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The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain. At least five different types of Int280, designated α, β, γ, δ, and ɛ, have been described based on sequence variation in this region. Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium , which normally displays intimin β, to express intimin γ from enterohemorrhagic Escherichia coli . We show that intimin γ binds to the translocated intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing intimin γ could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.

Adhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton. Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion. A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation. In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence. Intimin appears to play a key role in MLP elongation. At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected. In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of 'cage-like' structures engulfing the bacteria. Inoculating HEp-2 cells with intimin-coated latex spheres induced similar 'cage-like' structures. Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred. Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e. elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre. These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.

The hallmark of enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) adhesion to cultured human host cells is intimate attachment and the formation of attaching and effacing (A/E) lesions. Recently, EHEC O157:H7 was shown to induce A/E lesions on human intestinal explants. Unlike EPEC, which colonized the small intestine, EHEC adhesion was restricted to follicle-associated epithelium (FAE) of ileal Peyer's patches. This study tested the hypothesis that the bacterial adhesin intimin contributes to tissue specificity. Complementing the eae gene mutation in CVD206 (derived from EPEC strain E2348/69) with EPEC eaealpha (encoding intimin-alpha) restored the ability to colonize small intestinal mucosa like the parent strain. In contrast, complementing with EHEC eaegamma (encoding intimin-gamma) resulted in the strain adhering and inducing A/E lesion on Peyer's patches, similar to EHEC. An intimin-gamma-positive O55:H7 EPEC also targeted FAE. Thus, intimin contributes to the tissue specificity of A/E lesion-forming microbial pathogens.

A new easily scalable approach to the recovery of biologically active oligosaccharides from milk has been developed which relies on the combination of enzymatic treatment of defatted milk using beta-galactosidase and nanofiltration. It was shown that enzymatic hydrolysis of lactose significantly improves the efficiency and selectivity of membrane-based separations. With the best membrane, as much as 6.7 g of oligosaccharides (containing very little contaminating lactose) could be obtained from one liter of defatted human milk in just four nanofiltration cycles. The human milk oligosaccharides recovered by this method were shown to inhibit binding of intimin, an adhesion molecule of enteropathogenic Escherichia coli, to epithelial cells in vitro. No significant difference in the oligosaccharide profile between samples prepared by this method and conventional gel-permeation chromatography was found. The developed approach is also suitable for the recovery of substantial quantities of tri- and tetra-saccharides from caprine milk.

