Expression of Escherichia coli virulence usher protein attenuates wild-type Salmonella

Abstract

Generation of a live attenuated vaccine for bacterial pathogens often requires prior knowledge of the pathogen's virulence factors. We hypothesized an alternative approach of heterologous gene expression would make a wild-type (wt) pathogen more susceptible to host cell killing, thus, resulting in immunization. As proof of concept, the heterologous expression of enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I) was tested to attenuate Salmonella. The overexpression of CFA/I resulted in significant attenuation of wt Salmonella. In-depth studies revealed the attenuation depended on the co-expression of chaperone (CfaA) and usher (CfaC) proteins. Remarkably, the CfaAC-attenuated Salmonella conferred protection against wt Salmonella challenge. Mechanistic study indicated CfaAC made Salmonella outer membranes permeable, causing Salmonella to be vulnerable to host destruction. Thus, enhancing bacterial permeability via CfaAC represents an alternative method to attenuate pathogens despite the presence of unknown virulence factors.

Figure 1. Evaluation of CFA/I fimbriae expression by protein gel blot analysis and AFM imaging. (A) Schematic physical maps of asd-based plasmids. The cfa/I operon is regulated by a tripartite fusion promoter in P1-pHC, a single promoter in P1-pC and no cfa/I operon is harbored in control P1-pY. (B) Protein gel blot analysis shows CFA/I fimbria expression by P1-pHC (Lane 1) and -pC (Lane 2) and no fimbriae expression was detected for -pY (Lane 3). Prestained molecular weight standards are shown in Lane M. (C) AFM images indicate expression of CFA/I fimbriae for strains (1) P1-pHC and (2) -pC, but no fimbriae were observed for control strain (3) -pY.

Figure 2. Overexpression of cfa/I attenuates Salmonella and confers protective immunity. (A) Strains P1-pHC, -pC and -pY were assessed for their survival in RAW264.7 macrophages at varying bacteria to macrophage ratios of (1) 1:1, (2) 10:1 or (3) 100:1. While initial levels of infection (t = 0 h) were significantly different for each strain, at 4 or 24 h post-infection, survival rates were augmented for each tested strain. In fact, at 4 and 24 h post-infection, P1-pHC was cleared at or below the detection limit. While P1-pC was not cleared as efficiently, it still was impaired relative to -pY. Values are the mean ± SEM of three independent experiments. Differences in macrophage colonization were calculated using Tukey Kramer multiple comparisons test by P1-pY vs. (vs) -pHC or -pC are indicated as: **p < 0.01, ***p < 0.001. (B) Overexpression of cfa/I reduces tissue inflammation and colonization. BALB/c mice were orally dosed with 1 × 10⁹ CFUs of P1-pHC, -pC, -pY, or wt strain H71 and at 5 d post-infection, (1) splenic weights and total CFUs/tissue were determined for (2) spleens, (3) PPs and (4) livers. Results are mean ± SEM of two independent experiments. Differences in tissue weight and colonization burden were calculated using Tukey Kramer multiple comparisons test. Values representing significant differences in weight or colonization in mice infected with wt H71 vs P1-pHC, -pC, or -pY are indicated as: *p < 0.05, **p < 0.01 and ***p < 0.001; ND, none detected. (C) cfa/I-attenuated Salmonella confers protection against wt Salmonella challenge. Groups of BALB/c mice were orally dosed with 1 × 10⁹ CFUs of (1) P1-pHC (n = 15), -pC (n = 15), -pY (n = 15), wt H71 (n = 10), or 5 × 10⁹ CFUs of H647 (n = 10); all mice given P1-pY, -pC, or wt H71 succumbed to infection. The Kaplan-Meier method was used to obtain the survival fractions for wt H71, P1-pHC, -pC and H647 administered mice. Survival fractions of wt H71 were compared with mice dosed with P1-pHC, -pC and H647 and significance was determined: **p < 0.01; and mice administered with P1-pY were compared with mice dosed with P1-pHC, -pC and H647 and significance was determined: ^¶¶¶p < 0.001. Data are the mean from three experiments. (2) P1-pHC-immunized mice were challenged with 5 × 10⁷ CFUs wt H71 strain and 60% of the mice survived with similar survival to H647-immunized mice at 70%. All sPBS-dosed mice succumbed to challenge. The Kaplan-Meier method was used to obtain the survival fractions after challenge for sPBS-, P1-pHC- and H647-immunized mice. Survival fractions of sPBS group were compared with P1-pHC and H647-immunized groups and significance was determined: ***p < 0.001. Data are the mean from two experiments. (D) A kinetic analysis of serum IgG and fecal IgA (1) anti-CFA/I fimbriae and (2) anti-heat-killed S. typhimurium (HKST) Ab titers were performed for BALB/c mice vaccinated with P1-pHC. Differences were calculated by Student t-test. Anti-CFA/I fimbriae IgG titers by P1-pHC immunized mice were significantly different from IgG titers by sPBS-dosed mice and indicated as: *p < 0.05, **p < 0.01 and ***p < 0.001; and anti-CFA/I fimbriae IgA titers by P1-pHC immunized mice vs sPBS-dosed mice are indicated as: ^¶p < 0.05, ^¶¶p < 0.01 and ^¶¶¶p < 0.001. H647 induced no anti-CFA/I fimbriae titers, but it induced significantly higher anti-heat killed Salmonella typhimurium (HKST) IgG titers than sPBS-dosed mice and indicated as: ^†p < 0.05, ^††p < 0.01 and ^†††p < 0.001; and IgA titers by H647 vs sPBS-dosed mice are indicated as: ^‡p < 0.05, ^‡‡p < 0.01 and ^‡‡‡p < 0.001. Data are the mean ± SEM of two independent experiments.

