At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm. EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.

Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones.It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus. The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system. We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC). Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN. We showed by affinity chromatography that EscN and Tir bind CesT specifically. Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT. Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC. These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.Numerous substrates of protein transport systems, ranging from the sec export pathway in bacteria to the mitochondrial import system in eukaryotic cells, interact with components of the transport machinery during transit (37, 51). These types of interactions are anticipated for the type III secretion system (TTSS), which facilitates delivery of many bacterial effectors from the cytoplasm of gram-negative bacterial pathogens into host cells. Many protein-protein interactions have been demonstrated for the type III-related flagellar assembly apparatus, in terms of both components and substrates interacting with the flagellar assembly apparatus (6,19,23,29,42,44,46,54,61).The ATPase FliI in the flagellar assembly apparatus appears to play a pivotal role in interacting with other components of the apparatus and substrates of the system. FliI is required for the export of flagellin and other export substrates (41, 54). The substrates of the flagellar assembly apparatus, such as flagellin and other filament-type substrates, interact with both cytosolic components (ATPase FliI, FliH, and FliJ) and membranebound components (FlhA and FlhB) of the apparatus (44,54,61). FliI is present in the cytoplasm and in association with the inner membrane in Caulobacter crescentus and Salmonella enterica serovar Typhimurium (4, 55). The ATPase activity of S. enterica serovar Typhimurium FliI, localized to the carboxy terminus, is required for flagellar assembly (16,41). FliI also interacts with a number of components of the flagellar export app...

SummaryEnterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria. EHEC and a related pathogen, enteropathogenic E. coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation. An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host. In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC. In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir. The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC. In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC. EHEC forms pedestals with both wild-type EPEC Tir and the nontyrosine-phosphorylatable EPEC Tir Y474F. Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell. These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.

The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by enteropathogenic Escherichia coli (EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC.The delivery of virulence factors directly into host cells to interfere with and alter host processes is a crucial step in bacterial virulence for several significant animal and plant pathogens (38, 41). The type III secretion system (TTSS) facilitates delivery of many bacterial effectors directly from the cytoplasm of gram-negative bacteria into host cells, thereby crossing bacterial inner membrane, peptidoglycan, and outer membrane and the host plasma membrane (reviewed in reference 16). Development of ways to interfere with this fundamental pathogenic mechanism could lead researchers to novel means for combating a variety of important gram-negative pathogens (reviewed in references 12 and 32).The TTSS is present and highly conserved in many diseasecausing gram-negative bacteria (16). TTSSs are also involved in the symbiotic relationship between Rhizobium species and legumes (35) and in the insect endosymbiont Sodalis glossinidius (4). Although not strictly pathogens, symbiotic bacteria establish intimate relationships with their hosts that resemble those that arise during pathogenesis, although the outcome is different. TTSS have not been found in nonpathogenic bacteria or in members of normal microbial flora of humans. While the TTSS has similarities to the flagellar assembly and export apparatus, there are substantial differences, supporting the concept of specifically targeting the type III apparatus for inhibition. A recent paper used a gene probe to detect type III genes as an indicator of virulence, and there was no interference due to genetic similarity between type III systems and the flagellar assembly apparatus (40).Enteropathogenic Escherichia coli (EPEC) is a h...