The predicted massive growth in the world overall population and the proportion of the elderly together with climate change and water shortages pose major global challenges in ensuring food security, both in terms of availability and safety. The geographic and demographic changes are likely to force unprecedented changes in land usage and agricultural practices (i.e. bringing closer together animal and crop production and the need to recycle water), which might increase the risk of contamination. Fresh produce, particularly salad leaves that are consumed raw, is becoming an increasingly important source of human infections. Despite the increasing risk to public health, little is currently known about the mechanism through which human pathogens bind to leaf surfaces or the plant traits that facilitate bacterial attachment. Infections with enteric pathogens, such as enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 and Salmonella enterica, which are ranked as key food‐borne pathogens of humans, are often attributed to consumption or handling of contaminated bovine or chicken products. However, recent outbreaks of food poisoning have been associated with consumption of contaminated vegetable or salad produce and evidence suggests that such outbreaks are increasing (reviewed in Little and Gillespie, 2008; Holden et al., 2009). Examples of international outbreaks of S. enterica and EHEC O157 linked to ready‐to‐eat plant produce include a Scandinavian/UK outbreak of S. Thompson infection associated with consumption of rocket leaves, a Danish outbreak of S. Anatum infection linked to imported basil, an outbreak of S. Typhimurium DT204b in several European countries associated with consumption of lettuce and an outbreak of S. Senftenberg infection associated with imported Israeli basil affecting the UK, Denmark, the Netherlands and the USA (Pezzoli et al., 2007). The largest EHEC O157 outbreak to date, in 1996 in Sakai City, Osaka, Japan, was traced to consumption of white radish sprouts. In 2005 an outbreak affecting 120 people on the west coast of Sweden was linked to iceberg lettuce and in 2006 a severe outbreak that affected many parts of the USA was traced to contamination of pre‐packed spinach from California (reviewed in Little and Gillespie, 2008; Holden et al., 2009). Numerous studies have investigated the potential sources of vegetable contamination both pre harvest in the field and post harvest in the food chain. During the pre‐harvest phase, the potential exists for persistent pathogen populations to establish on growing crops and then the risk to be amplified post harvest either by further direct contamination or by proliferation of existing pathogen populations during processing and handling procedures. Contamination in the field may occur because of application of inadequately composted manures or sewage, insect transmission, water run‐off from nearby animal pastures or exposure to contaminated feces of wild animals. The risk associated with using water from a range of sources and microbiological qualities for irrigation of salads and vegetables has been assessed and the need for improved guidelines recognized (Hamilton et al., 2006). Certainly in lettuce plants, surface irrigation and spray irrigation with suspensions of E. coli O157 : H7 lead to recovery of the pathogen from lettuce tissue 20 days later. The lettuce remained contaminated with E. coli O157 : H7 even after washing, indicating that irrigation of food crops with water of unknown microbiological quality poses significant hazards. In the UK and Europe, legislation has moved responsibility for fresh produce safety away from government and into the supply chain, and retailers and producers have made significant progress in driving safety in production procedures through use of their own or other recognized standards (Monaghan, 2006). However, despite these efforts significant food‐poisoning incidents related to consumption of fresh produce are still reported, indicating that under certain conditions inoculation and survival of human pathogens continue to occur. There have been a very large number of studies of the persistence of human pathogens on plants. Most studies have focused on the survival of E. coli (largely E. coli O157 : H7) and Salmonella sprayed or applied directly on to foliage of plants in some way, or applied on seeds, roots or into soil. With so many different experimental systems, it is difficult to make general statements concerning population dynamics and survival of human pathogens on crop plants. Nevertheless, when applied directly to foliage or into soil, both E. coli and Salmonella are able to survive for extensive periods of time: > 100 days on foliage and > 5 months in soil (reviewed in Holden et al., 2009). Importantly, standard post‐harvest decontamination procedures with solutions containing approximately 20–200 µg ml−1 free chlorine for various lengths of time can reduce bacterial numbers, but do not eliminate either the natural microbial population or human pathogens completely. The phyllosphere is characterized by a number of extreme and often fluctuating environmental conditions combined with unique physio‐chemical characteristics, to which typical phyllosphere microorganisms show a number of adaptations, allowing them to grow in these habitats. Human pathogens are not normally considered a part of the phyllosphere microbial population, but it is clear that they do occur, as evidenced by the outbreaks of food poisoning described above. We recently reviewed the ecology of human pathogens in relation to the phyllosphere (Whipps et al., 2008) and concluded that human pathogens can survive successfully in the phyllosphere of crop plants. We identified a need to understand the bacterial factors controlling attachment to intact and cut plant surfaces, the role of genetic variation in plants for a propensity for colonization of the phyllosphere by human pathogens and to identify those plant characteristics that could be manipulated through crop genetics to decrease pathogenic bacteria attachment and survival. While environmental and genetic factors play a critical role in determining patterns of phyllosphere colonization by bacteria, including human pathogens, much less is known of plant‐related factors that determine the potential for human pathogens to colonize, grow or survive as epiphytes or endophytes on or in leaves. Plant defence mechanisms have been shown to respond to the presence of such pathogens. Genomewide analysis of the transcriptional response of Arabidopsis thaliana to a plant pathogen and E. coli O157 : H7 infiltrated into the leaf tissues identified many genes in common, suggesting the presence of common effector molecules in both bacteria (Thilmony et al., 2006). Development of the microbial population in the phyllosphere will also be determined by phenotypic characteristics of the plant; certainly, there are ‘hot‐spots’ of microbial growth on the leaf associated with specific sites. Gross plant morphology is known to influence the size of phyllosphere bacterial populations and variation in bacterial populations between different species has been attributed to a range of plant characteristics, including leaf water content, leaf P content, amounts of bacteria‐inhibitory phenolics and leaf and mesophyll thickness (Yadav et al., 2005). There have been very few studies of plant cultivar effects on human pathogens; however, a recent report demonstrated a differential interaction between lettuce cultivars and S. enterica serovars (Klerks et al., 2007). However, such a complex interaction is likely to be under the control of multiple plant and bacterial genes. An understanding of the mechanisms involved could lead to a reduction in pathogenic microorganisms on fresh produce and thereby a decrease in the risk of food‐borne illness. A powerful approach to understanding and exploiting the underlying plant genetics would be quantitative genetic analysis on selected plant species using appropriate experimental mapping populations. Genetic analysis to identify quantitative trait loci (QTL) allows the dissection of continuous phenotypic variation into contributions from discrete genetic factors based on the use of DNA markers. To do this a suitable mapping population is required with a clear differential parental response to the trait(s) of interest for construction of a genetic linkage map and also for the collection of ‘robust’ phenotypic data for the traits through properly designed replicated experiments. It is possible to determine whether QTL are specific to certain plant tissues, developmental stages or structures. For example, a study on shelf life of ‘baby leaf’ lettuce identified QTL for properties, such as cell wall strength, stomatal index and epidermal cell area, and suggested that bacterial colonization could play a role in post‐harvest performance. As discussed earlier, plant characteristics such as these could indeed have a significant effect on pathogenic bacteria. In conclusion, in face of the continuing potential risk to human health from consumption of contaminated fresh produce, there is a need to investigate microbe–plant interactions at both the phenotypic, genetic and molecular levels. Performing parallel complementary studies on the pathogen and the host crop will allow a better understanding of the factors affecting colonization of the phyllosphere by human pathogens, which will ultimately form the basis of development of better control and decontamination strategies leading to production of safer produce.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q 65 -K 199 ) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N -acetylglucosamine, and N -acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan.

