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Identification of the endogenous insoluble host targets of the Salmonella effectors SSeK3

ABSTRACT: Strains of Salmonella utilise two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effector SseK3 is a arginine glycosyltransferase that modify mammalian proteins with N-acetyl glucosamine (GlcNAc). Here, we used Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry appraoch to identify host proteins GlcNAcylated by endogenous levels of SseK3 during Salmonella infection. Focusing on the insoluble proteome we demostrate multiple typically insoluble proteins are targetted by SseK3 during infections. Surpisingly unlike SseK1 and the EPEC homologue NleB1 which target death domain contain proteins SseK3 appears to modify a number of proteins lacking death domains. These results demostrate that under endogenous infection non-death domain containing proteins can be modified by Salmonella.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Nichollas Scott

LAB HEAD: Elizabeth L Hartland

PROVIDER: PXD015082 | Pride | 2020-07-28

REPOSITORIES: pride

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			Action	DRS
	MQ_search.zip	Other
	Nsco_Arg_GlcNAc_Ssek3_pulldown_20170415_membrane_delta_SseK1_2_3_B1.raw	Raw
	Nsco_Arg_GlcNAc_Ssek3_pulldown_20170415_membrane_delta_SseK1_2_3_B2.raw	Raw
	Nsco_Arg_GlcNAc_Ssek3_pulldown_20170415_membrane_delta_SseK1_2_3_B3.raw	Raw
	Nsco_Arg_GlcNAc_Ssek3_pulldown_20170415_membrane_delta_SseK1_2_B1.raw	Raw

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Project description:Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins directly into infected host cells. A subset of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of human proteins by catalyzing the addition of unusual post-translational modifications. T3SS effectors play essential roles in bacterial virulence and their modes of action have provided great insight into the functions and the components of the innate immune system. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. Arginine glycosylation is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are chaperoned in the bacterium to keep the effectors partially unfolded and competent for secretion, as well as for targeting the effectors to the T3SS sorting platform. The chaperones are then stripped from their effector substrates at the sorting platform and the effectors are secreted in an unfolded conformation. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected into host cells. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the E. coli glutathione synthetase (GshB) is glycosylated on an arginine residue (R256) by NleB. NleB glycosyltransferase activity is essential to C. rodentium survival in oxidative stress conditions because glycosylation of GshB results in enhanced glutathione production. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that effector activities thought to be restricted to host cell compartments additionally play important roles in regulating bacterial physiology.

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Identification of the endogenous insoluble host targets of the Salmonella effectors SSeK3

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