Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway

Immigration Federalism: A Reappraisal

This Article identifies how the current spate of state and local regulation is changing the way elected officials, scholars, courts, and the public think about the constitutional dimensions of immigration law and governmental responsibility for immigration enforcement. Reinvigorating the theoretical possibilities left open by the Supreme Court in its 1875 Chy Lung v. Freeman decision, state and local offi- cials characterize their laws as unavoidable responses to the policy problems they face when they are squeezed between the challenges of unauthorized migration and the federal government’s failure to fix a broken system. In the October 2012 term, in Arizona v. United States, the Court addressed, but did not settle, the difficult empirical, theoretical, and constitutional questions necessitated by these enactments and their attendant justifications. Our empirical investigation, however, discovered that most state and local immigration laws are not organic policy responses to pressing demographic challenges. Instead, such laws are the product of a more nuanced and politicized process in which demographic concerns are neither neces- sary nor sufficient factors and in which federal inactivity and subfederal activity are related phenomena, fomented by the same actors. This Article focuses on the con- stitutional and theoretical implications of these processes: It presents an evidence- based theory of state and local policy proliferation; it cautions legal scholars to rethink functionalist accounts for the rise of such laws; and it advises courts to reassess their use of traditional federalism frameworks to evaluate these sub federal enactments

Ability of enteroaggregative Escherichia coli strains to adhere in vitro to human intestinal mucosa

A collection of 44 enteroaggregative Escherichia coli (EAggEC) strains isolated from infants with diarrhea in India and the United Kingdom were examined for their ability to adhere in vitro to human intestinal mucosa and by electron microscopy for production of putative adherence factors. None of the strains adhered to human duodenal mucosa, and six strains tested did not adhere to ileal mucosa; all 44 strains, however, adhered to human colonic mucosa in localized aggregates. Electron microscopy of infected colonic mucosa indicated fimbrially mediated adhesion of the EAggEC strains. Four morphologically distinct kinds of fimbriae, including a new morphological type of E. coli fimbriae consisting of bundles of fine filaments, were identified among the EAggEC strains; this new type of fimbria was observed in 43 of the 44 EAggEC strains. Forty-three of the 44 EAggEC strains were positive with a DNA probe developed to identify EAggEC, and most of the strains belonged to serotypes unrelated to the other major classes of diarrheic E. coli. These results suggest that EAggEC may be a large-bowel pathogen and colonize the colon by a fimbrially mediated adhesion mechanism

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

Atypical Enteropathogenic Escherichia coli Infection and Prolonged Diarrhea in Children

Infection of children with atypical EPEC is associated with prolonged diarrhea