Enteropathogenic Escherichia coli (EPEC) produces a plasmid-encoded type IV pilus, called the bundle-forming pilus (BFP), involved in the formation of the localized adhesion onto epithelial cells. In this study, we demonstrate that clinical isolates of serotypes O128ab:H2 and O119:H2 contain a ca. 13-kb deletion in the bfp operon, resulting in a lack of expression of these pili. An IS sequence with homology to the IS66 of Agrobacterium tumefaciens replaced the deleted bfp genes. These results suggest that the bfp operon was deleted through a transpositional event and that other adherence factors may mediate attachment of these bacteria to the host cells.

In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics.

Enteropathogenic Escherichia coli (EPEC) induce gross cytoskeletal rearrangement within epithelial cells, immediately beneath the attached bacterium. The C-terminal 280 amino acid residues of intimin (Int280; 30.1 kDa), a bacterial cell-adhesion molecule, mediate the intimate bacterial host-cell interaction. Recently, interest in this process has been stimulated by the discovery that the bacterial intimin receptor protein (Tir) is translocated into the host cell membrane, phosphorylated, and after binding intimin triggers the intimate attachment. Using multidimensional nuclear magnetic resonance (NMR) and combining perdeuteration with site-specific protonation of methyl groups, we have determined the global fold of Int280. This represents one of the largest, non-oligomeric protein structures to be determined by NMR that has not been previously resolved by X-ray crystallography. Int280 comprises three domains; two immunoglobulin-like domains and a C-type lectin-like module, which define a new family of bacterial adhesion molecules. These findings also imply that carbohydrate recognition may be important in intimin-mediated cell adhesion.

