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Cross-reacting Surface Proteins Between Different Escherichia Coli O157:h7 Strains and Their Immune Responses in Animal Models

Ashikun Nabi
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh; Department of Molecular, Cellular and Developmental Biology, Yale University, 219 Prospect St, New Haven, CT 06511, USA

Fatema Moni Chowdhury
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh; Department of Microbiology, Jagannath University, 9-10 Chittaranjan Road, Dhaka 1100, Bangladesh

Zeenat Jahan
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh; Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia

Md Murshed Hasan Sarkar
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh, Bangladesh Council of Scientific and Industrial Research, Rajshahi, Bangladesh

Fazle Rabbi
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Chowdhury Rafiqul Ahsan*
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Abstract/ Executive Summary:

Surface proteins of Escherichia coli O157:H7, those that are prominent in antigen-antibody reactions among different strains, were found to provide protection against E. coli O157 challenge in mice. Three strains such as E. coli O157:H7 NCTC reference strain and two other environmentally isolated strains have been used in this study. New Zealand rabbits were immunized with surface proteins of NCTC reference strain and immunoblot analysis was performed against the surface proteins of all three strains. Immunoblot analysis revealed that a 94 kDa surface protein of E. coli O157:H7 could be the possible candidate for the protective activity experiment. Group of mice receiving the 94 kDa surface protein through both intraperitoneal and intranasal routes survived the challenge experiment. Whereas, all the control mice died within a couple of days. Mice challenge experiment clearly demonstrated the strong potential of the 94 kDa protein in the immunized mice. The data of this study provide us with a basis for further characterization of 94 kDa surface protein of E. coli O157:H7 as a protective antigen.

Keywords: Surface protein, E. coli O157:H7, Immune response

Location: Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Animal

Objectives:

To demonstrate various cross reacting surface proteins of different E. coli O157:H7 strains and the immune responses induced by these surface proteins in protecting animals against live cell challenge.

Methodology:

