Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.

The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-κB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-κB activation. We show that inhibition of NF-κB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-κB activation by preventing activation of IKKβ and consequently the degradation of the NF-κB inhibitor, IκB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-κB signaling pathways.

The mechanisms by which pathogens sense the host and respond by remodeling gene expression are poorly understood. Enteropathogenic (EPEC), the cause of severe intestinal infection, employs a type III secretion system (T3SS) to inject effector proteins into intestinal epithelial cells. These effectors subvert host cell processes to promote bacterial colonization. We show that the T3SS also functions to sense the host cell and to trigger in response posttranscriptional remodeling of gene expression in the bacteria. We further show that upon effector injection, the effector-bound chaperone (CesT), which remains in the EPEC cytoplasm, antagonizes the posttranscriptional regulator CsrA. The CesT-CsrA interaction provokes reprogramming of expression of virulence and metabolic genes. This regulation is likely required for the pathogen's adaptation to life on the epithelium surface.

Highlights d Host-attached EPEC extract nutrients directly from infected host cytoplasm d Host nutrient extraction (HNE) requires CORE proteins of the EPEC injectisome d CORE supports the formation of protruding membranous nanotubes to enable HNE d Distant CORE genes restored HNE capacity in EPEC lacking its native CORE

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.Enterohemorrhagic Escherichia coli (EHEC) is an emerging pathogen causing outbreaks of food-borne gastroenteritis manifested by bloody diarrhea, which may progress to the potentially fatal hemolytic-uremic syndrome. The latter involves severe complications, such as renal impairment, hypertension, and central nervous system manifestations mainly caused by SLT toxins (3,22). EHEC belongs to the family of the attaching and effacing (AE)-inducing pathogens, which includes the closely related species enteropathogenic E. coli (EPEC), Citrobacter rodentium, and rabbit EPEC. When colonizing the gut, these pathogens form AE lesions on the intestinal epithelial cell surface. AE lesions are characterized by localized destruction of the brush border microvilli, intimate bacterial attachment to host cells, and the formation of actin structures, termed pedestals, beneath the attached bacteria (24). This histopathology is dependent upon a type III protein secretion system (TTSS), which functions as a molecular syringe to translocate effector proteins from the bacterial cytoplasm directly into the cytoplasm of host epithelial cells (15). These effectors subvert normal host cell functions and are required for efficient host colonization (15,34,35). One of these effectors, Tir, is inserted into the host cell membrane to form a binding site for an outer membrane adhesin, intimin. Interaction of intimin with translocated Tir promotes tight bacterial attachment to the ho...