Enteropathogenic Escherichia coli (EPEC) encode a type III secretion system located on a pathogenicity island known as the locus for enterocyte effacement. Four proteins are known to be exported by this type III secretion system – EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor protein (Tir) required for intimin-mediated intimate attachment and attaching and effacing lesion formation. The espA gene is located within the locus for enterocyte effacement and the EspA polypeptide from the prototype EPEC strain E2348/69 (O127:H6) has recently been shown to be a component of a filamentous structure involved in bacteria-host cell interaction and locus for enterocyte effacement-encoded protein translocation involved in attaching and effacing lesion formation. In this study we have extended our investigation of EspA to strains belonging to other classical EPEC serotypes. DNA sequencing demonstrated that the espA gene from the different EPEC strains share at least 65% DNA identity. In addition, we detected morphologically and antigenically similar EspA filaments in all but one of the bacterial strains examined including recombinant, non-pathogenic E. coli expressing espA from a cloned locus for enterocyte effacement region (HB101(pCVD462)).

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 ( eae deficient), CVD206(pCVD438) ( eae -complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a Tn phoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206(pCVD438) (both Int + BFP + ) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int + BFP − ) adhered and caused A/E lesions although bacteria adhered in “flat,” two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int − BFP + ) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium . Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium . These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions in the intestinal mucosa. The intimate bacterial adhesion associated with A/E lesion formation is promoted by intimin, a 94-kDa EPEC surface protein. Anti-intimin antisera raised in rabbits by using the purified 280-amino-acid cell binding domain of intimin as the immunogen were employed in immunofluorescence and immunoelectron microscopical studies to investigate the expression of intimin by classical EPEC strain E2348/69 (O127:H6) and defined E2348/69 derivatives during culture growth and A/E bacterium adhesion to cultured HEp-2 cells. In stationary-phase broth cultures, only a small fraction of E2348/69 bacteria expressed intimin, and of those that did, immunolabelling revealed a uniform distribution of intimin over the bacterial surface; increased numbers of bacteria expressing intimin were detected when E2348/69 was grown in tissue culture medium, an effect not seen with strain JPN15, a virulence plasmid-cured derivative of E2348/69. Strain CVD206, an eaeA mutant of E2348/69, did not stain with the anti-intimin antisera, but strain CVD206(pCVD438), containing a functional eaeA gene, stained uniformly. After a 3-h incubation of HEp-2 cells with strain E2348/69, double immunofluorescence labelling of intimin and cellular actin revealed strong intimin expression by all A/E bacteria, but after 6 h of incubation, intimin expression by most E2348/69 bacteria was greatly reduced or not detected. This effect on intimin expression was not observed with strain JPN15 but was restored for strain JPN15(pCVD450) harboring the virulence plasmid-encoded per genes. These results indicate that surface expression of intimin is regulated by environmental factors during bacterial growth and following A/E lesion formation and that virulence plasmid-encoded genes participate in these regulation processes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella -containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD 471-626 , which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD 471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol ( myo )-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella , and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector that has a role in intracellular bacterial replication.

Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions.

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated μCT (DLIT-μCT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-μCT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-μCT imaging and bacterial enumeration from feces for 8 days. The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and μCT scans using a single imaging platform. The low dose μCT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

Summary Mucus production by goblet cells of the large intestine serves as a crucial antimicrobial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here, we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and biogeographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucus secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies an innate immune regulatory pathway governing goblet cell mucus secretion, linking nonhematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism. PaperClip

Fresh fruit and vegetables are important components of a healthy and balanced diet. However, they are increasingly being recognized as important vehicles for transmission of human pathogens that were traditionally classified as zoonotic. There is a significant gap in our knowledge and understanding of the mechanisms by which human pathogens colonize and survive on or in fruits and vegetables. In this study we investigated the binding of Salmonella enterica to tomato fruits (Solanum lycopersicum), which is becoming a major source of human infection. We report that Salmonella enterica serovars Typhimurium and Senftenberg bound to the surface of unripe tomatoes in an aggregative pattern, while serovar Thompson adhered diffusely. We found that while flagella did not have a role in binding, bcsC S. Typhimurim mutants, deficient in cellulose production, exhibited significantly reduced level of attachment to tomatoes. Trans complementation of the mutation restored adhesion to the wild-type level.

