Type III secretion systems (T3SSs) are nanomachines that are dedicated to protein export in Gram-negative bacteria. T3SSs share the same morphology and overall structure and can be functionally classi?ed into two evolutionary-related classes: the ?agellar T3SS, which promotes bacterial locomotion and motility enabled by the ?agellum, and the pathogenic T3SS, which uses the injectisome to transport virulence proteins into human or animal host cells. Over the past decade signi?cant progress has been made in understanding the structure, assembly and the mode of operation of T3SS. The principal structural building proteins of the ?agellum and the injectisome, from the basal body embedded in the inner and outer bacterial membrane to the tip of the ?laments protruding from the cell surface, and the cytosolic components have been extensively characterized. Flagellar proteins and virulence factors (effectors, needle proteins and translocators) form tight complexes with T3S-dedicated chaperones in the cytosol and are subsequently targeted speci?cally to the export apparatus located at the membrane. Powered by ATP and the proton motive force, the ?agellar proteins and bacterial effectors are then translocated through the channel. Fundamental questions about the functional mechanisms underpinning these processes remain unaddressed. We propose to use an integrated approach combining structural, dynamic, thermodynamic, kinetic, biochemical and in vitro and in vivo functional assays to provide insight into the early events of the translocation process that involve the recognition mechanisms by chaperones, targeting mechanisms to the ATPase and the sorting platform, selection mechanisms that control the hierarchical transport of the ?lament-forming proteins and the effectors and ultimately the assembly of the cytosolic part of the machinery. We have extensively characterized over the last years T3S protein components from the enteropathogenic Escherichia coli (EPEC), the major cause of infantile diarrhea and child mortality worldwide, as well as from Salmonella sp. commonly associated with food poising. We present here novel ?ndings supporting very intriguing hypotheses about the mechanisms used by T3SSs to carry out their function. The speci?c aims are designed to provide atomic-resolution insight into (i) the mechanisms of speci?c interactions between and among key T3S proteins, (ii) the mechanistic basis for targeting of T3S proteins to the export gate, (iii) the ?recognition? and ?secretion? signal, and (iv) the assembly and operation mechanisms of the export gate and ultimately of the entire T3S machinery.

Public Health Relevance

Type III secretion system (T3SS) is encoded by many bacterial pathogens, including Shigella, Salmonella, Bordetella, Pseudomonas, and pathogenic E. coli that are pathogenic for humans and cause diseases such as plague, typhoid fever, bacterial dysentery and common food poisoning.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Macromolecular Structure and Function C Study Section (MSFC)
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Baqar, Shahida
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St. Jude Children's Research Hospital
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Xing, Qiong; Shi, Ke; Portaliou, Athina et al. (2018) Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system. Nat Commun 9:1773
Portaliou, Athina G; Tsolis, Konstantinos C; Loos, Maria S et al. (2017) Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli. EMBO J 36:3517-3531
Xia, Youlin; Rossi, Paolo; Tonelli, Marco et al. (2017) Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments. J Biomol NMR 69:45-52
Monneau, Yoan R; Rossi, Paolo; Bhaumik, Anusarka et al. (2017) Automatic methyl assignment in large proteins by the MAGIC algorithm. J Biomol NMR 69:215-227
Xia, Youlin; Rossi, Paolo; Subrahmanian, Manu V et al. (2017) Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated ? pulses. J Biomol NMR 69:237-243
Monneau, Yoan R; Ishida, Yojiro; Rossi, Paolo et al. (2016) Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications. J Biomol NMR 65:99-108
Khanra, Nandish; Rossi, Paolo; Economou, Anastassios et al. (2016) Recognition and targeting mechanisms by chaperones in flagellum assembly and operation. Proc Natl Acad Sci U S A 113:9798-803
Rossi, Paolo; Xia, Youlin; Khanra, Nandish et al. (2016) 15N and 13C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [1H,13C]-labeled proteins. J Biomol NMR 66:259-271
Chen, Li; Ai, Xuanjun; Portaliou, Athina G et al. (2013) Substrate-activated conformational switch on chaperones encodes a targeting signal in type III secretion. Cell Rep 3:709-15
Zuiderweg, Erik R P; Bagai, Ireena; Rossi, Paolo et al. (2013) EZ-ASSIGN, a program for exhaustive NMR chemical shift assignments of large proteins from complete or incomplete triple-resonance data. J Biomol NMR 57:179-91

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