)/pETGA+pUTaBE (10⁷ CFU/fish), 20 fish with similar weights from each group were randomly taken at 4, 9, and 24 h postinjection. The tissues surrounding and including the abdominal cavities were collected, and the weight of each sample was kept at between 6 and 7 g. The tissue samples were then homogenized in 15 ml PBS. (A) Bacterial survival in zebrafish. Each homogenate sample was diluted serially in PBS and plated in triplicate on LB agar containing kanamycin and ampicillin to count the CFU. (B) Antigen release by bacterial cells in zebrafish. Each homogenate sample, collected at defined time intervals, was centrifuged to harvest the supernatant, and the concentrated supernatant was analyzed by Western blotting to detect the antigen GAPDH.

Fig. 6.

Protection by vaccine candidate BL21(DE3)/pETGA+pUTaBE in turbot against A. hydrophila challenge. The fish (30 fish in each group, divided into three parallel groups) were vaccinated i.p. with E. coli BL21(DE3)/pETGA+pUTaBE (10⁷ CFU per fish), E. coli BL21(DE3)/pETGA+pUTa (10⁷ CFU per fish), purified GAPDH antigen with an equal volume of Freund's complete adjuvant (Sigma) (20 μg per fish), or PBS as a control (0.1 ml per fish). Four weeks after immunization, the four groups were injected i.p. with wild-type A. hydrophila LSA34 (5.0 × 10⁷ CFU per fish). The significant difference and the relative percent survival (RPS) were calculated by using Fisher's exact test and the formula RPS = [1 − (% mortality in vaccinated fish/% mortality in control fish)] × 100, respectively.

Fig. 7.

A novel antigen delivery and release system in E. coli. (A) Iron-rich conditions. In iron-rich medium, the iron-Fur complex binds to the Fur box of P_viuB and represses the transcription of the E gene, while the antigen gene is expressed under control of the IPTG-inducible T7 promoter. (B) Iron-limiting conditions. In the host organism, antigen gene expression is repressed by LacI in the absence of IPTG. Meanwhile the iron-Fur complex is dissociated and the transcription of the E gene initiated. Bacterial cells are rapidly lysed by protein E to achieve antigen release and biological containment.