Deciphering the role of SsaE in biofilm regulation and T3SS-dependent virulence of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is a major causative agent of foodborne diseases around the world, possessing a versatile arsenal of virulence factors that ensure its survival under different environmental conditions and within various host cells. A central element of its intracellular pathogenicity is the Type III Secretion System (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI-2), where ssa encodes a conserved chaperone-like protein involved in effector protein stabilization. This study aimed to elucidate broader roles of ssaE, particularly in biofilm formation, motility, and epithelial interactions. A deletion mutant (∆ssaE) of S. Typhimurium ATCC 14028 was constructed and complemented using the plasmid pBAD24::ssaE. Growth analysis revealed a delayed entry into the exponential phase, suggesting a role in stress adaptation. This mutant presented dramatically reduced biofilm biomass, cellulose production, and pellicle formation, and altered colony morphology, all of which were partially restored upon complementation. Motility assays at different temperatures indicated the impaired of swimming and swarming. The aggregation assays indicated lower auto-aggregation and co-aggregation with lactic acid bacteria. Adhesion to human epithelial Caco-2 and HEp-2 cells was drastically reduced in the mutant strain as supported by confocal microscopy. Gene expression analysis via qPCR indicated downregulation of virulence and biofilm-associated genes such as ssaB, invA, hilA, csgD, and yaiC. Protein network and docking analyses indicated that SsaE communicates with the regulatory systems controlling both SPI-2 and SPI-1, alongside environmental sensing signaling cascades. These findings reveal that SsaE is a multifunctional regulator bridging secretion system activity with adaptive and virulence-related phenotypes, thereby contributing to the environmental persistence and pathogenic potential of S. Typhimurium.