Enterotoxigenic Escherichia coli (ETEC) causes child and travelers' diarrhea and is presumed to be water- and food-borne. Sporadic outbreaks were traced to consumption of contaminated fresh produce, particularly salad leaves as lettuce and parsley. Importantly, the mechanism by which ETEC binds salad leaves is not known. In this study we investigated the ability of clinical ETEC isolates to adhere to Eruca vesicaria (commonly known as rocket). Towards this end we inoculated pieces of cut E. vesicaria leaves with clinical ETEC isolates grown in Luria broth at 20°C, conditions that are not permissive for expression of the plasmid-encoded colonization factors and hence mimic the actual transmission pathways of ETEC through intake of contaminated food. We found that ETEC strains bind E. vesicaria at various efficiencies. Examination of representative strains by scanning electron microscopy revealed that they adhere to the E. vesicaria surface in a diffuse pattern by extended filaments resembling flagella. Using the prototype ETEC strain H10407 we found that it also binds to lettuce, basil and spinach leaves. Binding of H10407 was dependent on flagella as a fliC mutant attached to leaves at a much lower efficiency. Interestingly, under the employed environmental conditions EtpA, which forms a flagellar tip structure, and colonization factor I are dispensable for leaf attachment. The results show that ETEC can bind specifically to salad leaves, which might represent an important, yet less recognized, source of infection.

Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions.

Injection of effector proteins by a type III secretion system (T3SS) is a common infection strategy employed by many important human pathogens, including enteric Escherichia coli, Salmonella, Yersinia, and Shigella, to subvert cell signaling and host responses. In recent years, great advances have been made in understanding how the T3SS effectors function and execute the diverse infection strategies employed by these pathogens. In this review, we focus on effectors that subvert signaling pathways that impact on endosomal trafficking, cell survival, and innate immunity, particularly phagocytosis, nuclear factor-κB (NF-κB), and mitogen-activated protein (MAP) kinase pathways and the inflammasome.

Enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and Shigella use a type 3 secretion system (T3SS) to inject dozens of effector proteins into the host cell. The effectors manipulate host cell processes including the host cytoskeleton, immune response, cell survival, and gut integrity. EPEC and EHEC share a large number of common effectors and some homologous effectors can be found in Shigella, however there is also considerable variability within and between pathotypes. For many effectors detailed molecular mechanisms of action have been described by identifying host interacting partners, homologous proteins, or enzymatic activities. However, understanding effector biology within the context of infection with multiple effectors, various cell populations, and host genetic differences remains a challenge.

Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin.

The formation of attaching and effacing ( A E ) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A E lesion by both epec and C. rodentium. In this study we found similar ultrastructural damage in small intestinal biopsies from an EPEC-infected child and large bowel specimens from C. rodentium-infected mice. The C. rodentium-infected large bowel biopsies revealed massive hyperplastic reactions and the infected human small intestinal biopsies showed an increase in total crypt cell number and mitotic index. EPEC-infected small intestinal organ cultures revealed bacteria adhering in a localized pattern and evidence of A E lesions. Covaspheres® coated with a biologically active cell-binding domain of intimin also adhered to cells in a localized fashion but did not induce the characteristic A E lesions.

