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

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a leading global cause of foodborne illnesses. The emergence of multidrug-resistant strains has exacerbated clinical treatment challenges and public health threats. The tricarboxylic acid (TCA) cycle is central to bacterial carbon metabolism, with key enzyme-encoding genes linked to environmental adaptation and pathogenicity. However, the biological role of ΔgltA, which encodes citrate synthase, the rate-limiting enzyme initiating the TCA cycle, in S. Enteritidis, and its potential as an attenuated vaccine target remain poorly understood.

We constructed a gltA deletion mutant (ΔgltA) and its complemented strain (ΔgltA+gltA) from S. Enteritidis C50336of using λ-Red homologous recombination. We systematically characterized the effects of ΔgltA deletion on bacterial growth, antimicrobial susceptibility, and stress tolerance. Transcriptomics and qRT-PCR were employed to investigate virulence regulation mechanisms, while mouse models were used to evaluate attenuation and immunoprotective efficacy.

ΔgltA deletion did not alter growth in nutrient-rich LB or nutrient-poor M9 media, but significantly impaired motility and biofilm formation by inhibiting curli fimbriae and cellulose synthesis, and increased susceptibility to 15 antimicrobial agents. The ΔgltA mutant also showed reduced tolerance to acidic, alkaline, oxidative, thermal, and osmotic stresses. Notably, intraperitoneal infection revealed >10⁶-fold attenuation in Kunming mice (wild-type LD₅₀ = 3.16 × 10³ CFU; no mortality at 1 × 10⁹ CFU ΔgltA), with decreased organ bacterial loads and global downregulation of 24 key virulence genes. A single oral immunization with ΔgltA elicited robust humoral and cellular immunity, conferring promising protection against lethal wild-type challenge.

Deletion of ΔgltA is associated with altered metabolic homeostasis, impaired environmental adaptability and attenuated pathogenicity in S. Enteritidis. The ΔgltA mutant conferred effective protective immunity in the mouse model used here, serving as a promising candidate strain and molecular target for further development of live attenuated S. Enteritidis vaccines.