SummaryType III secretion is a widespread method whereby Gram-negative bacteria introduce toxins into eukaryotic cells. These toxins mimic or subvert a normal cellular process by interacting with a specific target, although how toxins reach their site of action is unclear. We set out to investigate the intracellular localization of a type III toxin of Pseudomonas aeruginosa called ExoU, which has phospholipase activity and requires a eukaryotic factor for activity. We found that ExoU is localized to the plasma membrane and undergoes modification within the cell by addition of two ubiquitin molecules at lysine-178. A region of five amino acids at position 679-683 near the C-terminus of the ExoU protein controls both membrane localization and ubiquitinylation. Site-directed mutagenesis identified a tryptophan at position 681 as crucial for these effects. We found that the same region at position 679-683 was also required for cell toxicity produced by ExoU as well as in vitro phospholipase activity. Localization of the phospholipase ExoU to the plasma membrane is thus required for activation and allows efficient utilization of adjacent substrate phospholipids.

Vibrio cholerae, the pathogen that causes cholera, also survives in aqueous reservoirs, probably in the form of biofilms. Quorum sensing negatively regulates V. cholerae biofilm formation through HapR, whose expression is induced at a high cell density. In this study, we show that the concentration of the quorum-sensing signal molecule CAI-1 is higher in biofilms than in planktonic cultures. By measuring hapR expression and activity, we found that the induction of quorum sensing in biofilm-associated cells occurs earlier. We further demonstrate that the timing of hapR expression is crucial for biofilm thickness, biofilm detachment rates, and intestinal colonization efficiency. These results suggest that V. cholerae is able to regulate its biofilm architecture by temporal induction of quorum-sensing systems.Vibrio cholerae is a gram-negative, facultative pathogen that is the causative agent of cholera, a devastating diarrheal disease that affects millions of people in the developing world each year (5). Between epidemics, V. cholerae organisms live in marine, estuarine, and freshwater environments in association with zooplankton, phytoplankton, crustaceans, insects, and plants (3,12). Various studies have suggested that biofilm-mediated attachment to abiotic and biotic surfaces may be important for V. cholerae survival in the environment (28,29,33).Biofilm formation in V. cholerae is a multistep developmental process that is controlled by several regulatory pathways (28). The surface attachment of V. cholerae activates the transcription of the vps (Vibrio polysaccharide synthesis) genes that are responsible for synthesis of the VPS exopolysaccharide, the major component of the biofilm matrix (13,24,33). The regulation of VPS synthesis has been partially elucidated through the work of several groups. Environmental signals, such as monosaccharides, nucleosides, and bile salts, have been identified as activators of vps gene transcription and biofilm formation (10,11,13). VpsT, VpsR, and VieA are additional regulators of biofilm formation that respond to as-yet-unidentified environmental signals (2, 26, 31). In addition, quorum sensing also negatively regulates biofilm formation by repressing the expression of the vps operon (9, 34). Quorum sensing is a signaling process by which single-celled bacteria are able to produce and respond to small diffusible molecules called autoinducers, which accumulate as cell density increases and regulate the expression of a range of genes that control various physiological functions (6,20,27). The quorum-sensing system in V. cholerae has been shown to respond to at least two autoinducer molecules (21, 23, 34): CAI-1 and AI-2. CAI-1, whose structure is yet to be solved, is produced by CqsA and plays a major role in the regulation of biofilm formation. AI-2 is a furanosyl borate diester synthesized by LuxS that is also produced by many other bacteria (30). In contrast to CAI-1, AI-2 is largely dispensable in biofilm regulation (30). The accumulation of these autoinducers modulates the...

Microbial interactions with host cell signaling pathways are key determinants of the host cell response to infection. Many toxins secreted by bacterial type III secretion systems either stimulate or inhibit the host inflammatory response. We investigated the role of type III secreted toxins of the lung pathogen Pseudomonas aeruginosa in the inflammatory response of human respiratory epithelial cells to infection. Using bacteria with specific gene deletions, we found that interleukin-8 production by these cells was almost entirely dependent on bacterial type III secretion of exotoxin U (ExoU), a phospholipase, although other bacterial factors are involved. ExoU activated the c-Jun NH 2 -terminal kinase pathway, stimulating the phosphorylation and activation of mitogen-activated kinase kinase 4, c-Jun NH 2 -terminal kinase, and c-Jun. This in turn increased levels of transcriptionally competent activator protein-1. Although this pathway was dependent on the lipase activity of ExoU, it was independent of cell death. Activation of mitogen-activated kinase signaling by ExoU in this fashion is a novel mechanism by which a bacterial product can initiate a host inflammatory response, and it may result in increased epithelial permeability and bacterial spread.

Pseudomonas aeruginosa secretes a number of toxins by a type III system, and these are important in virulence. One of them, ExoS, is a bifunctional toxin, with a GTPase-activating protein domain, as well as ADP ribosyltransferase (ADPRT) activity. These two domains have numerous potential cellular targets, but the overall mechanism of ExoS action remains unclear. The effects of ExoS in a simple eukaryotic system, the yeast Saccharomyces cerevisiae, using a tetracycline-regulated expression system were studied. This system allowed controlled expression of ExoS in yeast, which was not possible using a galactose-induced system. ExoS was found to be an extremely potent inhibitor of yeast growth, and to be largely dependent on the activity of its ADPRT domain. ExoS produced a dramatic alteration in actin distribution, with the appearance of large aggregates of cortical actin, and thickened disorganized cables, entirely dependent on the ADPRT domain. This phenotype is suggestive of actin stabilization, which was verified by showing that the cortical aggregates of actin induced by ExoS were resistant to treatment with latrunculin A, an agent that prevents actin polymerization. ExoS increased the numbers of mating projections produced following growth arrest with mating pheromone, and prevented subsequent DNA replication, an effect that is again dependent on the ADPRT domain. Following pheromone removal, ExoS produced altered development of the mating projections, which became elongated with a swollen bud-like tip. These results suggest alternative pathways for ExoS action in eukaryotic cells that may result from activation of small GTPases, and this yeast expression system is well suited to explore these pathways.