Gram-negative pathogens such Yersinia, Shigella, Pseudomonas, enteropathogenic/enterohemorrhagic E. coli (EPEC/EHEC) and Salmonella, as well as Erwinia, Ralstonia and Xanthomona, are causative agents for many diseases in animals, humans, and plants. They range from mild to deadly outcomes, and include food-borne diseases such as diarrhoea or bubonic plaque, or induced cell necrosis in plants. A central aspect of pathogenicity are bacterial toxins (‘effectors’) which are delivered via the type-III secretion system, a large membrane-embedded machinery, from the bacterium to its host-cell. As a consequence, translocated effector proteins have the remarkable capacity to modulate various host-cell pathways, including endocytic trafficking, gene expression, programmed cell death, or cytoskeleton dynamics that induce membrane ruffling and subsequently render the host accessible to bacterial infection.
Unfolded protein transport across membranes?
The hallmark function of all type-III secretion is the safe and directional transport of effector proteins across membranes. Our recent structural analysis (Schraidt and Marlovits, Science 2011) of the injectisome, the most prominent and cylindrical structure of the type-III secretion system, revealed a potential secretion path through the central part of the membrane-embedded complex. However, the inner diameter of this path is too small to accommodate a fully folded effector protein, suggesting that either the injectisome must undergo large conformational changes during transport, or effector proteins need to be unfolded.
Thus, during the last year we focused on the following: a) determining the secretion path of injectisomes, b) understanding the mechanism of transport, and c) visualizing protein transport in situ.
To address these questions, we first analysed the requirements for substrate association with, transport through, and exit from the injectisome. To our surprise we found that the size and length of novel substrates do not have a major impact on their secretability. We learned that the fusion of thermodynamically stable protein domains to otherwise secreted substrates does not influence successful engagement to the injectisome, but prevents complete transport across membranes. Such designed and trapped substrates are highly associated with injectisomes.
We discovered that such substrates are inserted into the secretion path in a polar fashion –N-terminal regions first – suggesting that other substrates with a similar domain organization follow the same principle. Our structural analysis of trapped substrates clearly revealed for the first time that they are in an unfolded state during transport, suggesting that the type-III-specific ATPase acts as an unfoldase. In contrast, injectisomes stay largely invariant during protein transport. To understand whether such behaviour is in fact observed in situ, we performed cryo-electron tomography.
This method permits the investigation of molecular structures within cells in a spatiotemporal manner and in a near-native state. For the first time we were able to visualize pathogenic type-III secretion systems from Salmonella in action and – more generally – protein transport across several membranes.
By understanding the molecular mechanism of TTSS-mediated protein transport, we hope to provide a basis for the development of novel therapeutic strategies that will either inhibit its activity or modify the system for targeted drug delivery.
- Beckham, KS., Ciccarelli, L., Bunduc, CM., Mertens, HD., Ummels, R., Lugmayr, W., Mayr, J., Rettel, M., Savitski, MM., Svergun, DI., Bitter, W., Wilmanns, M., Marlovits, TC., Parret, AH., Houben, EN. (2017) Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nat Microbiol. 2:17047
- Song, M., Sukovich, DJ., Ciccarelli, L., Mayr, J., Fernandez-Rodriguez, J., Mirsky, EA., Tucker, AC., Gordon, DB., Marlovits, TC., Voigt, CA. (2017) Control of type III protein secretion using a minimal genetic system. Nat Commun. 8:14737
- DiMaio, F., Song, Y., Li, X., Brunner, MJ., Xu, C., Conticello, V., Egelman, E., Marlovits, TC., Cheng, Y., Baker, D. (2015). Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat Methods. 12(4):361-5
- Radics, J., Königsmaier, L., Marlovits, TC. (2014). Structure of a pathogenic type 3 secretion system in action. Nat Struct Mol Biol. 21(1):82-7
- Schraidt, O., Marlovits, TC. (2011). Three-dimensional model of Salmonella's needle complex at subnanometer resolution. Science. 331(6021):1192-5