Bacterial strains The E. coli O157:H7 (NCTC strain Ref. no. 12079) and two locally isolated environmental E. coli O157:H7strains5, were all obtained from Department of Microbiology, University of Dhaka. Animal maintenance New Zealand white rabbits (1.5-2 kg body weight) and Swiss albino mice (5-7 weeks old) were obtained from the Animal Division of International Centre for Diarrheal Disease Research. Bangladesh (icddr,b). The mice were randomly selected and kept in plastic cages with wood-cobe bedding. After five days of acclimation, the animals were divided into different groups for the experiments. All the experiments and procedures using these animals were undertaken following the ethical issues set by the Faculty of Biological Sciences, University of Dhaka, Bangladesh. Preparation of water extracted materials (WEM) Bacterial strains (NCTC strain and other two E. coli O157:H7 strains) were grown in Brain Heart Infusion broth medium (500 ml) for overnight at 37ºC in a shaker incubator. Cells were harvested by centrifugation, washed with normal saline (0.85% NaCl solution) for three times. The washed pellet was then resuspended in around 5-7 ml of autoclaved distilled water and the mixture was placed in room temperature shaker for 6 h. Finally, the mixture was centrifuged at 12000 ×g for 15 min. Supernatant was then filtered through the 0.45 µm milipore membrane filter and stored at -20ºC until use. The amount of protein in the WEM was estimated by Bio-Rad protein assay. The same lot of WEM from NCTC 12079 and other two environmental strains was used throughout the whole study. Immunization of rabbits and serum collection Four New Zealand white rabbits were immunized with prepared WEM of NCTC strain, containing surface proteins at a ratio of 50 µg per kg body weight. Water extracted materials were injected with incomplete and complete adjuvant via intramuscular route. Sera collected before immunization was served as preimmunized control sera. The rabbits were immunized with two doses of WEM at 15 days intervals and sacrificed 7 days after administering final booster dose. Serum was collected at 7 days intervals to measure the antibody titer. Enzyme Linked Immunosorbent Assay (ELISA) An ELISA method was used to quantitate serum antibody recognizing E. coli O157:H7 surface proteins preparation and to determine the cross-reactivity among the surface proteins of different E.coli O157:H7 clinical and environmental strains6. In brief, WEM of different strains (NCTC 12079 and two other environmental strains) was used as antigens. Flat bottom polystyrene plate was coated with diluted WEM (1µg of surface protein to each well) in bicarbonate coating buffer (pH 9.6) and 100 µl of rabbit serum, serially diluted in 0.5% BSA (w/v) in PBS, were added to each well. After incubation and washing four times, 100 µl of alkaline-phosphatase conjugated anti-rabbit IgG (diluted 1:1000) were added to each well and the plate was incubated for further 2 h at room temperature. After final washing, 100 µl of Á-nitrophenylphosphate disodium salt (1mg/ml) in glycine buffer (pH 9.5) was added to each well. Reaction was stopped by adding 25 µl of 3 M NaOH and the absorbance was measured at 405 nm in micro-ELISA reader. Gel electrophoresis and immunoblot analysis Gels consisting of 5% (w/v) acrylamide-stacking gel and 12.5% (w/v) acrylamide-separation gel in a discontinuous polyacrylamide gel electrophoresis (PAGE) system with Laemmli buffers were used for the separation of different surface proteins7. Electrophoresis was performed using the mini-protean III system (Bio-Rad Laboratories Ltd., Richmond, Calif.) with a current of 12 mA for first 12-15 min then 15 mA for 1 h. Presence of different surface proteins was visualized by coomassie blue staining. For immunoblotting, instead of staining with coomassie blue, separated surface proteins in the gels were transferred onto nitrocellulose paper following the method described by Laemmli8. Primary antibody of rabbit sera was added in 1:100 dilutions for 1.5 h at room temperature after blocking the strip with 3% skim milk in PBS. Strips were then washed with 0.1% Tween 20 in PBS for three times. Secondary antibody (alkaline phosphatase conjugated goat anti-rabbit antibody, whole molecule, Sigma, USA) in 3% skim milk at 1:10000 dilutions were added for 1.5 h at room temperature. After final washing as stated above, blot strips were placed in substrate solution (containing 0.1 M TrisHCl solution, 0.09 M NaCl solution, 0.15 M MgCl2, BCIP, NBT) until the bands had appeared. Molecular weight of different surface proteins reacted with immunized rabbit (WEM of NCTC) sera was determined by calculating the relative mobility (Rf) value of the standard marker proteins. Separation of the specific surface proteins from number of protein mixture in WEM A 10% separating gel was made in combination with a 5% stacking gel and approximately 300 µl of WEM of different E. coli O157:H7 strains were loaded on the gel. The separated proteins in gel were transferred to nitrocellulose membrane. The membrane was washed with 0.1% Amido black stain to locate the position of protein bands. The membrane was cut carefully and subjected to immunoblot analysis to locate the position of different proteins that gave immune reaction with antisera raised in rabbits immunized with the WEM of NCTC strain. Membrane was cut and placed into PBS after locating the position of the protein and were sonicated until the membrane reduced to fine powder form to pass through a 25-gauge hypodermic needle. Immunization of mice To demonstrate the protective efficacy of the different surface proteins in mice, a group of sixteen Swiss albino mice were used in each group. Each mouse was administrated with certain molecular weight of surface proteins (20-25µg protein/mice) via intraperitoneal or intranasal route at 15 days intervals. Sera were taken at 7 days intervals and 30 days after last immunization and titer of antibodies was checked in the pooled sera by the ELISA. Immunoblot analysis was also used to check the homogeneity of the separated surface proteins. Groups of 16 mice in case of both intraperitoneal and intranasal route were used as negative controls receiving only sonicated nitrocellulose membrane (same turbidity as the experimental sonicated membrane) in PBS buffer. Determination of 50% Lethal Dose (LD50) of E. coli O157:H7 (NCTC, Ref no. 12079) and challenge experiment LD50dose was determined by the method described by Boyd10, where each group of Swiss albino mice was administered with 100 µl of bacterial suspension (E. coli O157:H7 NCTC, Ref. no. 12079) containing 106, 107 and 108 CFU/ml live cells through intraperitoneal route. For the challenge experiment, 16 mice (67 weeks old) were included in each group, where immunized mice were challenged with the LD50 dose of the organism. Death of mice were recorded daily for 21 days and all mice alive after 21 days were considered to have survived the challenge.

Source of Information: Bangladesh J Microbiol, Volume 36, Number 1, June 2019, pp 11-15

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

For the development of a successful vaccine two important facts that should be considered are the choice of candidate immunogen and route of immunization14. In this study, we have included 94 kDa and 143 kDa surface proteins separately as immunogens and intraperitoneal and intranasal routes as a way of immunization. Immunization of mice with both the surface proteins has elicited IgG antibody response as compared with the control group mice. However, the groups of mice receiving 94 kDa surface protein through intraperitoneal and intranasal routes have demonstrated better response than the groups of mice receiving 143 kDa surface protein. Data of ELISA and immunoblot analysis after immunization has been later supported by the mice challenge experiment with live E. coli O157:H7 strain, where the group of mice receiving 94 kDa protein through intraperitoneal or intranasal route, has survived for 21 days where as nonimmunized control group died. The protection rate of intraperitoneal and intranasal group immunized with 94 kDa protein is 93% and 87% respectively, which is highly significant (pÂ0.05), whereas in case of 143 kDa protein it is 68% and 62.5% respectively. Absence of contamination with other surface proteins in immunized group and IgG antibodies against 94 kDa surface protein in control non-immunized group but the presence of this particular surface protein specific IgG antibody in immunized group, as has been checked by immunoblot, clearly demonstrate that the protection is provided by the 94 kDa specific IgG antibody.

Knowledge Management: Journal