Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7

P>Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

Role of NleH, a Type III Secreted Effector from Attaching and Effacing Pathogens, in Colonization of the Bovine, Ovine, and Murine Gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157: H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappa B to the nucleus. In this study we investigated the role of NleH during EHEC O157: H7 infection of calves and lambs. We found that while EHEC Delta nleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157: H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappa B reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappa B response elements, we found that NleH causes an increase in NF-kappa B activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Type III secretion homologs are present in Brucella melitensis, B-ovis, and B-suis biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.

EspJ is a prophage-carried type III effector protein of attaching and effacing pathogens that modulates infection dynamics

Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5' end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission.

Characterization of two non-locus of enterocyte effacement-encoded type III-translocated effectors, NleC and NleD, in attaching and effacing pathogens

Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5) P). We show that PtdIns(5) P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns(5) P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5) P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns(5) P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns(5) P production that plays an important role in host cell responses such as survival.

Interaction of enterohemorrhagic Escherichia coli O157 : H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for competitive fitness against bacteria and yeasts

When analysing the secretome of the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, we identified hemolysin co-regulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the Pst DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of hcp genes and tested the fitness of hcp knock-out mutants in host plant colonization and in inter-microbial competition. We found that the hcp2 gene is expressed, most actively at the stationary growth phase, and that the Hcp2 protein is secreted via T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and it does not contribute to virulence or colonisation in tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition against yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive in conditions where it has to compete with other micro-organisms for resources.

Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC)

BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:147 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7. (c) 2005 Elsevier SAS. All rights reserved.

Transcriptomic analysis of Enterohaemorrhagic Escherichia coli O157:H7 in response to plant extracts

Enterohaemorrhagic Escherichia coli (EHEC) are a group of food and contact-borne pathogens responsible for haemorrhagic colitis. The bacteria can be transmitted by contaminated meat, but importantly, also by plants. The bacteria can use plants as an alternative host, where they associate with both the leaves and the roots. Colonisation in the rhizosphere of plants is thought to be the main habitat for colonisation. Four different plant species, commonly associated with EHEC outbreaks, were infected with EHEC O157:H7 isolates Sakai and TUV 93-0 over ten days to assess the colonisation potential of the bacteria in both the phyllosphere and rhizosphere of plants. The rhizosphere was found to sustain a higher population level of bacteria over time in comparison to the phyllosphere, yet both strains were unable to utilize root exudates for growth. Global gene expression changes of EHEC O157:H7 strain Sakai were measured in response to plant extracts such as leaf lysates, root exudates and leaf cell wall polysaccharides from spinach cultivar Amazon and lettuce cultivar Salinas. Microarrays analysis showed a significant change in expression of 17 % of genes on exposure to leaf lysates of spinach. A more specific response was seen to spinach leaf cell wall polysaccharides with only a 1.5 % change. In contrast, when exposed to lettuce leaf cell wall polysaccharides a higher change of 4.8 % was seen. Genes that were differentially expressed belonged to multiple functional groups, including metabolism, indicating the utilization of plant-specific polysaccharides. Several areas of further investigation have been determined from this project, including the importance of culturing bacterial strains at a relevant temperature, the proposed lack of the type III secretion system in plant colonization by EHEC O157:H7 and the utilization of plant components for growth and persistence in the plant environment.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.