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

Translocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal “translocation stop” activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation., IMPORTANCE Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins NHERF2 (Na+/H+ exchanger regulatory factor 2) and AnxA2 (annexin A2), which are involved in regulation of intestinal ion channels, are recruited to the bacterial attachment sites. Using a stable HeLa-NHERF2 cell line, we found partial co-localization of AnxA2 and NHERF2; in EPEC-infected cells, AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerizes and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial effector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.

Enteric Escherichia coli (E. coli) are both natural flora of humans and important pathogens causing significant morbidity and mortality worldwide. Traditionally enteric E. coli have been divided into 6 pathotypes, with further pathotypes often proposed. In this review we suggest expansion of the enteric E. coli into 8 pathotypes to include the emerging pathotypes of adherent invasive E. coli (AIEC) and Shiga-toxin producing enteroaggregative E. coli (STEAEC). The molecular mechanisms that allow enteric E. coli to colonize and cause disease in the human host are examined and for two of the pathotypes that express a type 3 secretion system (T3SS) we discuss the complex interplay between translocated effectors and manipulation of host cell signaling pathways that occurs during infection.

The 5′ end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognized gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whereas two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears to be critical. Hence mutations that destroy the UAG stop codon, or short deletions between the UAG stop codon and the ler translation initiation region, result in big effects on ler expression. In the context of the full-length LEE1 operon leader sequence, translation of this very short two-codon open reading frame is necessary for optimal expression of the ler gene and for the subsequent interactions of enterohaemorrhagic Escherichia coli with host target cells.

Enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium colonize their respective hosts while forming attaching and effacing lesions. Their infection strategy relies on translocation of a battery of type III secretion system effectors, including Map, EspM and EspT, which belong to the WxxxE/SopE family of guanine nucleotide exchange factors. Using the C. rodentium mouse model we found that EspT triggers expression of KC and TNFα in vivo. Indeed, a growing body of evidence suggests that, in addition to subversion of actin dynamics, the SopE and the WxxxE effectors activate signalling pathways involved in immune responses. In this study we found that EspT induces expression of the pro-inflammatory mediators cyclooxygenase-2 (COX-2) an enzyme involved in production of prostaglandin E(2) (PGE2), interleukin (Il)-8 and Il-1β in U937 human macrophages by activating the nuclear factor kappa-B (NF-κB), the extracellular signal-regulated kinases 1 and 2 (Erk1/2) and c-Jun N-terminal kinase (JNK) pathways. Since EspT modulates the activation of Cdc42 and Rac1, which mediates bacterial invasion into epithelial cells, we investigated the involvement of these Rho GTPases and bacterial invasion on pro-inflammatory responses and found that (i) Rac1, but not Cdc42, is involved in EspT-induced Il-8 and Il-1β secretion and (ii) cytochalasin D inhibits EspT-induced EPEC invasion into U937 but not Il-8 or Il-1β secretion. These results suggest that while EPEC translocates a number of effectors (i.e. NleC, NleD, NleE, NleH) that inhibit inflammation, a subset of strains, which encode EspT, employ an infection strategy that also involves upregulation of immune mediators.

The enteric pathogens enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Shigella flexneri all translocate at least one effector protein of the EspG protein family into host cells via a type III secretion system (T3SS). The EspG family comprises EspG, EspG2 and VirA. From a Y2H screen, we identified the Golgi matrix protein GM130 as a potential binding partner of EspG. We confirmed EspG:GM130 protein interaction by affinity co-purification. In co-immunoprecipitation experiments EspG was co-precipitated with GM130 while both GM130 and tubulins were co-precipitated with EspG. When expressed ectopically in HeLa cells, the EspG protein family all localized to the Golgi and induced fragmentation of the Golgi apparatus. All EspG family proteins were also able to disrupt protein secretion to a greater extent than the T3SS effector NleA/EspI, which has previously been shown to localize to the Golgi and interact with SEC24 to disrupt COPII vesicle formation. We hypothesize that EspG:GM130 interaction disrupts protein secretion either through direct disruption of GM130 function or through recruitment of other EspG interacting proteins to the Golgi.

