Recombinant Saccharomyces cerevisiae EBY100/pYD1-FaeG: a candidate for an oral subunit vaccine against F4+ ETEC infection

Abstract

Diarrheal diseases attributable to multidrug-resistant F4+ enterotoxigenic Escherichia coli (ETEC) are escalating in severity, posing significant risks to the health and safety of both humans and animals. This study used Saccharomyces cerevisiae EBY100 to display the FaeG subunit of F4 colonizing factor as an oral vaccine against F4+ ETEC infection. Mice were orally immunized twice with 10⁸ CFU of EBY100/pYD1-FaeG, followed by a challenge with F4+ ETEC EC6 on day 7 post-immunization. The results showed that the recombinant strain EBY100/pYD1-FaeG orally enhanced the growth of the small intestine villi, significantly boosted the expression of tight junction proteins (ZO-1, Occludin, MUC2, and Claudin) (P < 0.05), and modulated the gut microbiota composition. Additionally, immunization with EBY100/pYD1-FaeG also upregulated the levels of IL-2, IL-4, and IFN-γ in the intestines of mice (P < 0.01), while serum IgG and fecal sIgA titer significantly increased (P < 0.05). These immune responses enhanced the capacity to fight against ETEC, leading to an increased survival rate of mice and relieved damage to tissues and organs of mice infection. In summary, the study suggested that the recombinant Saccharomyces cerevisiae EBY100/pYD1-FaeG could effectively stimulate the immune response and generate specific antibodies against F4+ ETEC, showing its potential to serve as a subunit oral vaccine candidate for preventing F4+ ETEC infection.IMPORTANCEThe multidrug-resistant F4+ enterotoxigenic Escherichia coli (ETEC) strains are the primary clinical pathogens responsible for post-weaning diarrhea in piglets, resulting in substantial economic losses in the pig farming industry. In the study, we developed an oral vaccine candidate, Saccharomyces cerevisiae EBY100/pYD1-FaeG, to prevent diarrhea caused by multidrug-resistant F4+ ETEC. Oral administration of EBY100/pYD1-FaeG significantly enhanced immune responses, improved intestinal health, and provided protection against F4+ ETEC infection in mice. This approach offers a potential application prospect for preventing F4+ ETEC infections that lead to post-weaning diarrhea in clinical settings and provides a promising solution for addressing the growing threat of antibiotic resistance in bacterial pathogens.

Keywords: F4+ ETEC; Saccharomyces cerevisiae; oral vaccine.

Fig 1

Construction and expression of the recombinant S. cerevisiae EBY100/pYD1-FaeG. (A) Diagram of the workflow for constructing recombinant yeast. After the FaeG fragment was digested with MluI and NheI, the purified FaeG fragment was ligated with T4 ligase with plasmid pYD1. Finally, the recombinant plasmid pYD1-FaeG was transformed into S. cerevisiae EBY100 strain by the LiAc method, and positive clones were screened by the minimal dextrose plates. (B) Analysis of the expression of the FaeG protein in S. cerevisiae EBY100 via western blot. From left to right lanes are M (Protein Maker), EBY100/pYD1-FaeG, EBY100/pYD1, and EBY100. (C) The morphology of recombinant S. cerevisiae EBY100/pYD1-FaeG under a positive fluorescence microscope. The bar represents 10 µm. (D) The expression profile of recombinant protein was also evaluated through flow cytometry.

Fig 2

The fermentation and expression kinetics of the FaeG protein in EBY100/pYD1-FaeG. (A) The growth curve of the S. cerevisiae strains. The OD_600nm value of EBY100, EBY100/pYD1, and EBY100/pYD1-FaeG in YPD medium with galactose was measured at various time points. (B) The number of cells in YPD medium containing galactose was detected using the plate counting method at the same time points as the OD_600nm value measured for the growth curve. (C) The expression levels of the FaeG protein at 12, 24, 36, 48, 60, and 72 h in EBY100/pYD1-FaeG were determined by western blotting, with GAPDH as the internal control and the EBY100 strain as the negative control. (D) The relative gray value of protein bands was analyzed using the Image J software. All experiments were repeated three times. The data represent the mean ± SD.

Fig 3

Immune effect of the EBY100/pYD1-FaeG strain on mice. (A) Immunization process. Forty-five mice were randomly divided into three groups (15 per group) and were orally immunized twice on days 1–7 and 15–21, respectively. The corresponding samples were collected at different time points (black solid circles), as illustrated in the diagram. (B) The changes in body weight in mice at 0, 7, 14, 21, and 28 days (n = 10). (C) The visceral index of mice on day 28 after immunization. The wet weight to body weight ratio of the heart, liver, spleen, lungs, and kidneys (n = 5). (D) The specific IgG antibody titers of FaeG protein in the sera of mice after immunization. (E) Antibody titers of secretory IgA in feces of mice at days 7, 14, 21, and 28 after immunization. All experiments were performed in triplicate. The data in the figure represent the mean ± SD, and the significance of the difference between groups was assessed via the two-way ANOVA (*P < 0.05; **P < 0.01; ns, no significant difference).

Fig 4

Effects of the EBY100/pYD1-FaeG strain on the intestinal tract of mice following oral immunization. (A) The histopathological changes in the jejunum of mice in different immunization groups after H-E staining. The bar represents 100 µm. Quantitative analysis of villus length (B) and crypt depth (C) in the small intestine of mice (n = 3). Six intestinal villi and crypts were taken from each section for measurement. (D) The ratio of villus length to crypt depth (the V/C ratio) in mice. (E) Effect of immunization of mice on the expression of tight junction proteins in the small intestine. The relative expression of the tight junction proteins ZO-1, Occludin, MUC2, and Claudin in the small intestine after immunization was detected by quantitative real-time PCR. (F) The expression levels of cytokines (IL-2, IL-4, IL-10, and IFN-γ) in the intestines of mice after immunization. The experiments were repeated three times, and the data in the figure are represented as mean ± SEM. The significance of the difference between the data in each group was determined by the t-test and the one-way ANOVA (*P < 0.05; **P < 0.01; and ***P < 0.001).

Fig 5

Effects of oral immunization with the recombinant S. cerevisiae strain EBY100/pYD1-FaeG on gut microbiota in mice. α-diversity of gut microbiota among the groups of mice after immunization is shown by Sobs (A), Chao1 (B), Shannon (C), and Simpson indices (D). (E) The PCoA plots show the β-diversity of gut microbial composition based on Bray-Curtis distance at the OTU level. (F) The Venn diagram shows the unique or shared OTUs in different samples. (G) Histogram of differences in species distribution of gut microbiota across phyla in each group of mice. (H) At the genus level (top 30 species), mouse gut microbial community composition was different. Data are shown as mean ± SEM (n = 5).

Fig 6

Immunoprotective effect of the recombinant strain on mice against ETEC EC6 infection after oral immunization. (A) Survival curve. On day 28 post-immunization, mice were challenged with LD₅₀ of ETEC EC6 by i.p. (10⁸ CFU, 200 µL/mouse) and continuously monitored for 7 days. (B) Bacterial load in tissues and organs of mice 7 days post-infection. (C) Observation of histopathological changes in mouse tissues and organs 7 days post-infection with ETEC EC6. The pathological changes in liver, spleen, jejunum, ileum, and colon were observed by H-E staining. The black arrow points to inflammatory cells; the yellow arrow indicates cytoplasmic vacuolization; and the red arrow points to nuclear fragmentation. The bar represents 100 µm. All data were obtained from three independent replicates and presented as mean ± SEM. Statistical analysis was performed by t-test and one-way ANOVA (*P < 0.05 and **P < 0.01).