Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

Background

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.

Methodology/Principal Findings

When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).

Significance/Conclusions

The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.     

Widening the scope of constriction

JGP modeling study suggests that selectivity filter constriction is a plausible mechanism for C-type inactivation of the Shaker voltage-gated potassium channel.

In response to prolonged activation, many K⁺ channels spontaneously reduce the membrane conductance by undergoing C-type inactivation, a kinetic process crucial for the pacing of cardiac action potentials and the modulation of neuronal firing patterns. In the pH-activated bacterial channel KcsA, C-type inactivation appears to involve constriction of the channel’s selectivity filer that prohibits ion conduction, but whether voltage-gated channels like Drosophila Shaker use a similar mechanism is controversial (1). In this issue of JGP, a computational study by Li et al. suggests that filter constriction is indeed a plausible mechanism for the C-type inactivation of Shaker (2).(Left to right) Jing Li, Benoît Roux, and colleagues use computational modeling to show that selectivity filter constriction, allosterically promoted by opening of the intracellular activation gate, is a plausible mechanism for the C-type inactivation of voltage-gated K⁺ channels such as Drosophila Shaker. The selectivity filter is conductive (left) when the intracellular gate is partially open, but adopts a constricted conformation (right) when the gate is open wide.Various structural approaches have shown that C-type inactivation of KcsA channels is associated with the symmetrical constriction of all four channel subunits at the level of the central glycine residue in the selectivity filter. Benoît Roux and colleagues at The University of Chicago used MD simulations to show that the KcsA pore can transition from the conductive to the constricted conformation on an appropriate timescale, and that this transition is allosterically promoted by the wide opening of the pore’s intracellular gate (3). Modeling by Roux and colleagues suggests that C-type inactivation of cardiac hERG channels could also involve selectivity filter constriction, though in this case it appears to be an asymmetric process in which only two of the channel’s subunits move closer together (4).“In view of the high similarity between the pore domains of Shaker and KcsA (almost 40% sequence identity), we wanted to examine if it’s possible for the Shaker selectivity filter to constrict and, if so, how similar it is to KcsA,” Roux explains. Led by first author Jing Li—now an assistant professor at the University of Mississippi—Roux and colleagues developed several homology models of the Shaker pore domain with the intracellular gate open to various degrees (2).MD simulations and free energy calculations revealed that the Shaker selectivity filter can dynamically transition from a conductive to a constricted conformation, and that this transition is allosterically coupled to the intracellular gate; the constricted conformation is stable when the gate is wide open. “Our computations strongly suggest that constriction is a plausible mechanism for the C-type inactivation of Shaker,” Roux says. “There’s no reason based on the currently available information to reject the existence of a constricted state in Shaker channels.”As with KcsA, Shaker channels appear to constrict symmetrically at the level of the selectivity filter’s central glycine. But Li et al.’s simulations revealed some small variations between the two channels, including differences in the number of water molecules bound to each channel subunit and the arrangement of the hydrogen-bond network they form to stabilize the constricted state.Li et al. also modeled the pore domain of the Shaker W434F mutant, which is widely assumed to be trapped in a C-type inactivated state. The simulation suggests that the mutant channel’s filter adopts a stable constricted conformation even when the intracellular gate is only partially open, although the constriction is asymmetric and occurs at the level of a different filter residue (2).Constriction may therefore be a universal mechanism of C-type inactivation, even if the exact conformation varies from channel to channel. But, says Roux, confirming this will require more experimental work using the right conditions and mutations to capture the structure of inactivated channels.     

The Molecular Basis of Phospholipase D2-Induced Chemotaxis: Elucidation of Differential Pathways in Macrophages and Fibroblasts

