The effect of O-antigen length determinant wzz on the immunogenicity of Salmonella Typhimurium for Escherichia coli O2 O-polysaccharides delivery

Abstract

Attenuated Salmonella Typhimurium is a promising antigen delivery system for live vaccines such as polysaccharides. The length of polysaccharides is a well-known key factor in modulating the immune response induced by glycoconjugates. However, the relationship between the length of Lipopolysaccharide (LPS) O-antigen (OAg) and the immunogenicity of S. Typhimurium remains unclear. In this study, we assessed the effect of OAg length determined by wzz_ST on Salmonella colonization, cell membrane permeability, antimicrobial activity, and immunogenicity by comparing the S. Typhimurium wild-type ATCC14028 strain to those with various OAg lengths of the Δwzz_ST mutant and Δwzz_ST::wzz_ECO2. The analysis of the OAg length distribution revealed that, except for the very long OAg, the short OAg length of 2-7 repeat units (RUs) was obtained from the Δwzz_ST mutant, the intermediate OAg length of 13-21 RUs was gained from Δwzz_ST::wzz_ECO2, and the long OAg length of over 20 RUs was gained from the wild-type. In addition, we found that the OAg length affected Salmonella colonization, cell permeability, and antibiotic resistance. Immunization of mice revealed that shortening the OAg length by altering wzz_ST had an effect on serum bactericidal ability, complement deposition, and humoral immune response. S. Typhimurium mutant strain Δwzz_ST::wzz_ECO2 possessed good immunogenicity and was the optimum option for delivering E. coli O2 O-polysaccharides. Furthermore, the attenuated strain ATCC14028 ΔasdΔcrpΔcyaΔrfbPΔwzz_ST::wzz_ECO2-delivered E. coli O2 OAg gene cluster outperforms the ATCC14028 ΔasdΔcrpΔcyaΔrfbP in terms of IgG eliciting, cytokine expression, and immune protection in chickens. This study sheds light on the role of OAg length in Salmonella characteristics, which may have a potential application in optimizing the efficacy of delivered polysaccharide vaccines.

Keywords: Escherichia coli O2; O-antigen chain length; Salmonella Typhimurium; immune response; lipopolysaccharide.

Figure 1

Analysis of LPS profiles from S. Typhimurium with various OAg lengths. A Identification of gene replacement mutants by silver-stained SDS-PAGE. The lower band was unsubstituted lipid A-core, whereas the upper cluster of bands showed LPS with different lengths of OAg. Lane 1, 2 and 3 represents ST03 (Δwzz_ST), ST04 (Δwzz_ST::wzz_ECO2) and ATCC14028, respectively. ST03 (Δwzz_ST) was assigned into short OAg length group (< 15 RUs), ST04(Δwzz_ST::wzz_ECO2) was assigned into intermediate OAg length group (15–20 RUs), and the RUs of WT strain ATCC14028 was assigned into long OAg length group (20–35 RUs). B Western blotting detection against anti-Salmonella O₄ serum. C LPS profiles of attenuated S. Typhimurium with different OAg lengths. D Identification of panel C by Western blotting. Lane 4, 5, 6 represents ST06 (ATCC14028Δwzz_STΔcrpΔcya), ST07 (ATCC14028Δwzz_ST::wzz_ECO2ΔcrpΔcya) and ST05 (ATCC14028ΔcrpΔcya), respectively. E LPS profiles of E. coli O2 synthesized in S. Typhimurium ST08 (ATCC14028Δwzz_STΔcrpΔcyaΔasdΔrfbP) and ST09 (ATCC14028Δwzz_ST::wzz_ECO2ΔcrpΔcyaΔasdΔrfbP). Lane 7, 8, 9 represents ATCC14028, E. coli O2, O2-ST08, and O2-ST09, respectively. F Western blotting of LPS with rabbit antiserum against serotype O2 E. coli.

Figure 2

The impacts of OAg length on bacterial colonization in mice, cell permeability and antibiotic resistance. Colonization of ATCC14028, ST03 and ST04 in mice intestines (A), spleens (B), and livers (C). D Membrane permeability of ATCC14028, ST03 and ST04. The study was performed by recording the fluorescence of Ethidium influx into wzz substitution mutant strains at 600 nm with excitation at 545 nm. Blank control is ATCC14028 suspended in 50 mM potassium phosphate buffer without Ethidium bromide, PC control is ATCC14028 treated with 75% ethanol for 1 h. E Antibiotic sensitivity assay. The inhibitory zone diameter for each strain was measured. Error bars represent the standard deviation of 3 replicate samples.

Figure 3

Antibody immune responses induced by S. Typhimurium with variable OAg lengths in mice. Serum IgG (A), IgA (B), IgG1 (C), and IgG2a (D) specific for S. Typhimurium LPS were measured by ELISA. Error bars represent the standard deviation of 5 replicate samples. *P < 0.05, **P < 0.01 assessed by two-way ANOVA. E Serum bactericidal assay. Serum collected from BSG, ST06, ST07, and ST05-immunized groups and commercial rabbit serum against salmonella O4 (PC) were reacted with wild-type ATCC14028 to determine the survival rate of S. Typhimurium. Error bars represent the standard deviation of 3 replicate samples. F Flow cytometry histograms of C3 complement deposition. Antibody deposition on S. Typhimurium in 10% pooled antiserum from each group of attenuated S. Typhimurium differing for OAg length immunized mice. Wild-type S. Typhimurium incubated with sera from the BSG-inoculated group was set as the negative control (black line), incubated with sera from the ST05 (ATCC14028ΔcrpΔcya)-inoculated group was set as the positive control (green-yellow line). The blue line indicates incubation with sera from ST06 (ATCC14028Δwzz_STΔcrpΔcya)-inoculated group, and the purple line indicates the incubation with sera from mice immunized with ST07 (ATCC14028Δwzz_ST::wzz_ECO2ΔcrpΔcya).

Figure 4

Cytokine expression in CD4⁺ T cells stimulated with S. Typhimurium LPS ex vivo. After immunization in mice, the cytokine stimulation index (SI) was expressed relative to the percentage of IFN-γ (A) or IL-4 (B) cell subsets in the BSG-treated group. Data from two independent experiments were pooled and analyzed (n = 4) by t-test and two-way ANOVA. NS = not significant. Error bars represent the standard deviation of 4 replicate samples.

Figure 5

Immune protective evaluation in birds. A Antibody level detection, B Survival rate, C mRNA level of cytokines elicited in immunization group, the cytokine stimulation index (SI) was expressed relative to the percentage of IL-2, IL-4, IL-10 and IFN-γ mRNA level in the BSG-treated group. Data were analyzed (n = 5) by t-test. *P < 0.05, **P < 0.01, NS = not significant. Error bars represent the standard deviation of 5 replicate samples.

Figure 6

Representative images of histopathological changes in birds. Sections were stained with HE for pathological examination. Gross lesions and immune infiltrate were visually assessed. Scales bars = 50 μm.

Figure 7

Representative images of immunohistochemical staining of E. coli O2 after challenge in birds. Immunohistochemical staining were performed on paraffin-embedded section of chicken liver and spleen tissues. The hematoxylin-stained nucleus are blue, DAB positive expression is brownish yellow. Scales bars = 100 μm.