The type III secretion system (TTSS) of Gram-negative bacterial pathogens delivers effector proteins required for virulence directly into the cytosol of host cells. Delivery of many effectors depends on association with specific cognate chaperones in the bacterial cytosol. The mechanism of chaperone action is not understood. Here we present biochemical and crystallographic results on the Yersinia SycE-YopE chaperone-effector complex that contradict previous models of chaperone function and demonstrate that chaperone action is isolated to only a small portion of the effector. This, together with evidence for stereochemical conservation between chaperone-effector complexes, which are otherwise unrelated in sequence, indicates that these complexes function as general, three-dimensional TTSS secretion signals and may endow a temporal order to secretion.

A number of clinical isolates of Pseudomonas aeruginosa are cytotoxic to mammalian cells due to the action of the 74-kDa protein ExoU, which is secreted into host cells by the type III secretion system and whose function is unknown. Here we report that the swift and profound cytotoxicity induced by purified ExoU or by an ExoUexpressing strain of P. aeruginosa is blocked by various inhibitors of cytosolic (cPLA 2 ) and Ca 2؉ -independent (iPLA 2 ) phospholipase A 2 enzymes. In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A 2 enzymes or by a number of inhibitors of signal transduction pathways. This suggests that phospholipase A 2 inhibitors may represent a novel mode of treatment for acute P. aeruginosa infections. We find that 300 -600 molecules of ExoU/cell are required to achieve half-maximal cell killing and that ExoU localizes to the host cell plasma membrane in punctate fashion. We also show that ExoU interacts in vitro with an inhibitor of cPLA 2 and iPLA 2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA 2 and iPLA 2 enzymes. Mutation of either the serine or the aspartate renders ExoU non-cytotoxic. Although no phospholipase or esterase activity is detected in vitro, significant phospholipase activity is detected in vivo, suggesting that ExoU requires one or more host cell factors for activation as a membrane-lytic and cytotoxic phospholipase.

The helical regions of RNA are generally very stable, but the single-stranded and loop regions often exist as an ensemble of conformations in solution. The theophylline-binding RNA aptamer forms a very stable structure when bound to the bronchodilator theophylline, but the theophylline binding site is not stably formed in the absence of ligand. The kinetics for theophylline binding were measured here by stopped-flow fluorescence spectroscopy to probe the mechanism for theophylline binding in this RNA aptamer. The kinetic studies showed that formation of the RNA-theophylline complex is over 1000 times slower than a diffusion-controlled rate, and the high affinity of the RNA-theophylline complex arises primarily from a slow dissociation rate for the complex. A theophylline-independent rate was observed for formation of the theophylline-RNA complex at high theophylline concentration, indicating that a conformational change in the RNA is the rate-limiting step in complex formation under these conditions. The RNA-theophylline complex requires divalent metal ions, such as Mg2+, to form a high-affinity complex, and there is a greater than 10000-fold reduction in affinity for theophylline in the absence of Mg2+. This decrease in binding affinity in the absence of Mg2+ results primarily from an increased dissociation rate for the complex. The implications of an ensemble of conformations in the free state of this theophylline-binding RNA are discussed and compared with mechanisms for formation of protein-ligand complexes.