We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body''s innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by ∼60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid [PA]) and another that involves protein-protein interactions. The first is evidenced by partial abrogation of chemotaxis with lipase activity-defective constructs (PLD2-K758R) and by n-butanol treatment of cells. The second is evidenced by PLD association with the growth factor receptor-bound protein 2 (Grb2) through residue Y¹⁶⁹, located within a Src homology 2 (SH2) consensus site. The association Grb2-PLD2 could be visualized by fluorescence microscopy in RAW/LR5 macrophages concentrated in actin-rich membrane ruffles, making possible that Grb2 serves as a docking or intermediary protein. The Grb2/PLD2-mediated chemotaxis process also depends on Grb2''s ability to recognize other motility proteins, like the Wiskott-Aldrich syndrome protein (WASP). Cell transfection with WASP, PLD2, and Grb2 constructs yields the highest levels of cell migration response, particularly in a macrophage cell line (RAW/LR5) and only modestly in the fibroblast cell line COS-7. Further, RAW/LR5 macrophages utilize for cell migration an additional pathway that involves S6 kinase (S6K) through PLD2-Y²⁹⁶, known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.Normalcy of migration is found in the ability of leukocytes to move toward foreign invaders of the body in phagocytic and immunogenic responses. Fibroblasts and endothelial cells migrate to aid wound repair and deposit collagen around the wounded area (32, 41). Migration is also important in embryogenesis and angiogenesis (37). Cellular migration may be involved not only in the normal physiological state of some cells but also in the pathological state, as in metastasis, whereby tumor cells migrate from the initial tumor site into the circulatory system and establish new colonies (32). Before migration can occur, cells must orient themselves to establish a distinct cell front and rear. The orientation of the cell depends on the inflammation site and location of the chemical stimuli or chemoattractants being released (32, 35). A localized enrichment of filamentous actin (F-actin) under the cell membrane occurs shortly after cell stimulation and is responsible for the formation of the lamellipodia and other morphological changes, such as membrane protrusions or ruffles that occur right before chemotaxis (28).Monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), and colony-stimulating factor (CSF-1, also known as macrophage colony-stimulating factor [M-CSF]) are chemoattractants found to increase cell motility of monocytes and macrophages (52). MIP-1α acts through G-protein-coupled cell surface receptors that are highly abundant on those cells (42) and activate the phosphatidylinositol 3-kinase (PI3K) pathway and phospholipase C (PLC) (49). CSF-1 plays essential roles in the ability of monocytes to survive, proliferate, differentiate, and mature (49) and is also a monocyte/macrophage chemoattractant. CSF-1 acts by binding to cell surface receptors (CSF-1R) encoded by the c-fms proto-oncogene (48). Upon the binding of M-CSF, the CSF-1R dimerizes, and the tyrosine kinase domain is activated, resulting in transphosphorylation of the receptor. Grb2 and PI3K bind to two of the four phosphotyrosyl residues created, and the signal is transmitted to the cell interior (8).Epidermal growth factor (EGF) triggers proliferation and differentiation in fibroblasts (6, 37). EGF also serves other biological functions, such as cell rounding, ruffling, actin cytoskeletal reorganization, filopodium extension, and cell motility (37). Tyrosines that are autophosphorylated at the EGF receptor (EGFR) C terminus upon ligand binding enable binding to Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains (37). Receptor kinase activity, along with at least one of the C-terminal tyrosine autophosphorylation sites, is required for cell movement (9). In addition, PLCγ and protein kinase C (PKC) have been linked to EGF and its ability to enhance cell motility (9, 10).As for the downstream signaling mechanism of these cell surface receptors, the recognized key players are the small GTPases, Rho, Rac, and cdc42. They are activated during actin cytoskeleton rearrangement and in cellular migration (7). New evidence has implicated other signaling proteins. Phospholipase D (PLD) has been found to play a role in leukocyte chemotaxis and adhesion (35). PLD is also involved in the regulation of essential cellular functions, largely due to the production of second messengers such as phosphatidic acid (PA) and ultimately diacylglycerol (DAG) (2, 11, 21, 22, 25, 38, 47, 53). Once produced, PA is involved in many cellular functions, including cytoskeletal rearrangement, phagocytosis, vesicle trafficking, exocytosis, and neuronal and cardiac stimulation (1, 11, 21, 30, 38). PA mediates chemotaxis, as increasing concentrations of PA enhanced the rate of cell migration of phagocytes (35). In the murine lymphoma cell line EL4, Knoepp et al. found that activated PLD2 promotes phosphorylation of FAK and Akt, leading to cell-substrate adhesion (7, 29). However, while inactivated PLD2 inhibits adhesion, migration, proliferation, and tumor invasion, it does not alter the basal level of FAK and Akt phosphorylation (29). Although PA does play a role in cell migration, the specific mechanisms involved are not completely understood, and more precise structure-function studies are needed.We have reported earlier an association of PLD2 and Grb2 important for DNA synthesis/cell proliferation at the level of Y¹⁷⁹ (13) and Y⁵¹¹ (23). This study uncovers that migrating cells also use PLD2 and Grb2 but through a different mechanism, involving residue Y¹⁶⁹ and the presence of the Wiskott-Aldrich syndrome protein (WASP). We also present new evidence implicating PLD2-Y²⁹⁶, known to be phosphorylated by EGFR kinase (24), in a pathway utilizing S6 kinase (S6K). This additional pathway serves to boost the ability of RAW/LR5 cells to migrate more readily than the other type of cell utilized in this study, COS-7 fibroblasts.     

Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster

Post-mating reduction in immune defence is common in female insects, and a trade-off between mating and immunity could affect the evolution of immunity. In this work, we tested the capacity of virgin and mated female Drosophila melanogaster to defend against infection by four bacterial pathogens. We found that female D. melanogaster suffer post-mating immunosuppression in a pathogen-dependent manner. The effect of mating was seen after infection with two bacterial pathogens (Providencia rettgeri and Providencia alcalifaciens), though not after infection with two other bacteria (Enterococcus faecalis and Pseudomonas aeruginosa). We then asked whether the evolution of post-mating immunosuppression is primarily a ‘female’ or ‘male’ trait by assaying for genetic variation among females for the degree of post-mating immune suppression they experience and among males for the level of post-mating immunosuppression they elicit in their mates. We also assayed for an interaction between male and female genotypes to test the specific hypothesis that the evolution of a trade-off between mating and immune defence in females might be being driven by sexual conflict. We found that females, but not males, harbour significant genetic variation for post-mating immunosuppression, and we did not detect an interaction between female and male genotypes. We thus conclude that post-mating immune depression is predominantly a ‘female’ trait, and find no evidence that it is evolving under sexual conflict.     

Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.     

Isolation of the North American strain of viral hemorrhagic septicemia virus (VHSV) associated with epizootic mortality in two new host species of Alaskan marine fish

Thousands of dead Pacific herring Clupea pallasi, Pacific hake Merluccius productus and walleye pollock Theragra chalcogramma were reported in Lisianski Inlet near Pelican, Alaska, USA, on August 1, 1998. The Pacific hake and pollock continued to die through the end of September. Virological examinations of dead fish identified the North American strain of viral hemorrhagic septicemia virus (VHSV) from all 3 species of fish as well as associated high virus titers and possible histopathological lesions. No other primary fish pathogens were detected and there were no apparent environmental causes for fish mortality. This is the first report of VHSV in 2 new Alaskan fish host species and of a natural epizootic associated with VHSV in which progressive mass mortality was observed simultaneously in herring and 2 other species of free-ranging marine fish.     

Evaluating legume species as alternative trap crops to chickpea for management of Helicoverpa spp. (Lepidoptera: Noctuidae) in central Queensland cotton cropping systems

Mounting levels of insecticide resistance within Australian Helicoverpa spp. populations have resulted in the adoption of non-chemical IPM control practices such as trap cropping with chickpea, Cicer arietinum (L.). However, a new leaf blight disease affecting chickpea in Australia has the potential to limit its use as a trap crop. Therefore this paper evaluates the potential of a variety of winter-active legume crops for use as an alternative spring trap crop to chickpea as part of an effort to improve the area-wide management strategy for Helicoverpa spp. in central Queensland's cotton production region. The densities of Helicoverpa eggs and larvae were compared over three seasons on replicated plantings of chickpea, Cicer arietinum (L.), field pea Pisum sativum (L), vetch, Vicia sativa (L.) and faba bean, Vicia faba (L.). Of these treatments, field pea was found to harbour the highest densities of eggs. A partial life table study of the fate of eggs oviposited on field pea and chickpea suggested that large proportions of the eggs laid on field pea suffered mortality due to dislodgment from the plants after oviposition. Plantings of field pea as a replacement trap crop for chickpea under commercial conditions confirmed the high level of attractiveness of this crop to ovipositing moths. The use of field pea as a trap crop as part of an area-wide management programme for Helicoverpa spp. is discussed.