Membrane-associated processes are a fundamental characteristic of all living cells. They ensure that the cells are able to effectively communicate with, and adapt to, their environment. The cells achieve this by either physically translocating molecules to the opposite site of a membrane or by receiving, transmitting, and amplifying incoming signals. Our laboratory is interested in understanding the molecular mechanism underlying such processes. Specifically, we focus on machineries capable of translocating bacterial toxins into eukaryotic cells.
Gram-negative pathogens such as Yersinia, Shigella, Pseudomonas, enteropathogenic/enterohemorrhagic E. coli (EPEC/EHEC) and Salmonella in animals and Erwinia, Ralstonia and Xanthomonas in plants employ type III secretion systems (T3S systems) for the infection process and are required for persistence inside the host. Human diseases in which type III secretion (T3S) is involved range from mild, such as diarrhea, to deadly, such as bubonic plague.
T3S systems are multi-component macromolecular machineries that are usually encoded on specific pathogenicity islands (Figure 1). Their function is to inject proteinacous toxins, referred to as "effectors", into the host cell upon intimate contact. 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 make the host accessible to bacterial infection.
The T3S system in Gram-negative bacteria has evolved to a complex molecular machine that achieves protein translocation across three membranes - the inner and outer membrane of the bacterial cell and the plasma membrane of the eukaryotic host cell. It consists of many components, its most prominent one being the needle complex, a large hetero-oligomeric membrane protein complex with a molecular weight of about 3.5 megadalton (Figure 2). The name stems from the needle-like protrusion visible in electron micrographs of whole bacterial cells. These protrusions are protein filaments that engage with the host cell and are believed to serve as a conduit for the secretion substrate. The needle filament is linked to the membrane-embedded basal body, which in Gram-negative bacteria spans the inner and outer membrane (about 30x30nm) including the periplasmic space. The basal body has a cylindrical shape, defining a central space within which the inner rod and the socket/cup are localized. The inner rod presumably connects the socket/cup with the needle filament and may help to stably anchor the filament into the basal body.
Recently, our lab was the first to provide an experimentally validated map of the topology of the proteins within the complex (Schraidt et al 2010). We subsequently determined the structure of this large organelle to sub-nanometer resolution by cryo EM and single particle analysis (Schraidt & Marlovits, 2011). The structure serves as a model to further understand the structural determinants required for protein translocation across several membranes and thus bacterial infection, and may also be used to design small molecules that interfere with the assembly pathway.
The needle complex from Salmonella (SPI-1) is composed of multiple copies of approximately ten proteins (PrgH/K/I/J, InvG, SpaP/Q/R/S, InvA). A system at this level of complexity requires defined and controlled steps during the assembly. It is initially dependent on the cellular sec-machinery, in particular during the early ring-forming events of assembly. The export apparatus, a group of essential and conserved inner membrane proteins in T3S systems, plays a critical role during the initial phase of the NC assembly. It generates sub-complexes that may serve as nucleation points for the subsequent concentric ring organization of the two inner membrane rings (Wagner et al., 2010) (Figure 3) and is thus localized in the center of assembled needle complexes.
In the past year, we have set out to understand the role of the individual members of the export apparatus using structural and biochemical approaches. We have learned that the very early steps of needle complex assembly require only three export apparatus proteins, all of which are essential to arrive at functional complexes. Due to its central position within complexes, we speculate that the export apparatus proteins may also play a role during protein transport. Thus, in the future we will address how substrates engage with the needle complex. 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.