Towards capture of dynamic assembly and action of the T3SS at near atomic resolution.

The T3SS is a syringe-shaped nanomachine essential for the progression of many Gram-negative bacterial infections including plague, typhoid fever, and dysentery. It spans both bacterial membranes and that of the host allowing delivery of proteins that modulate cell function to aid bacterial survival. Its structure has been the focus of scrutiny for 20 years; however, limitations in purification and structure determination techniques have restricted understanding to atomic structures of individual components and subcomplexes or lower resolution information of the more complete assembly. The recent cryo-EM resolution revolution has facilitated dramatic advances in our structural understanding of the T3SS with complimentary techniques of single particle cryo-EM and cryo-ET revealing structures of isolated complexes to near-atomic resolutions or the architecture of the entire T3SS in its native cellular environment. Here we present an overview of these advances and discuss how these structures further understanding of the dynamic process of injectisome assembly.

Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.

The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents.

Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria.

The acquisition and evolution of customized and often highly complex secretion systems allows Gram-negative bacteria to efficiently passage large macromolecules across both inner and outer membranes and, in some cases, that of the infected host. Essential to the virulence and ultimate survival of the many pathogenic species that encode them, secretion systems export a wide variety of effector proteins and DNA as well as the downstream extracellular filaments of the secretion apparatus themselves. Although these customized secretion systems differ in their cytosolic and inner membrane components, several commonly rely on the secretin family of giant pores to allow these large substrates to traverse the outer membrane. Recently, several near-atomic resolution cryo-EM secretin structures have unveiled the first insights into the unique structural motifs required for outer membrane localization, assembly, hallmark ultrastable nature, spontaneous membrane insertion, and mechanism of action-including the requisite central gating needed to prevent deleterious passage of periplasmic contents to the extracellular space.

Piecing together the type III injectisome of bacterial pathogens.

The Type III secretion system is a bacterial 'injectisome' which allows Gram-negative bacteria to shuttle virulence proteins directly into the host cells they infect. This macromolecular assembly consists of more than 20 different proteins put together to collectively span three biological membranes. The recent T3SS crystal structures of the major oligomeric inner membrane ring, the helical needle filament, needle tip protein, the associated ATPase, and outer membrane pilotin together with electron microscopy reconstructions have dramatically furthered our understanding of how this protein translocator functions. The crucial details that describe how these proteins assemble into this oligomeric complex will need a hybrid of structural methodologies including EM, crystallography, and NMR to clarify the intra- and inter-molecular interactions between different structural components of the apparatus.