Bacterial Ghosts of Pseudomonas aeruginosa as a Promising Candidate Vaccine and Its Application in Diabetic Rats

Abstract

Infections with Pseudomonas aeruginosa (PA) pose a major clinical threat worldwide especially to immunocompromised patients. As a novel vaccine network for many kinds of bacteria, bacterial ghosts (BGs) have recently been introduced. In the present research, using Sponge-Like Reduced Protocol, P. aeruginosa ghosts (PAGs) were prepared to maintain surface antigens and immunogenicity. This is the first study, to our knowledge, on the production of chemically induced well-structured bacterial ghosts for PA using concentrations of different chemicals. The research was carried out using diabetic rats who were orally immunized at two-week intervals with three doses of PAGs. Rats were subsequently challenged either by the oral route or by the model of ulcer infection with PA. In challenged rats, in addition to other immunological parameters, organ bioburden and wound healing were determined, respectively. Examination of the scanning and transmission electron microscope (EM) proved that PAGs with a proper three-dimensional structure were obtained. In contrast to control groups, oral PAGs promoted the generation of agglutinating antibodies, the development of IFN-γ, and the increase in phagocytic activity in vaccinated groups. Antibodies of the elicited PAGs were reactive to PA proteins and lipopolysaccharides. The defense against the PA challenge was observed in PAGs-immunized diabetic rats. The resulting PAGs in orally vaccinated diabetic rats were able to evoke unique humoral and cell-mediated immune responses and to defend them from the threat of skin wound infection. These results have positive implications for future studies on the PA vaccine.

Keywords: Pseudomonas aeruginosa; bacterial ghost; diabetic ulcer; oral immunization; sponge-like reduced protocol (SLRP); vaccine.

Figure 1

An illustration of the experimental design on animals. Experimental protocols are shown for rats challenged orally (A) or through ulcer induction and infection (B). Negative control groups (not shown) were also included and were given saline instead of the vaccine. Each experimental group included six rats. Blood samples were collected on day 34 to determine the agglutinating antibody titers, phagocytic and killing activities, and serum IFN-γ levels. The timeline is not to scale.

Figure 2

Characterization of Pseudomonas aeruginosa ghosts (PAGs). (A) Agarose gel electrophoresis of prepared PAGs. Lane PA: P. aeruginosa cells showing intense DNA bands; Lane M: DNA marker; Lanes 1–4: P. aeruginosa ghost prepared by Scheme 1; Lanes 5–7: P. aeruginosa ghost prepared by Scheme 2. (B) SDS-PAGE of prepared PAGs. M: Protein ladder; Lane 1: PAGs preparation; Lane 2: P. aeruginosa cells. Samples were boiled in sample buffer for 3 min and loaded into gel.

Figure 3

Microscopical examination of PAGs. (A) Light microscope pictures following crystal violet simple staining using oil immersion lens (×100): (1) P. aeruginosa untreated cells, (2) cells after the basic step, (3) cells after the H₂O₂ step, and (4) P. aeruginosa ghost after the ethanol step. External structure integrity is observed in all smears. (B) Scanning electron micrographs at different magnification powers. (1) Normal P. aeruginosa cells showing intact cell walls. (2), (3), and (4) show BGs after chemical treatment showing a 3D non-deformed cell wall where the arrows show the pores formed.

Figure 4

PAGs increase immunological responses in diabetic rats after oral vaccination. (A) Agglutinating antibody titers as determined by the slide agglutination test. Percentage phagocytic (B) and NBT reduction (C) activity was also evaluated. (D) Serum IFN-γ was determined by ELISA. All tests were carried out on samples recovered on day 34 of the vaccination regimen. All experiments were repeated twice. (*) p < 0.05; (**) p < 0.001 using Mann–Whitney test (A) and Student’s t test (B–D) (N = 6).

Figure 5

The bioburden of organs in challenged rats. Diabetic animals were orally vaccinated with 10⁸ PAGs for a total of three doses at two-week intervals. On day 35, both vaccinated and unvaccinated groups were challenged orally with 10⁸ CFU of P. aeruginosa, and organ bioburden was determined 5 days post-challenge. (*) p < 0.05 using Student’s t test (N = 6).

Figure 6

Following PA infection of artificial ulcers, a Kaplan–Meier survival curve was created. Artificial ulcers of PAGs orally immunized diabetic rats were infected with PA and animal survival was observed for 6 days. (***) p < 0.001 by Log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests.

Figure 7

The rate of ulcer healing in various PA-infected animal groups. Artificial ulcers in vaccinated and unvaccinated diabetic rats were infected with PA and healing was observed for 6 days. All unvaccinated rats died by the fifth day post-challenge.

Figure 8

Immune responsiveness of PAGs antibodies to LPS and proteins from P. aeruginosa. (A) Coomassie blue-stained gel of the purified extracted P. aeruginosa LPS. (B) The silver-stained gel of purified LPS. (C) Western blot of purified LPS using serum from PAGs immunized rats as the primary antibody. (D) Coomassie blue-stained gel of prepared PAGs. (E) Western blot against PAGs proteins using serum from PAGs-immunized rats as the primary antibody.