Deletion of gltA attenuates virulence and confers immune protection against Salmonella Enteritidis

Salmonella enterica serovar Enteritidis is the leading cause of foodborne diseases worldwide. The prevalence of multidrug-resistant strains has significantly increased the challenges of clinical treatment and public health risks. The tricarboxylic acid (TCA) cycle serves as the central hub of bacterial central carbon metabolism, and genes encoding its key enzymes are closely associated with the environmental adaptability and pathogenicity of bacteria. However, the biological function of gltA, the gene encoding citrate synthase—the rate-limiting enzyme initiating the TCA cycle—in S. Enteritidis, as well as its potential for use in attenuated vaccines, has not yet been systematically elucidated. In this study, the λ-Red homologous recombination technique was used to construct a gltA-deleted strain (ΔgltA) and its complement (ΔgltA+gltA) from S. Enteritidis strain C50336. We systematically evaluated the effects of gltA deletion on the basic biological characteristics, antimicrobial susceptibility, and environmental stress tolerance of the strains. The molecular mechanism underlying its regulation of virulence was elucidated by combining transcriptomics and quantitative real-time PCR (qRT-PCR), and the attenuation effect and immunoprotective efficacy of the deletion mutant were assessed using a mouse model. The results showed that gltA deletion did not affect the growth characteristics of the strain in nutrient-rich LB medium or nutrient-poor M9 minimal medium, but significantly reduced its motility and biofilm formation ability, inhibited the synthesis of core biofilm components Curli fimbriae and cellulose, and simultaneously markedly increased the susceptibility of the strain to 15 antimicrobial agents. Furthermore, the ΔgltA mutant exhibited significantly decreased tolerance to various extreme environments, including acidic, alkaline, oxidative stress, high temperature, and high osmolarity. Mouse infection studies revealed more than 10⁶-fold attenuated virulence in Kunming mice via intraperitoneal injection compared with the wild-type strain (wild-type LD₅₀ = 3.16 × 10³ CFU; no mortality was observed in mice challenged with 1 × 10⁹ CFU of the deletion mutant). The bacterial loads in the liver and spleen in vivo were significantly decreased, and the expression levels of 24 key virulence genes were comprehensively downregulated. Immunization and challenge experiments demonstrated that a single oral immunization with the ΔgltA mutant induced strong humoral and cellular immune responses in mice, providing 100% immunoprotection against lethal challenge with wild-type S. Enteritidis. This study demonstrates that gltA is a key functional gene regulating metabolic homeostasis, environmental adaptability, and pathogenicity in S. Enteritidis. The ΔgltA mutant displays displays promising immunoprotective efficacy, providing an ideal molecular target and candidate strain for the development of novel live attenuated vaccines against S. Enteritidis.