The most prominent substructure of the TTSS is known as the “needle complex”: a cylindrical, needle-shaped and membrane-embedded organelle protruding from the bacterial envelope (Figure 1). The needle complex is believed to serve as a conduit for safe transport of virulence factors from the bacterial cytoplasm through a number of natural barriers into eukaryotic cells.  In Salmonella typhimurium, which serves as our model for bacterial delivery systems, this complex is formed by multiple copies of only five proteins:  PrgH, PrgK, and InvG constitute the membrane-associated base structure, PrgJ the inner rod, and PrgI the needle filament extending into the extracellular environment (Figure 2).

In order to investigate the molecular mechanism of type III secretion, we first set out to determine structural components of the TTSS (Figure 3).  We were challenged by the mega-dalton size of the complex, its natural composition (membrane and soluble proteins), and its limited availability.  Nevertheless, we were able to purify sufficient quantities of the entire ‘needle complex’ and its precursor, the ‘base’, by a combination of detergent extraction and size separation by velocity gradient centrifugation. A detailed structural analysis by three-dimensional electron cryo-microscopy and single-particle analysis finally revealed a new structural component, the inner rod, which is located at the center of the needle complex. It extends the secretion path from the base into the needle filament and also serves as an anchor to stably connect the needle filament into the base. During assembly, the inner rod and the needle filament are added as new structural components to the base (Figure 2, 3). As a consequence, it must undergo large conformational rearrangements which demonstrates the flexible but also stable qualities of the base.  Functionally, this dynamic behavior is a crucial event in the assembly phase during which the secretion machine is reprogrammed to become competent for the secretion of virulence factors. Structurally, it underlines the importance of specific interaction epitopes critical for the assembly into a functional unit.

Recent crystallographic analyses of individual separated domains which are predicted to be periplasmically located, revealed a common structural motif organized in repeating modules. Attempts have been made to “dock” these protein domains into the needle complex structure, which resulted in different and mutually incompatible locations. We used a combination of methods, including bacterial genetics, biochemistry, mass spectrometry and cryo-electron microscopy/single-particle analysis, to experimentally determine which specific protein domains correspond to different substructures of the needle complex. In addition, we identified specific interaction sites among components of the needle complex, which are critical for stable assembly and consequently functional complex. In combination, this analysis provides the first experimentally validated topographic map of different components of the needle complex of the S. Typhimurium TTSS.

Although the design of the TTSS appears to be conceptually simple, several questions remain unanswered:  What nucleates the assembly of the TTSS? How dynamic is the entire assembly process?  And how are substrates recognized and translocated? We have started to address some of these questions. Understanding the molecular mechanism of TTSS-mediated protein transport should provide a basis for the development of novel therapeutic strategies to either inhibit its activity or modify the system for the purpose of achieving targeted drug delivery.