One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilized complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen’s benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants, but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies.

Type III secretion systems (TTSSs) mediate translocation of virulence factors into host cells. We report the 17-angstrom resolution structures of a central component of Salmonella typhimurium TTSS, the needle complex, and its assembly precursor, the bacterial envelope-anchored base. Both the base and the fully assembled needle complex adopted multiple oligomeric states in vivo, and needle assembly was accompanied by recruitment of the protein PrgJ as a structural component of the base. Moreover, conformational changes during needle assembly created scaffolds for anchoring both PrgJ and the needle substructure and may provide the basis for substrate-specificity switching during type III secretion.

Direct electron detectors have made it possible to generate electron density maps at near atomic resolution using cryo-electron microscopy single particle reconstructions. Critical current questions include how best to build models into these maps, how high quality a map is required to generate an accurate model, and how to cross-validate models in a system independent way. We describe a modeling approach that integrates Monte Carlo optimization with local density guided moves, Rosetta all-atom refinement, and real space B-factor fitting, yielding accurate models from experimental maps for three different systems with resolutions 4.5 Å or higher. We characterize model accuracy as a function of data quality, and present a model validation statistic that correlates with model accuracy over the three test systems.

A cryo–electron microscopy structure of a bacterial secretion complex reveals threefold symmetry.

Assembly of multi-component supramolecular machines is fundamental to biology, yet in most cases, assembly pathways and their control are poorly understood. An example is the type III secretion machine, which mediates the transfer of bacterial virulence proteins into host cells. A central component of this nanomachine is the needle complex or injectisome, an organelle associated with the bacterial envelope that is composed of a multi-ring base, an inner rod, and a protruding needle. Assembly of this organelle proceeds in sequential steps that require the reprogramming of the secretion machine. Here we provide evidence that, in Salmonella typhimurium, completion of the assembly of the inner rod determines the size of the needle substructure. Assembly of the inner rod, which is regulated by the InvJ protein, triggers conformational changes on the cytoplasmic side of the injectisome, reprogramming the secretion apparatus to stop secretion of the needle protein.

During kinetochore assembly in budding yeast, the key steps of CENP-A recognition and outer kinetochore recruitment are executed through different yeast CCAN subunits, potentially protecting against inappropriate kinetochore assembly.

Type III protein secretion systems are unique bacterial nanomachines with the capacity to deliver bacterial effector proteins into eukaryotic cells. These systems are critical to the biology of many pathogenic or symbiotic bacteria for insects, plants, animals, and humans. Essential components of these systems are multiprotein envelope-associated organelles known as the needle complex and a group of membrane proteins that compose the so-called export apparatus. Here, we show that components of the export apparatus associate intimately with the needle complex, forming a structure that can be visualized by cryo-electron microscopy. We also show that formation of the needle complex base is initiated at the export apparatus and that, in the absence of export apparatus components, there is a significant reduction in the levels of needle complex base assembly. Our results show a substantial coordination in the assembly of the two central elements of type III secretion machines.bacterial pathogenesis | cryo-electron microscopy | membrane proteins | organelle assembly | protein secretion

The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis.