|Abstract:||Salmonella induces inflammatory diarrhea and epithelial invasion using a Type Three Secretion System (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The SPI1 T3SS directly injects several effector proteins into host cytosols, resulting in host actin rearrangement and engulfment of the bacteria. HilA activates transcription of the SPI1 structural components and effector proteins. Three AraC-like regulators, HilD, HilC, and RtsA, form a feed-forward regulatory loop that activates transcription of hilA. Many environmental signals and regulatory systems are integrated into this circuit to precisely regulate SPI1 expression. Our previous genetic analyses suggest that many of these upstream regulatory inputs are fed into hilD or hilA at the level of translation, but the exact mechanisms are unknown. Through bioinformatic and genetic analyses, I identified a number of sRNAs that feed into hilD or hilA translation to contribute to SPI1 expression. Among them, I demonstrate that two oxygen-dependent sRNAs, FnrS and ArcZ, repress hilD translation. Genetically, the sRNAs base pair with hilD mRNA to regulate translational inhibition, rather than to destabilize the mRNA. I suggest that the two oxygen-dependent sRNAs act to define an ‘oxygen window’ for optimal activation of SPI1 in the intestine. In vivo, deletion of the sRNAs showed altered invasion capacity in both SPI1-dependent and -independent manners. In a second study, I show that the sRNA, PinT, regulated by the PhoPQ two-component system, regulates both hilA and rtsA translation. PinT basepairs with hilA mRNA to repress its translation. PinT also directly interacts with 5’ UTR of rtsA transcript to cause both translational inhibition and degradation of the rts transcript. PinT regulates flhD expression by repressing crp expression, resulting in downregulation of HilD protein activity through FliZ. In addition to the PhoP-mediated transcriptional repression of hilA expression, PinT acts to efficiently repress hilA expression at the posttranscriptional level through these multiple pathways. This PinT-mediated regulation of SPI1 expression is important for shutting the system off when it is no longer required, such as in the intra-phagosomal environment. I observed fitness advantage conferred by deletion of pinT during systemic infection in mice, presumably due to the previously characterized regulatory effects on the SPI2 T3SS, induced when Salmonella is replicating in macrophages. I propose that the virulence function of PinT is to control the transition of virulence gene expression from invasion to systemic stages of infection by controlling expression of SPI1, SPI2 and flagellar genes. In a third study, I suggest that the sRNA InvR acts as a feedback regulator of hilA translation. HilD activates invR transcription, and InvR directly binds to the hilA 5’ UTR for translational inhibition of hilA. The InvR binding region on the 5’ UTR of hilA is far upstream from the ribosome binding site (RBS), and we assume that InvR binding at the 5’ UTR of hilA induces structural changes in the hilA UTR to form an inhibitory structure that leads to translational repression. I finally define several regulatory functions at the hilD 3’UTR. A sRNA SdsR increases expression of hilD expression via the 3’UTR. RNase E and ProQ also affect the hilD expression via the 3’ UTR. Deletion of the 3’ UTR increases the hilD expression, causing overactivation of the SPI1. I suggest that the hilD 3’ UTR functions as destabilizing element of the hilD mRNA. Although I identified several additional regulatory sRNAs that affect SPI1 expression, some of them still are not fully characterized, and the exact regulatory role of the hilD 3’ UTR is not fully understood. However, this work provides a key insight into SPI1 T3SS regulation at the post-transcriptional level.