Functional Characterization of Novel Salmonella Effectors and Small RNAs

Salmonella Typhimurium is a pathogenic, Gram-negative bacterium that is a leading cause of food borne illness, and a major public health problem worldwide. The Salmonella genome has two type III secretion systems (T3SSs) encoded within Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are responsible for the delivery of bacterial effectors into host cells. These effectors play an essential role in bacterial infection, as they function to manipulate host signal transduction pathways. In addition to effector proteins, it was reported that small RNAs (sRNAs) are involved in the regulation of Salmonella infection. Many effectors have been identified and elucidated to date, but mechanisms responsible for Salmonella invasion and replication are not yet fully understood. Hundreds of Salmonella proteins have their function annotated as hypothetical , as they have never been experimentally characterized. Moreover, hundreds of sRNAs have been identified in Salmonella, but only a fraction of published sRNAs are functionally characterized. This study focuses on the identification and functional characterization of novel Salmonella effectors and Salmonella sRNAs. An in silico screen was performed to identify putative T3SS effector candidate proteins. Using the β-lactamase protein translocation reporter assay, nine novel effectors were found to translocate into host cells during Salmonella infection. Nonpolar deletions of these novel effectors were constructed to test their role in Salmonella infection. Among these newly identified effectors, STM4491 exhibits serine protease activity that is essential for mediating down-regulation in Salmonella invasion. Another effector, STM1239, was found to be essential for Salmonella replication in macrophages. Further study demonstrates that STM1239 effector is required to maintain the integrity of Salmonella-containing vacuole (SCV) membrane. In addition to the effectors, this work also surveyed the function of recently identified Salmonella sRNAs. The sRNA InvS was found to be required for efficient Salmonella invasion into non-phagocytic cells. Proteomic analysis was performed and the result showed that InvS null mutants have decreased effector secretion. InvS activates the expression of prgH, a type III secretion apparatus protein, and down regulates the expression of fimZ, a negative regulator of Salmonella invasion. This study identified and characterized molecular factors involved in Salmonella infection, and paves way to further knowledge in the molecular/biochemical mechanism of Salmonella pathogenesis