Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach. These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease. D iarrheagenic enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium (CR) are attaching͞effacing (A͞E) bacterial pathogens that attach to host intestinal epithelium and efface brush border microvilli, forming A͞E lesions (1, 2). EHEC and EPEC represent a significant threat to human health. Sequencing the genome of EHEC O157:H7, the causative agent of ''Hamburger disease'' and the most common serotype associated with food and water poisoning, has identified many putative virulence factors (3). These factors are often encoded by pathogenicity islands (PAI) present in the genomes of pathogenic, but not closely related nonpathogenic, strains (4). However, the functions of the PAIs in virulence have not been systematically analyzed.Many key virulence factors shared by A͞E pathogens reside in the locus of enterocyte effacement (LEE), a PAI essential for A͞E lesion formation (5-8). The LEE contains 41 genes and encodes a type III secretion system (TTSS), a common virulence mechanism for many human and plant pathogens (4, 9, 10). TTSSs are conserved organelles that deliver bacterial effector proteins capable of modulating host functions into host cells. The LEE encodes proteins for forming such an organelle (2), but the LEE genes involved in assembling and regulating this apparatus have not been defined.The LEE also encodes a regulator (Ler), an adhesin (intimin) and its receptor (Tir) responsible for intimate attachment, several secreted proteins, and their chaperones (1, 2). The secreted proteins consist of effectors as well as translocators (EspA, EspD, and EspB) required for translocating effectors into host cells. Five LEEencoded effectors (Tir, EspG, EspF, Map, and EspH) have been identified, which are involved in modulating host cytoskeleton (2, 11). However, nearly half of the LEE genes have no homologs and have not been functionally studied.Because EHEC and EPEC are human pathogens, efforts aimed at elucidating the function of the LEE have primarily been restricted to in vitro studies. Animal models, including neonatal...

14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

Summary EnterohaemorrhagicEscherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium , and was absent from nonpathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.