Marlovits Group Research

Molecular Machines

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.

Microbial Pathogenesis

Figure 1 (Click to view legend)

Many animal and plant pathogens share the same principles of infecting host cell organisms: they translocate specific bacterial toxins (collectively referred to as “effector proteins”), which originate from the bacterial cytoplasm, directly into the cytoplasm of a eukaryotic host cell. As a result, 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. At the heart of this process is the type-3 secretion system (T3SS), a protein-delivery machine that establishes intimate contact between the microorganism and the host cell, and permits safe and unidirectional passage of specific effectors. These systems are widespread among Gram-negative animal pathogens, including Yersinia, Pseudonomas, Shigella, enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively), or Salmonella, and the plant pathogens Erwinia, Ralstonia or Xanthomonas. They are essential for the onset of a variety of diseases ranging from diarrhea, bubonic plaque, even with fatal outcomes, to fire blight and bacterial wilt. While the task of translocating proteins from one compartment to the other has been basically solved in nature (for example the targeting and/or secretion of proteins through the Sec-system or the Tat-system), the contextual situation is complicated by the fact that the translocation must occur through a number of environments, which includes two bacterial membranes and one eukaryotic membrane, the periplasmic and the extracellular space. Consequently, the nature of a T3SS system is complex in terms of specific mechanistic details as well as the organization of all involved components. Using Salmonella typhiumurium, we are investigating the molecular mechanisms and structural framework required to translocate effector proteins specifically and safely into eukaryotic cells.

Architecture of the needle complex of the T3SS:

Figure 2 (Click to view legend)

The core, and probably the most prominent structure of the T3SS (SPI-1), is the needle complex. It is a ‘syringe’-like multi-component system. Overall, the needle complex is a large (approximately 30x80nm) cylindrical complex. In its native environment it is embedded in the inner as well as outer membranes, spans the periplasmic space, and protrudes into the extracellular environment with a needle filament. Its overall architecture provides a structural framework for a direct connection of bacterial and host cell cytoplasm, and delineates the secretion pathway through the needle complex. Although the needle complex is about 3.5 MDa in size, its overall shape is dictated by only five proteins. Nevertheless, mutually exclusive models of the individual protein organization have been described in the past. These models were rendered complex by a paucity of positional information, incorrect assumptions about the symmetry and stoichiometry of ring-forming base proteins, and consequent difficulties of modeling. Our laboratory 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 will serve as a basis to further understand the structural determinants required to form ring-like structures in membrane-embedded systems, and may also be used to design small molecules that interfere with the assembly pathway.

Assembly of the T3SS:

Figure 3 (Click to view legend)

Our topological analysis revealed that additional proteins must be present. These constitute the cup/socket structure which is located in the center of the needle complex (export apparatus). Using mass spectrometry, we were able to identify five additional candidate proteins that co-fractionate in marginal quantities with purified needle complexes. Subsequent structural analysis revealed the absence of the cup/socket, suggesting that one or more of these proteins are required to build up the cup/socket (Figure 3). We were also able to show that these proteins nucleate the coordinated assembly of the needle complex (Wagner et al., 2010)

Structural Plasticity of the needle filament

Efficient effector protein translocation is known to occur only after host cell contact. Therefore, it is conceivable that the extracellular filament is a key player in the transmission of this information, probably due to small conformational changes throughout the filament. This hypothesis is supported by mutations found in the homologous Shigella needle filament, which convert the system into a constitutively “on” state. If this is true it would be justified to presume that the filament is provided with a certain degree of structural heterogeneity in order to accommodate the required conformational plasticity for signal transmission. We therefore analyzed the structure of the needle filament by cryo electron microscopy (Figure 3) and discovered that the structure is, indeed, highly variable (Galkin et al., 2010).

Although the design of the TTSS appears to be conceptually simple, many questions remain unanswered: How dynamic is the entire assembly process? How are substrates recognized by the needle complex? What is the molecular mechanism of protein translocation? We have started to address some of these questions. 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.