bioRxiv2026
Human-restricted typhoidal Salmonella enterica serovars cause systemic infections known as enteric fever, whereas most non-typhoidal serovars cause self-limiting gastroenteritis. Both groups rely on flagella-mediated motility during intestinal colonization and invasion, yet their intracellular flagellar programs appear to diverge after host cell entry. Non-typhoidal Salmonella are thought to rapidly silence flagellar expression intracellularly, whereas human-restricted S. Paratyphi A maintains flagellar gene expression and uses motility to evade xenophagy. However, the fate and function of flagella assembled before invasion have remained unclear. Here, we used fluorescence microscopy to follow preassembled flagella during epithelial cell invasion across typhoidal and non-typhoidal Salmonella. We show that intracellular bacteria remain flagellated during invasion across epithelial cell lines, serovars, and invasion conditions, indicating that Salmonella do not universally shed or lose preassembled flagella upon host cell entry. We demonstrate that internalized flagella are disassembled within Salmonella-containing vacuoles, whereas cytosolic flagella are targeted by autophagy. We further show that S. Paratyphi A uses flagella assembled before invasion to power intracellular motility and promote escape from the Salmonella-containing vacuole, and that intracellular motility is a phenotype shared by typhoidal Salmonella. Together, these findings reveal that human-restricted Salmonella can exploit preassembled flagella inside host cells as part of an intracellular pathogenic strategy. We propose that intracellular motility represents an adaptation to the human epithelial environment that may promote vacuolar escape and host restriction. ImportanceFlagella are best known for driving bacterial motility before host cell invasion, but their fate and function after intracellular entry remain poorly understood. This study shows that Salmonella do not universally lose their flagella during epithelial cells invasion. Instead, both typhoidal and non-typhoidal Salmonella serovars enter host cells with intact preassembled flagella, but only human-restricted typhoidal serovars use them for intracellular motility. This distinction separates flagellar presence from flagellar function and identifies intracellular motility as a serovar-specific behavior associated with human-restricted systemic disease. The findings suggest that typhoidal Salmonella have adapted to the human epithelial intracellular environment by maintaining a motile state that may promote escape from the Salmonella-containing vacuole and favor cytosolic lifestyle. More broadly, this work reframes flagella not only as extracellular colonization factors but also as intracellular determinants of pathogen behavior, host adaptation, and disease outcome.