Burkholderia pseudomallei , the causative agent of melioidosis, exploits the Bsa type III secretion system (T3SS) to deliver effector proteins into host cells. These effectors manipulate host cell functions; thus, contributing to the ability of the bacteria to evade the immune response and cause disease. Only two Bsa-secreted effectors have been conclusively identified to date. Here, we report the identification of the third B . pseudomallei type III secreted effector protein, designated BopC. BopC is encoded by the bpss1516 gene abutting, bpss1517 , which encodes its putative chaperone. The genes are located in the close proximity to the bsa T3SS gene cluster of B. pseudomallei K96243 (Fig. 1). BopC was secreted into culture supernatant by the wild-type B . pseudomallei strain, but its secretion was abolished in the bsaZ T3SS mutant. Using pull down and co-purification assays, we confirmed that BopC interacts with its putative chaperone, BPSS1517, in vitro . Furthermore, the first 20 N-terminal amino acids of BopC were found to be sufficient to mediate the T3SS-dependent translocation of a reporter protein from a heterologous enteropathogenic Escherichia coli host into mammalian cells. Finally, bopC mutant was found to be less invasive than the wild-type strain in the epithelial cells.

The human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection.

Bax inhibitor 1 (BI-1) was originally discovered as an inhibitor of Bax-induced apoptosis; this review highlights the fundamental importance of BI-1 in a wider context, including in tissue homeostasis and as a regulator of cellular stress. BI-1 has been shown to interact with a broad range of partners to inhibit many facets of apoptosis, such as reactive oxygen species production, cytosolic acidification and calcium levels as well as endoplasmic reticulum stress signalling pathways. BI-1's anti-apoptotic action initially enables the cell to adapt to stress, although if the stress is prolonged or severe the actions of BI-1 may promote apoptosis. This almost universal anti-apoptotic capacity has been shown to be manipulated during infection with enteropathogenic and enterohaemorrhagic Escherichia coli inhibiting host cell death through direct interaction between their effector NleH and BI-1. In addition, BI-1 activity is important in a large number of cancers, promoting metastasis by modulating actin dynamics, a process dependent upon the BI-1 C-terminus and BI-1:actin interaction. Manipulation of BI-1 therefore has the potential for significant therapeutic benefit in a wide range of human diseases.

Many pathogens have the ability to vary the antigenic composition of surface-associated antigens. Often, this variation is mediated by the regulation of gene expression. By varying its antigenicity, the pathogen is able to avoid host immune responses more efficiently; however, this makes the design of vaccines against pathogens that exhibit antigenic variation difficult. In this review, we use the pathogenic Neisseria as an example of antigenically variable bacteria and discuss some attempts to overcome the problems of vaccine design posed by such organisms.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are important human pathogens that rely on translocation of type III secretion system (T3SS) effectors for subversion of signal transduction pathways and colonization of the mammalian gut mucosa. While a core set of effectors is conserved between EPEC and EHEC strains, a growing number of accessory effectors that were found at various frequencies in clinical and environmental isolates have been recently identified. Recent genome projects identified espV as a pseudogene in EHEC but a putative functional gene in EPEC strains E110019 and E22 and the closely related mouse pathogen Citrobacter rodentium . The aim of this study was to determine the distribution of espV among clinical EPEC and EHEC strains and to investigate its function and role in pathogenesis. espV was found in 16% of the tested strains. While deletion of espV from C. rodentium did not affect colonization dynamics or fitness in mixed infections, expression of EspV in mammalian cells led to drastic morphological alterations, which were characterized by nuclear condensation, cell rounding, and formation of dendrite-like projections. Expression of EspV in yeast resulted in a dramatic increase in cell size and irreversible growth arrest. Although the role of EspV in infection and its target host cell protein(s) require further investigation, the data point to a novel mechanism by which the T3SS subverts cell signaling.

