Type VI Secretion System Immunity Protein Tldi1 Modulates Host Inflammatory Responses and Gut Microbiota Homeostasis in Chickens Infected with Salmonella Enterica Serovar Typhimurium

The type VI secretion system (T6SS) is an important fitness determinant of Salmonella enterica serovar Typhimurium (S. Typhimurium) during host colonization and bacterial competition. Tldi1 has been identified as an immunity protein associated with a T6SS antibacterial toxin-immunity module, but its contribution to avian infection remains unclear. This study investigated whether Tldi1 affects the infection outcome of S. Typhimurium in young chickens. Three-day-old Hy-Line Brown chickens were challenged with the wild-type strain SL1344, a tldi1 deletion mutant, or a complemented strain. Clinical signs, body weight, liver and spleen indices, splenic bacterial loads, tissue pathology, cecal mucin staining, inflammatory cytokine expression, intestinal barrier-related gene expression, and cecal microbiota composition were evaluated. Compared with wild-type infection, the tldi1 deletion mutant caused milder clinical signs, lower liver and spleen indices, reduced splenic bacterial loads, and attenuated histopathological lesions in the liver, spleen, ileum, and cecum. The mutant also induced lower expression of several pro-inflammatory cytokine genes and partially alleviated the downregulation of intestinal barrier-related genes. Complementation of tldi1 largely restored the wild-type infection phenotype. Cecal 16S rRNA sequencing showed that tldi1 deletion was associated with altered microbial community features and reduced inter-individual dispersion; however, overall beta-diversity differences were not statistically significant. These findings indicate that Tldi1 contributes to the in vivo fitness and infectionassociated pathology of S. Typhimurium in chickens. The results provide a basis for further investigation of T6SS toxin-immunity modules as potential targets for controlling Salmonella infection in poultry.