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Characterizing Salmonella pathogenicity island 2 effector interactions implicated in host endomembrane reorganization

University of British Columbia

The success of Salmonella Typhimurium as a pathogen relies on its ability to invade and survive within intestinal epithelial cells. S. Typhimurium thrives as an intracellular pathogen through the activity of a type III secretion system (T3SS2) whose secreted effectors create a unique intracellular replicative niche for S. Typhimurium through regulation of host immune pathways, host cell cytoskeleton rearrangement, regulating host cell ubiquitination events, and hijacking the host endosomal system. Significant efforts to elucidate T3SS2 effector molecular mechanisms through single effector studies have thus far been unable to fully explain the events leading to establishment of the S. Typhimurium intracellular niche. We undertook a systematic study to delineate the contribution of each effector associated with manipulation of the host endosomal system—namely SifA, SopD2, PipB2, SteA, SseJ, and SseFG by creating a deletion mutant library containing single-effector and multiple effector deletion mutants. It was shown that each of Salmonella-induced filament biogenesis, intracellular localization of the Salmonella-containing vacuole, intramacrophage replication, colonization, and virulence depends on the activities of multiple effectors. We demonstrate the complex interplay between effectors and highlight the necessity to study T3SS2-secreted effectors as groups, rather than as individual effectors A T3SS2 overexpression mutant (T3SS2⁺) was created to increase the abundance of secreted T3SS2 proteins and tagged effectors implicated to manipulate the host endosomal system. To identify novel host targets for these effectors while maintaining natural effector delivery throughout infection, a global mass spectrometry screen was performed using the SILAC (stable isotopic labelling with amino acids in cell culture)-labelling method to label host cells and infected them with T3SS2⁺ mutants. Using this method, the host protein annexin A2 was identified as a target for both SopD2 and PipB2. A shared host target for two effectors may explain the complex and nuanced phenotypes observed during S. Typhimurium infection. Together, the work presented here demonstrates that T3SS2-secreted effectors are dependant on each other’s activities during infection and shows binding of two effectors to a single host protein. This work contributes to our understanding of the intracellular lifestyle of S. Typhimurium and sets a new paradigm for future research.

Text

2022-04-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77858

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/