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Direct binding of type III secretion effectors to cytosolic sorting platform complexes in live bacteria indicates an effector shuttling mechanism

ABSTRACT: The bacterial type III secretion system, also called injectisome, translocates effector proteins into eukaryotic target cells through a hollow needle that is anchored in the bacterial membranes. The recruitment of effector proteins to the machinery and the resulting export hierarchy involve the sorting platform, a conserved set of interacting proteins at the cytosolic interface of the injectisome. To gain insight into these processes, we localized and measured the movement of different functional fluorescently labeled sorting platform components in live Yersinia enterocolitica. The sorting platform proteins were part of ~5.1 and ~17.9 injectisomes per bacterium under non-secreting and secreting conditions, respectively. However, they also displayed a considerable mobile cytosolic pool. Using single particle tracking photoactivated localization microscopy, we were able to show the interaction of different components of the sorting platform with effector proteins in the cytosol of live bacteria. We found that effector proteins directly bind to the sorting platform proteins SctQ and SctL. In wild-type bacteria these proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these complexes is modulated in presence of effector proteins and their chaperones, and upon initiation of secretion, which additionally leads to the emergence of large soluble complexes. Our quantitative data supports an effector shuttling mechanism, in which sorting platform protein bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Yersinia Enterocolitica Subsp. Enterocolitica Nbrc 105693

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD044214 | Pride | 2023-10-10

REPOSITORIES: Pride

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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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Direct binding of type III secretion effectors to cytosolic sorting platform complexes in live bacteria indicates an effector shuttling mechanism

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