Salmonella enterica serovar Typhimurium lacking hfq gene confers protective immunity against murine typhoid

Abstract

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+) T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

Figure 1. STMΔhfq is highly attenuated in vitro and in vivo (A) Generation of STMΔhfq by one step gene inactivation.

Confirmatory PCR showing the presence of Kanamycin cassette (1.5 Kb) in STMΔhfq. (B) Intracellular replication of STM-WT and STMΔhfq strains in INT-407, HT-29, CaCo-2 and RAW 264.7 cell lines. Cells were infected with the STMΔhfq or STM-WT at an MOI of 10. Data shows fold increase from 2 h to 16 h. All the experiments were done in triplicate. (C&D) Organ loads of STM-WT and STMΔhfq. Two groups of mice (5 each) were infected with 10⁷ CFU/mouse orally and sacrificed on day 4 (C) and day 7 (D) of post infection. Bacterial counts in spleen, MLN and liver were shown as CFU/gm.wt with standard errors. Plots are representative of two independent experiments with similar results. Statistical significance was defined as follows: (*p<0.05; ** p<0.005) (Student's t test & Mann-Whitney U test).

Figure 2. STMΔhfq mutant is highly safe for infection in murine model. (A & B) Survival curves.

Mice were infected orally (A) at a dose of 10⁸ or intraperitoneally (B) with a dose of 10⁴ bacteria per mouse (n = 10) and mice were observed twice daily for survival. (C) Reduced fecal shedding of the STMΔhfq. Mice were infected orally with the STM-WT or STMΔhfq mutant strains at a dose of 10⁷ bacteria/mouse. Uninfected mice were used as control. Fresh fecal pellets were collected at the indicated time points and shedding was determined by plating on culture media. Data is represented as the number of bacteria per gram weight of the fresh fecal pellets. Shown are the combined results from two experiments with 5 mice in each group. Statistical significance was defined as follows: (*p<0.05) (Student's t test & Fischer's exact test).

Figure 3. Multiple immunizations with STMΔhfq confer protection against virulent Salmonella.

Groups of 5 mice were immunized orally with 10³ (A&C), 10⁴ or 10⁵ (B&D) STMΔhfq for two booster doses followed by challenge with WT through oral route (for organ load: 10⁷ and for survival analysis: 10⁸ of WT) 7 days after the final booster dose. Control mice received PBS. 7 days after challenge, mice were sacrificed and bacterial load in different organs was enumerated using direct cultures of serially diluted homogenized samples. A&B depict bacterial load in different organs and C&D shows survival curves. Result presented is one of two independent experiments performed. Statistical significance was defined as follows * p<0.05; ** p<0.005 (Mann-Whitney U test & Fischer's exact test).

Figure 4. Single dose of vaccination imparts protection against WT Salmonella and induces long-term immunological memory.

BALB/c mice were vaccinated through oral route with 10⁸ CFU of STMΔhfq or PBS and challenged with either 10⁷ CFU of WT for assessing organ load (A) or 10⁸ of WT for assessing mortality (B). 7 days after challenge, mice were sacrificed and bacterial load was enumerated. For long-term immunological memory mice were challenged orally after 52 days of vaccination (C). Each group contains 5-6 mice. Statistical significance was defined as follows * p<0.05 (Mann-Whitney U test & Fischer's exact test).

Figure 5. Flow cytometric analysis of CD4⁺ T cell population in the spleen on 4^th day (A) and 7^th day (B) post infection.

Group of mice were inoculated with the STM-WT or STMΔhfq with dose of 10⁷ bacteria per mouse. Uninfected mice were used as control. Splenocytes were isolated on 4^th and 7^th day post infection from both infected and control mice and stained with FITC-conjugated anti-CD4 MAb. The relative levels of CD4⁺ T-lymphocytes were measured through FACS. Data was analyzed with BD Cell-Quest software and represented by dot plot. The results are representative of two independent experiments. Each group consisted of 4-5 mice. Statistical significance was defined as follows: (* p<0.05) (Student's t test).

Figure 6. Analysis of splenic CD4⁺ T cell population in vaccinated and unvaccinated mice with or without challenge.

Group of mice were orally given PBS or 10⁸ STM Δhfq bacteria and then challenged with 10⁷ CFU of the STM-WT per mouse. On 7^th day post vaccination and 7^th day post challenge, single cell suspension of splenocytes were prepared and stained with FITC-conjugated anti-CD4 MAb. The relative levels of CD4⁺ T-lymphocytes were measured through FACS. Data was analysed by BD Cell-Quest software and represented through dot plot (A) and bar graph (B). Statistical significance was defined as follows: (* p<0.05) (Student's t test). Each group consisted of 4-5 mice.

Figure 7. Analysis of serum cytokine levels with single dose of vaccination.

(A) IFN-γ (B) IL-6. Group of mice were orally given PBS or STMΔhfq (10⁸) and then challenged with STM-WT (10⁷ CFU/mouse) at 7^th post vaccination. Serum was collected on 7^th day post vaccination (from unchallenged mice) and 7^th day post challenge (from challenged mice) and cytokine levels were measured using ELISA. Data is representative of two independent experiments (n = 4-5). Statistical significance was defined as follows: (*p<0.05; ** p<0.005) (Student's t test).

Figure 8. Humoral immune response elicited by single dose of vaccination with STMΔhfq.

Group of mice were orally given PBS or STMΔhfq (10⁸) and then challenged with STM-WT (10⁷ CFU/mouse) at day 7 of post vaccination. Serum and intestinal mucus were collected on 7^th day post vaccination (from unchallenged mice) and 7^th day post challenge (from challenged mice). Serum IgG (B&D) and intestinal S-IgA (A&C) antibodies specific for LPS and OMP were measured by ELISA. The samples were assayed in triplicate and the antibody titer is expressed as the absorbance at 450 nm. Data is representative of two independent experiments. Statistical significance was defined as follows: (*p<0.05; ** p<0.005) (Student's t test). (n = 4-5).

Figure 9. Increased antigen presentation of the STMΔhfq by Denditic cells.

Dendritic cells were isolated from the BALB/C mice and were plated at 5×10⁴ cells/well in 96 well flat-bottomed tissue culture plates. C57BL/6 mice were infected with STM-WT for pre stimulation and T-lymphocytes were isolated on 5^th day aseptically. DCs were infected with the STM-WT and STMΔhfq. The infected DCs were then co-cultured with T-lymphocytes at a ratio of (1∶1) and (1∶10) for 72 h and then pulsed for 16 h with [³H] thymidine (1 µCi/well). The incorporation of [³H] thymidine in proliferating T cells were measured by liquid scintillation counter. T cell proliferation was represented counts/min using a beta-counter. The results are representative of two independent experiments in triplcate. Statistical significance was defined as follows: (*p<0.05) (Student's t test).