HU-Lacking Mutants of Salmonella enterica Enteritidis Are Highly Attenuated and Can Induce Protection in Murine Model of Infection

Abstract

Salmonella enterica infection is a major public health concern worldwide, particularly when associated with other medical conditions. The serovars Typhimurium and Enteritidis are frequently associated with an invasive illness that primarily affects immunocompromised adults and children with HIV, malaria, or malnutrition. These serovars can also cause infections in a variety of animal hosts, and they are the most common isolates in poultry materials. Here, we described S. Enteritidis mutants, where hupA and hupB genes were deleted, and evaluated their potential use as live-attenuated vaccine candidates. In vitro, the mutants behaved like S. Typhimurium described previously, but there were some particularities in macrophage invasion and survival experiments. The virulence and immunogenicity of the mutant lacking both hupA and hupB (PT4ΔhupAB) were evaluated in a BALB/c mice model. This mutant was highly attenuated and could, therefore, be administrated at doses higher than 10⁹ CFU/treatment, which was sufficient to protect all treated mice challenged with the wild-type parental strain with a single dose. Additionally, the PT4ΔhupAB strain induced production of specific IgG and IgA antibodies against Salmonella and TH1-related cytokines (IFN-γ and TNF-α), indicating that this strain can induce systemic and mucosal protection in the murine model. Additional studies are needed to better understand the mechanisms that lead to attenuation of the double-mutant PT4ΔhupAB and to elucidate the immune response induced by immunization using this strain. However, our data allow us to state that hupAB mutants could be potential candidates to be explore as live-attenuated vaccines.

Keywords: HU protein; Salmonella enterica Enteritidis; live-attenuated strains; non-typhoidal Salmonella; nucleoid-associated proteins.

FIGURE 1

In vitro phenotypes of HU-lacking mutants. The growth of all mutant strains and wild-type parental strain SEnPT4 was measured every hour for 12 h by OD₆₀₀ readings (A) or CFU counts (B). The growth curves represent the results of three independent experiments. (C) The motility capacity of the mutants was also evaluated in 0.35% agar plates, measured after 14 h of incubation. (D) J774A.1. macrophages were infected in vitro with HU mutants and SEnPT4 parental strains. Graphs show the number of colony forming units (CFUs) of intracellular bacteria recovered at 2 and 5 h after infection. Values of motility and macrophage assay are represented as mean ± SEM of quintuplicate samples of three independent experiments. ^∗P < 0.01 by Tukey’s test as compared to SEnPT4.

FIGURE 2

Bacterial cell counting in BALB/c mice organs and attenuation assay following oral inoculation with PT4ΔhupAB. (A) Mice were orally inoculated with 1 × 10⁴ or 1 × 10⁶ CFU of SEnPT4 to evaluate the virulence of the parental strain; another group was inoculated with 2 × 10⁹ CFU of PT4ΔhupAB that exhibited attenuation. Peyer’s patches (B), spleen (C), and liver (D) were collected from five animals at 3, 7, or 14 days after inoculation, homogenized in PBS, and plated onto MacConkey’s agar for bacterial counting. Data are representative of two experiments with similar results, and the values are represented by mean ± SD. Some colonies were randomly chosen for PCR confirmation (data not shown).

FIGURE 3

Protection assay, antibody production, and cytokine detection after immunization. BALB/c mice were orally immunized with one (on day 0) or two (on days 0 and 21) doses of 2 × 10⁹ CFU of PT4ΔhupAB or inoculated with PBS (control). (A) Mice were orally challenged with 1 × 10⁷ CFU of the wild-type strain SEnPT4 21 days after the last immunization. The graph shows the percentage of immunized animals that survived the challenge. Fecal and serum samples were obtained on days 0, 14, 31, and 49 after the first immunization to measure specific IgA (B) and total IgG (C) by ELISA, respectively. SEnPT4 antigens were used as the coating antigen (1 μg/mL). Results are expressed as the mean ± SEM of OD 450 nm values of seven mice per group and are a representative experiment of two assays. ^∗P < 0.01 compared to the control group with Bonferroni’s test. Cytokine detection in serum collected from groups of nine BALB/c mice after immunization with one (on day 0) or two (on days 0 and 21) doses of 2 × 10⁹ CFU of PT4ΔhupAB or with PBS (control). Serum samples were obtained on days 0, 14, 31, and 49 after the first immunization, and levels of IFN-γ (D), TNF-α (E) and IL-6 (F) were measured by Cytokine Bead Array. Results are expressed as the mean ± SEM of serum concentration (pg/mL). ^∗∗P < 0.05 compared to the control group with Tukey’s test.