E-MAP Directed Analysis of Effector Protein Function in Salmonella enterica serovar Typhimurium

Six effector proteins of Salmonella enterica serovar Typhimurium - SseC, SseG, SseI, SseK1, SteB, and SopD - were studied in order to determine their molecular contributions to virulence. Data collected from a high-throughput quantitative genetic interaction screen in budding yeast, called an E-MAP, was used to generate a list of GO terms for each effector protein. The E-MAP used in this analysis involved crossing yeast strains expressing the Salmonella effector proteins with single deletion mutant yeast from a deletion library of 4800 non-essential genes. To determine the validity of the proposed GO terms, unbiased experiments were conducted in mammalian cells. Immunoprecipitation was used to determine effector protein interacting partners, and the results were analyzed by Mass Spectrometry. Immunofluorescence Microscopy was used to observe localization patterns. It was found that SseC interacts with the retromer, a protein complex functioning in retrograde protein trafficking that assembles on endosomal membranes. The current hypothesis is that SseC is involved in promotion of complex disassembly from endosomes. SseG was shown to colocalize with the Golgi through Immunofluorescence data, supporting a hypothesis that SseG functions by interfering with host trafficking processes. The results from these unbiased mammalian experiments align with the GO terms generated from the E-MAP, providing support for the E-MAP as an efficient means of uncovering effector protein function in pathogenic bacteria