Figure 3. Schematic maps and AFM imaging of P1 strains bearing cfa/I gene components. (A) Schematic maps of five recombinant plasmids. (B) AFM images of P1 strains bearing the plasmid with (1) pHcfaABC, (2) pHcfaACE, (3) pHcfaAC, (4) pHcfaA or (5) pHcfaC. No fimbriae were detected in any of these strains.

Figure 4. The combination of cfaAC genes is required for Salmonella attenuation. (A) Evaluation of in vitro virulence in RAW264.7 macrophages was determined for P1 strains bearing the plasmids, -pHcfaABC, -pHcfaACE, -pHcfaAC, -pHcfaC, -pHcfaA or -pY. Macrophages were infected at a bacteria-to-macrophage ratio of 1:1 and the bacterial CFUs were enumerated at 0, 4 and 24 h post-infection. Differences in macrophage colonization were calculated using Tukey Kramer multiple comparisons test. Values depict the mean ± SEM of 4 independent experiments; ***p < 0.001 depicts significant differences in colonization vs P1-pY; ^¶¶¶p < 0.001 depicts significant differences at 4 and 24 h post-infection for grouped strains, P1-pY, -pHcfaA and -pHcfaC vs P1-pHcfaAC, -pHcfaACE and -pHcfaABC. (B) Evaluation of in vivo virulence of cfa/I gene-expression of Salmonella strains was determined in BALB/c mice. Groups of BALB/c mice (5/group) were orally infected with one of the six strains (1 x 10⁹ CFUs/strain/mouse) and at 5 d post-infection, (1) splenic weights and total CFUs/tissue from (2) spleens, (3) PPs and (4) livers were determined. Differences in tissue weight and colonization burden were calculated using Tukey Kramer multiple comparisons test. *p < 0.05, **p < 0.01 and ***p < 0.001 depict significant differences in splenic weights or splenic, PP and liver CFUs from mice infected with cfa/I gene-attenuated strains vs P1-pY infected mice. (C) To assess the lethality of the recombinant Salmonella strains, groups of mice were orally dosed with one of the 6 strains, as described above, in (B). Strains P1-pY (n = 10), -pHcfaA (n = 10) and -pHcfaC (n = 10) were lethal to BALB/c mice; the P1-pHcfaABC (n = 15) and -pHcfaAC (n = 15) infected mice survived and 90% of the P1-pHcfaACE (n = 10) infected mice survived. The Kaplan-Meier method was used to obtain the survival fractions. Values are the mean ± SEM of two (P1-pHcfaACE, -pHcfaA, -pHcfaC and -pY) or three (P1-pHcfaABC and -pHcfaAC) independent experiments; survival fractions obtained from mice dosed with P1-pHcfaABC, -pHcfaACE, or -pHcfaAC were compared with mice dosed with P1-pY, -pHcfaA, -pHcfaC and significance is shown: ***p < 0.001. Arrows indicate the time of infection.

Figure 5. CfaA and CfaC together facilitate erythromycin uptake but do not lead to enhanced sensitivity to polymyxin B. Eight strains, (1) P1-pHcfaA, (2) -pHcfaC, (3) -pHcfaAC, (4) -pHC, (5) -pHcfaABC, (6) -pHcfaACE, (7) -pC and (8) -pY, were analyzed for their sensitivity to erythromycin and polymyxin B. (A) The eight strains at 10⁵ CFUs/20 µl were used to inoculate LB agar plate containing 128 µg/ml erythromycin, incubated at 37°C and allowed to grow overnight. Depicted are representative images from one of three experiments. (B and C) Each of the eight strains was inoculated at ~4.5 × 10³ CFUs/ml to liquid LB media containing various concentrations of (B) erythromycin or (C) polymyxin B. Following 37°C overnight incubation, growth or no growth was determined to obtain MICs. Differences in MICs were calculated using Tukey Kramer multiple comparisons test. Values depict the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 depict significant differences of P1-pHcfaAC, -pHC, -pHcfaABC, -pHcfaACE and -pC vs control -pY.

Figure 6. Usher-attenuated P1-pHcfaACE and -pHcfaAC vaccines are protective against wt Salmonella challenge. (A) Four attenuated strains, P1-pHC (n = 10), -pHcfaABC (n = 15), -pHcfaACE (n = 10) and -pHcfaAC (n = 15), were used to orally immunize BALB/c mice, as done in Figure 4C; sPBS-dosed mice served as a control. Six weeks after the second immunization, mice were orally challenged with 5 × 10⁷ CFUs of wt H71. ***p < 0.001 depicts the survival fractions when compared with challenged, PBS-dosed mice. The data depict the mean of 2 to 3 experiments. (B) The same four attenuated strains used in (A) were used to orally immunize C57BL/6 mice, with sPBS-dosed mice as a control. Four weeks post-immunization, mice were orally challenged with 5 × 10⁷ CFUs of wt H71. *p < 0.05 and **p < 0.01 depict the survival fractions from two experiments when compared with challenged, PBS-dosed mice. The Kaplan-Meier method was used to obtain the survival fractions after challenge for both (A) and (B) experiments.