T he human-restricted, typhoidal Salmonella Paratyphi A (S. Paratyphi A) and S. Typhi are the major c auses of enteric (typhoid) fever and are endemic in regions with poor sanitation. Despite the recent i ncreased rate of S. Paratyphi A isolation from patients in Asia, its pathogenesis remains largely u nknown. Asymptomatic chronic carriage in the gallbladder is encountered in about 5% of patients a nd is facilitated by efficient immune evasion. In this study, we have shown that bile changes the e xpression of > 5% of genes in S. Paratyphi A, including both bile tolerance and virulence-associated g enes. S. Paratyphi A and S. Typhi may differentially regulate certain metabolic pathways in response t o bile. Furthermore, a clinical S. Paratyphi A isolate appears to exhibit distinct regulatory mechanisms. A s inflammasomes have been shown to play a key role in Salmonella infection, we also investigated t heir role following infection of macrophages with S. Paratyphi A, using S. Typhi and S. Typhimurium a s controls. This work demonstrates that S. Paratyphi A and S. Typhi induce pyroptosis, which is lower t han that triggered by S. Typhimurium. While the pathway activated during S. Typhi infection remains u nclear, S. Paratyphi A-triggered pyroptosis occurs via activation of caspase-1, caspase-4, caspase-8 a nd NLRP3. Both S. Paratyphi A and S. Typhi require their SPI-1 injectisome to enable inflammasome a ctivation. However, while the Vi antigen of S. Typhi is dispensable for limiting pyroptosis, the S.Paratyphi A FepE-mediated synthesis of very long O-antigen chains impairs macrophage cell death
a nd a ΔfepE mutant elicited enhanced inflammasome activation. Very long O-antigen chains can also a ct as an inflammasome dampening mechanism in S. Typhimurium, but reduced fepE expression i ndicates that this strategy is not exploited by this pathogen. Therefore, this work points towards distinct mechanisms of virulence by S. Paratyphi A, highlighting the need for a systematic c haracterisation of its molecular pathogenesis.