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...

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.