Gram-negative bacterial pathogens, like Salmonella, benefit from multi-protein appendages, called type III secretion systems (T3SS), serving as a conduit to introduce effector proteins into the host cell and concomitantly manipulate host cellular processes.
Over 50 Salmonella enterica serovar Typhimurium (STm) effectors have been reported to play a role in invading and steering the host cell to establish an intracellular replicative niche. Effectors are multifaceted and may fulfill multiple tasks, i.e. modulating host cell signaling, depending on their localization and timing during infection inside the host. While effector functions are starting to emerge, comprehensive functional characterization of each effector using different approaches is indispensable to illuminate effector interconnectivity and Salmonella virulence strategies. We demonstrate the utility of virus-like particle (VLP) trapping technology Virotrap as an effective approach to catalog STm effector-host protein complexes. Relying on the vesicle-forming properties of the HIV-1 Gag protein, Virotrap captures prey proteins inside VLPs lined with Gag-bait fusion proteins. Using mass spectrometry (MS)-based Virotrap analysis, we validate previously described host targets of STm effectors SspH2, SopD, PipB2 and SopB, along with tens of new candidate host targets for various effectors. Owing to its unique properties, Virotrap is highly adept at identifying membrane(-associated) proteins and thereby provides complementary data to the current state-of-the-art toolkit to study protein complexes. With its proven success, Virotrap will supplement the current and future knowledge on the multilayered host-pathogen interface to decipher bacterial pathogenesis.