Biogenesis and structure of a type VI secretion membrane core complex.

Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine-glycine repeat protein G (VgrG) spike and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)-TssJ, TssM and TssL-and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.

Structure of a type IV secretion system.

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.

Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA.

Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown. In this study, we provide experimental evidence that ComFA binds to single stranded DNA (ssDNA) and has ssDNA-dependent ATPase activity. We show that both ComFA and ComFC are essential for the transformation process in pneumococci. Moreover, we show that these proteins interact with each other and with other proteins involved in homologous recombination, such as DprA, thus placing the ComFA-ComFC duo at the interface between DNA uptake and DNA recombination during transformation.

A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae.

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor.

Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.

Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2.

There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway.

Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse; involvement of SNARE complexes.

The mechanism by which T cell antigen receptors (TCR) accumulate at the immunological synapse has not been fully elucidated. Since TCRs are continuously internalized and recycled back to the cell surface, we investigated the role of polarized recycling in TCR targeting to the immunological synapse. We show here that the recycling endosomal compartment of T cells encountering activatory antigen-presenting cells (APCs) polarizes towards the T cell-APC contact site. Moreover, TCRs in transit through recycling endosomes are targeted to the immunological synapse. Inhibition of T cell polarity, constitutive TCR endocytosis, or recycling reduces TCR accumulation at the immunological synapse. Conversely, increasing the amount of TCRs in recycling endosomes before synapse formation enhanced their accumulation. Finally, we show that exocytic t-SNAREs from T cells cluster at the APC contact site and that tetanus toxin inhibits TCR accumulation at the immunological synapse, indicating that vesicle fusion mediated by SNARE complexes is involved in TCR targeting to the immunological synapse.