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Title: Functional analysis of the SseK family of Salmonella SPI-2 type III secretion system effectors
Author: Günster, Regina Agnes
ISNI:       0000 0004 9350 1575
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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In humans, serovars of Salmonella enterica cause diseases ranging from gastroenteritis to systemic typhoid fever. Salmonella encodes numerous translocated virulence (effector) proteins that enable the bacterium’s complex intracellular lifestyle. Three poorly characterised Salmonella effectors are the SseK proteins: SseK1, SseK2 and SseK3. This work showed that all three effectors are translocated by the SPI‐2 type III secretion system into macrophages. Despite high amino acid similarity between the proteins, SseK1 is cytosolic and SseK2 and SseK3 associate with the Golgi network. When ectopically expressed in mammalian cells, SseK1, SseK2 and SseK3 suppressed NF‐κB pathway activation. This inhibition was specific for TNFα‐induced signaling but not TLR4‐, or interleukin‐induced NF‐κB pathway activation. After physiological delivery of the effectors into macrophages during Salmonella infection, SseK1 and SseK3 inhibited NF‐ κB activity in an additive manner and suppressed TNFα‐induced necroptotic host cell death. Despite stable interaction between SseK3 and the E3‐ubiquitin ligase TRIM32 in macrophages, SseK3‐mediated inhibition of NF‐κB activation and host cell death did not require TRIM32. In addition, bacterial burden in Trim32‐/‐ mice were indistinguishable from wild‐type mice after short‐term infection with Salmonella. Catalytic activity of the SseK effectors required a DxD motif, which in SseK1 and SseK3 is essential for arginine‐N‐acetylglucosamine (GlcNAc)‐ ylation of target proteins. In infected macrophages, SseK1 and SseK3 caused arginine‐ GlcNAcylation of a diverse range of distinct bacterial and host proteins, including auto‐ GlcNAcylation of SseK1 and SseK3. Both SseK1 and SseK3 induced arginine‐GlcNAcylation of TRADD, which is most likely the cause of the effector’s inhibitory effect on both NF‐κB activation and host cell death. In addition, a large number of Golgi network associated proteins were identified as potential SseK3 GlcNAcylation targets. This suggests additional, yet‐to‐be validated functions of the effectors during Salmonella infection besides inhibition of NF‐κB signalling and necroptosis in macrophages.
Supervisor: Holden, David W. ; Wigneshweraraj, Sivaramesh Sponsor: Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral