Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli

BACKGROUND: The λ Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of λ Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli. RESULTS: In this report, we have identified conditions that optimize the use of λ Red for recombineering in EHEC and EPEC. Using plasmids that contain a P(tac)-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (~1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC. CONCLUSIONS: The λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of λ Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development

Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts

Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly

Enterohemorrhagic Escherichia coli (EHEC) generate F-actin–rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspFU, a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspFU repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspFU are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspFU fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspFU. Whereas clustering of a single EspFU repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspFU derivatives promote actin assembly more efficiently. Moreover, the EspFU repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3–mediated signaling pathways

Enterohemorrhagic E. coli Requires N-WASP for Efficient Type III Translocation but Not for EspFU-Mediated Actin Pedestal Formation

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspFU (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspFU is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspFU into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspFU, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspFU were the only bacterial effectors required for pedestal formation, and the EspFU sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspFU, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization

Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance.

Enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) are closely related extracellular pathogens that reorganize host cell actin into "pedestals" beneath the tightly adherent bacteria. This pedestal-forming activity is both a critical step in pathogenesis, and it makes EPEC and EHEC useful models for studying the actin rearrangements that underlie membrane protrusions. To generate pedestals, EPEC relies on the tyrosine phosphorylated bacterial effector protein Tir to bind host adaptor proteins that recruit N-WASP, a nucleation-promoting factor that activates the Arp2/3 complex to drive actin polymerization. In contrast, EHEC depends on the effector EspFU to multimerize N-WASP and promote Arp2/3 activation. Although these core pathways of pedestal assembly are well-characterized, the contributions of additional actin nucleation factors are unknown. We investigated potential cooperation between the Arp2/3 complex and other classes of nucleators using chemical inhibitors, siRNAs, and knockout cell lines. We found that inhibition of formins impairs actin pedestal assembly, motility, and cellular colonization for bacteria using the EPEC, but not the EHEC, pathway of actin polymerization. We also identified mDia1 as the formin contributing to EPEC pedestal assembly, as its expression level positively correlates with the efficiency of pedestal formation, and it localizes to the base of pedestals both during their initiation and once they have reached steady state. Collectively, our data suggest that mDia1 enhances EPEC pedestal biogenesis and maintenance by generating seed filaments to be used by the N-WASP-Arp2/3-dependent actin nucleation machinery and by sustaining Src-mediated phosphorylation of Tir

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility.

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members

The actin pedestals assembled by KC12+EspF_U and EPEC Y474* promote motility and exploration of the host cell surface.

<p>(A) NIH3T3 cells stably expressing mCherry-actin were infected with EPEC expressing GFP for 3 h and imaged live for 45 min. Scale bar, 10 μm. (B) mCherry-actin expressing cells were infected with the indicated strains for 3 h and imaged live for 18–20 min. 15–20 bacteria per host cell for each strain were tracked, and data from representative cells were plotted such that points starting at t = 0 were centered at the origin. (C) Bacterial motility rates were quantified from cells infected as in (B). Each bar represents the mean speed (+/- SE) of bacteria on 6–20 host cells (95–293 total bacteria). ** p<0.01, *p<0.05 (ANOVA, Tukey post-hoc tests). (D) The fraction of pedestals that were considered moving was quantified, using the average speed of the pedestal deficient counterpart strain as a minimum cutoff to define movement. Each bar represents the mean (+/- SE) from 227–320 pedestals. p = 0.3 (Fisher’s exact test). (E) Directional persistence of pedestals was calculated by dividing the maximum displacement by the total path length for pedestals considered to be moving. Each bar represents the mean (+/- SE) for 205–297 pedestals on 19–20 cells. p = 0.1 (unpaired <i>t</i> test) (F) Cells were infected with EHEC strains with or without EspF_U and imaged live. Each bar represents the mean speed (+/- SE) of bacteria on 6 cells (60 bacteria). *p<0.05 (ANOVA, Tukey post-hoc test).</p

EHEC and EPEC pedestals can be compared directly using engineered EPEC strains.

<p>(A) EPEC Y474*, EPEC Y474F, KC12+EspF_U, and KC12+vector are all EPEC strains engineered to express HA-tagged versions of Tir and/or myc-tagged EspF_U to reflect the WT EPEC or WT EHEC pathways of pedestal assembly. Green and purple boxes represent EPEC and EHEC proteins, respectively. The asterisk indicates phosphotyrosine residue 474. (B) HeLa cells were infected for 3 h with the indicated strains, fixed, and stained to visualize LPS, HA-Tir, and F-actin. Scale bar, 10 μm.</p

KC12+EspF_U and EPEC Y474* form macrocolonies that grow over time on polarized epithelial cells.

<p>(A) Polarized Caco-2 monolayers were infected with KC12+EspF_U for 6 h, fixed, and stained for LPS, DNA, and F-actin. Scale bar, 25 μm; inset 2.5 μm. (B) Cells infected in (A) with KC12+EspF_U or EPEC Y474* were imaged at a lower magnification. Scale circles have areas of 100, 500, and 1000 μm². (C) Polarized Caco-2 monolayers were mock infected (top panels) or infected for 6 h with KC12+EspF_U (bottom panels), and visualized by scanning electron microscopy. The inset highlights a portion of a macrocolony. Scale bars, 10 μm; inset,1 μm. (D) Cells infected in parallel with those in (C) were visualized by transmission electron microscopy. The inset shows a cross-section of a pedestal. Scale bars, 2 μm. (E) Polarized Caco-2 monolayers were infected for 3, 5, or 7 h, fixed, and stained to visualize bacteria, DNA, and F-actin. Scale circles, 100, 500, 1000 μm². (F) Macrocolony sizes were quantified from cells infected as in (E). Each bar represents the mean (+/- SE) of macrocolony sizes calculated from 3–6 coverslips (85–2309 colonies). Macrocolonies over 25 μm² were included in quantification. *p<0.05, ***p<0.001 (unpaired <i>t</i> tests).</p

EspF_U can enhance macrocolony size using either the EHEC or EPEC version of Tir.

<p>(A) Polarized Caco-2 monolayers were infected for 6 h with the indicated KC12 and KC12Δ<i>tir</i> strains, fixed, and stained to visualize bacteria, DNA, and F-actin. (B) Experiments described in (A) were quantified. Each bar represents the mean macrocolony size (+/- SE) calculated from 6 coverslips (2025–3179 colonies). (C) The experiments in (B) were also used to quantify the % of monolayer area infected. Each bar represents the mean (+/- SE) from 59–60 FOVs. (D-E) Polarized Caco-2 monolayers were infected with EHEC strains for 8 h, fixed, and stained as in (A). Bars represent the mean macrocolony size (+/- SE) calculated from 315–617 macrocolonies. (F-G) Polarized Caco-2 monolayers were infected with WT EPEC strains with or without EspF_U. Each bar represents the mean macrocolony size (+/- SE) calculated from 1163–2722 macrocolonies. KC12+EspF_U is shown in purple. For all panels, scale circles, 100, 500, 1000 μm². ** p <0.01, *** p <0.001 (ANOVA, Tukey post-hoc tests). To allow for a sufficient number of Δ<i>tir</i> colonies that could be analyzed, colonies larger than 50 μm² were included in quantification, unlike <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g004" target="_blank">Fig 4</a> where 100 μm² was the lower limit.</p