Evaluation of Prevalence, Homology and Immunogenicity of Dispersin among Enteroaggregative Escherichia coli Isolates from Iran

Abstract

Background: Diarrhea, caused by enteroaggregative Escherichia coli (EAEC), is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin (antiaggregation protein, aap) is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients.

Methods: PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund's adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated.

Results: The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed.

Conclusion: Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections.

Keywords: Diarrhea; Enteroaggregative E. coli; Enzyme-linked immunosorbent assay.

Fig. 1

PCR amplification of aag gene. The genome of the enteroaggregative Escherichia coli (EAEC) isolates was extracted, and PCR amplification of aap gene was performed by PCR method. Lane 1, molecular weight marker (100 bp ladder DNA); Lane 2, E. coli K12 strain (negative control); Lane 3, EAEC strain 042 (positive control); Lanes 4-7 and 9-11, products of aap gene (about 348 bp); Lane 8, negative control (double-distilled water).

Fig. 2

Confirmation of cloning of aap gene in expression vector pBAD-gIIIA. The aap genes were cloned in pBAD-gIIIA vector and the confirmation of cloning was carried out by (A) electrophoresis (Lanes 1-3 and 5-7, pBAD-aap clones; Lane 4, pBAD-gIIIA); (B) PCR Lane 1, negative control; Lanes 2-6, pBAD-aap clones; Lane 7, 100 bp molecular weight marker; (C) Enzyme digestion with NcoI and XbaI restriction enzymes (Lane 1, molecular weight marker (1-kb ladder mix); Lanes 2 and 3, pBAD-aap clones).

Fig. 3

Analysis of the expressed products of aap gene by SDS-PAGE and Western-blotting. The expressed protein dispesin (aap) was evaluated by (A) SDS-PAGE (Lane 1, uninduced construct; Lanes 2-6, aap induced by arabinose 0.002%, 0.02%, 0.2%, 2% and 20%, respectively; Lane 7, protein marker); (B) Western-blot with His-tag monoclonal antibody (Lanes 1-5, aap induced by arabinose 0.002%, 0.02%, 0.2%, 2% and 20%, respectively; Lane 6, uninduced construct; Lane 7, protein marker).

Fig. 4

Analysis of purified dispersin protein by SDS-PAGE. Lanes 1 and 2, purified dispersin protein (elution 1 and 2, respectively); Lane 3, protein marker.

Fig. 5

Induction of anti-dispersin IgG responses in rabbits. Rabbits were immunized three times with dispersin and admixed with Freund’s adjuvat. The control groups were immunized with PBS. (A) The IgG response after the first, the second and the third immunization. (B) The dispersin-specific IgG profile after the third immunization at 1:100 serum dilution. * indicates statistical significance between the first injection and the control (P<0.05). ** indicates statistical significance of the second and the third injection in comparison to the control group (P<0.01). Preinjection is the result of anti-dispersin IgG response in serum collected before the first injection of dispersin and was used as a control serum. Results were expressed as mean and standard errors.

Fig. 6

Evaluation of dispersin and anti-dispersin antibody in HeLa cell. (A) EAEC 042 strain as a positive control; (B) E. coli K12 strain as a negative control; (C) An EAEC isolate in the absence of anti-dispersin antibody; (D) An EAEC isolate in the presence of anti-dispersin antibody.