Recent structural advances towards understanding of the bacterial type III secretion injectisome.

The bacterial injectisome is a structurally conserved, syringe-shaped nanomachine that spans the Gram-negative envelope and forms a continuous channel for type III secretion of protein effectors. The injectisome, and the host-modulating effectors it secretes, are essential for the pathogenesis of several Gram-negative bacterial species, and it is a key virulence factor associated with the progression of many clinical and community-based infectious diseases. The molecular structure of the injectisome has been the focus of intense research efforts over the past 30 years, and during this time significant progress has been made in determining the molecular structures of many components. In this review we present major advances in our structural and mechanistic understanding of the injectisome, as facilitated by cryoelectron microscopy approaches.

Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins.

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin.

The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex from the prototypical Salmonella Typhimurium SPI-1 type III secretion system, with local masking protocols allowing for model building and refinement of the major membrane spanning components of the needle complex base in addition to an isolated needle filament. This work provides significant insight into injectisome structure and assembly and importantly captures the molecular basis for substrate induced gating in the giant outer membrane secretin portal family.

Structural and biochemical characterization of SpoIIIAF, a component of a sporulation-essential channel in Bacillus subtilis.

Environmental stress factors initiate the developmental process of sporulation in some Gram-positive bacteria including Bacillus subtilis. Upon sporulation initiation the bacterial cell undergoes a series of morphological transformations that result in the creation of a single dormant spore. Early in sporulation, an asymmetric cell division produces a larger mother cell and smaller forespore. Next, the mother cell septal membrane engulfs the forespore, and an essential channel, the so-called feeding-tube apparatus, is formed. This assembled channel is thought to form a transenvelope secretion complex that crosses both mother cell and forespore membranes. At least nine proteins are essential for channel formation including SpoIIQ under forespore control and the eight SpoIIIA proteins (SpoIIIAA-AH) under mother cell control. Several of these proteins share similarity with components of Gram-negative bacterial secretion systems and the flagellum. Here we report the X-ray crystallographic structure of the soluble domain of SpoIIIAF to 2.7 Šresolution. Like the channel components SpoIIIAG and SpoIIIAH, SpoIIIAF adopts a conserved ring-building motif (RBM) fold found in proteins from numerous dual membrane secretion systems of distinct function. The SpoIIIAF RBM fold contains two unique features: an extended N-terminal helix, associated with multimerization, and an insertion at a loop region that can adopt two distinct conformations. The ability of the same primary sequence to adopt different secondary structure conformations is associated with protein regulation, suggesting a dual structural and regulatory role for the SpoIIIAF RBM. We further analyzed potential interaction interfaces by structure-guided mutagenesis in vivo. Collectively, our data provide new insight into the possible roles of SpoIIIAF within the secretion-like apparatus during sporulation.

Structural characterization of SpoIIIAB sporulation-essential protein in Bacillus subtilis.

Endospore formation in the Gram-positive bacterium Bacillus subtilis initiates in response to nutrient depletion and involves a series of morphological changes that result in the creation of a dormant spore. Early in this developmental process, the cell undergoes an asymmetric cell division that produces the larger mother cell and smaller forespore, the latter destined to become the mature spore. The mother cell septal membrane then engulfs the forespore, at which time an essential channel, the so-called feeding-tube apparatus, is thought to cross both membranes to create a direct conduit between the cells. At least nine proteins are required to form this channel including SpoIIQ under forespore control and SpoIIIAA-AH under the mother cell control. Several of these proteins share similarity to components of Type-II, -III and -IV secretion systems as well as the flagellum from Gram-negative bacteria. Here we report the X-ray crystallographic structure of the cytosolic domain of SpoIIIAB to 2.3 Šresolution. This domain adopts a conserved, secretion-system related fold of a six membered anti-parallel helical bundle with a positively charged membrane-interaction face at one end and a small groove at the other end that may serve as a binding site for partner proteins in the assembled apparatus. We analyzed and identified potential interaction interfaces by structure-guided mutagenesis in vivo. Furthermore, we were able to identify a remarkable structural homology to the C-subunit of a bacterial V-ATPase. Collectively, our data provides new insight into the possible roles of SpoIIIAB protein within the secretion-like apparatus essential to bacterial sporulation.

Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body.

The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.