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by β-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-β, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer’s patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.

Here we show that the type III secretion gatekeeper protein SepL resembles an aberrant effector protein in binding to a class 1 type III secretion chaperone (Orf12, here renamed CesL). We also show that short N-terminal fragments (≤70 amino acids) from SepL are capable of targeting fusion proteins for secretion and translocation.

Much research into food-borne human pathogens has focused on transmission from foods of animal origin. However, recent investigations have identified fruits and vegetables are the source of many disease outbreaks. Now believed to be a much larger contributor to produce-associated outbreaks than previously reported, norovirus outbreaks are commonly caused by contamination of foods from hands of infected workers. Although infections with Shiga toxin-producing E. coli O157 have been linked to beef more often than to any other food product, severe outbreaks have been traced to consumption of contaminated radish sprouts and pre-packaged spinach. Similarly, while infections with Salmonella have mainly been linked to consumption of foods of animal origin, many outbreaks have been traced to contaminated fresh produce. E. coli O157 binds to lettuce leaves by alternative mechanisms involving the filamentous type III secretions system, flagella and the pilus curli. Association of Salmonella with fresh produce appears to be serovar-specific involving flagella, curli, cellulose, and O antigen capsule. A better understanding of plant, microbiological, environmental, processing and food handling factors that facilitate contamination will allow development of evidence-based policies, procedures and technologies aimed at reducing the risk of contamination of fresh produce.

Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotidefree RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF. © 2010 Blackwell Publishing Ltd.

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air–liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

Background Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. Methods In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. Conclusion This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.

Summary Several lines of evidence indicate a relatively low genetic heterogeneity in the natural Salmonella typhi population. However, some S. typhi isolates found in Indonesia express, instead of the usual fliC-d flagellin gene, a different flagellar gene fliC-j. In addition, Indonesian strains may have a second flagellar antigen fliC-z66. We have previously suggested, on the basis of the flagellar antigen constitution, that S. typhi evolved in an isolated human population in Indonesia. In order to test this hypothesis, we have gathered S. typhi isolates from around the world and tested the genetic heterogeneity among them. In general, polymorphism was greater in isolates from the Far East, as was indicated by Southern hybridizations with rDNA and fliC DNA probes. Gene fliC-j was not found in S. typhi isolates, other than those from Indonesia. However, the one-clone origin of S. typhi was indicated by a common DNA fingerprint pattern and by the occurrence, in the 5′ end region of the fliC gene, of 10 scattered nucleotides that differ from the corresponding 10 nucleotides in other fliC alleles studied. These nucleotides were present in all isolates tested but did not change the amino acid sequence of the flagellin polypeptide.

Citrobacter rodentium (formally C itrobacter freundii biotype 4280) is a highly infectious pathogen that causes colitis and transmissible colonic hyperplasia in mice. In common with enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), C. rodentium exploits a type III secretion system (T3SS) to induce attaching and effacing (A/E) lesions that are essential for virulence. Here, we report the fully annotated genome sequence of the 5.3-Mb chromosome and four plasmids harbored by C. rodentium strain ICC168. The genome sequence revealed key information about the phylogeny of C. rodentium and identified 1,585 C. rodentium -specific (without orthologues in EPEC or EHEC) coding sequences, 10 prophage-like regions, and 17 genomic islands, including the locus for enterocyte effacement (LEE) region, which encodes a T3SS and effector proteins. Among the 29 T3SS effectors found in C. rodentium are all 22 of the core effectors of EPEC strain E2348/69. In addition, we identified a novel C. rodentium effector, named EspS. C. rodentium harbors two type VI secretion systems (T6SS) (CTS1 and CTS2), while EHEC contains only one T6SS (EHS). Our analysis suggests that C. rodentium and EPEC/EHEC have converged on a common host infection strategy through access to a common pool of mobile DNA and that C. rodentium has lost gene functions associated with a previous pathogenic niche.