Exploitation of host cells by enteropathogenic Escherichia coli

Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells. One such subversive microbe is enteropathogenic Escherichia coli (EPEC). A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions. Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir). Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells. The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria. This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions. Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

Effectiveness of treatments for infantile colic: systematic review

Objective: To evaluate the effectiveness of diets, drug treatment, and behavioural interventions on infantile colic in trials with crying or the presence of colic as the primary outcome measure.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin-binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death.

A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence

Enterotoxigenic Escherichia coli associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic E. coli strains that have serologically different pili.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na+ current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na+ channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na+ channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

The reversible condensation and expansion of the rotavirus genome

Understanding the structural organization of the genome is particularly relevant in segmented double-stranded RNA viruses, which exhibit endogenous transcription activity. These viruses are molecular machines capable of repeated cycles of transcription within the intact capsid. Rotavirus, a major cause of infantile gastroenteritis, is a prototypical segmented double-stranded RNA virus. From our three-dimensional structural analyses of rotavirus examined under various chemical conditions using electron cryomicroscopy, we show here that the viral genome exhibits a remarkable conformational flexibility by reversibly changing its packaging density. In the presence of ammonium ions at high pH, the genome condenses to a radius of ≈180 Å from ≈220 Å. Upon returning to physiological conditions, the genome re-expands and fully maintains its transcriptional properties. These studies provide further insights into the genome organization and suggest that the observed isometric and concentric nature of the condensation is due to strong interactions between the genome core and the transcription enzymes anchored to the capsid inner surface. The ability of the genome to condense beyond what is normally observed in the native virus indicates that the negative charges on the RNA in the native state may be only partially neutralized. Partial neutralization may be required to maintain appropriate interstrand spacing for templates to move around the enzyme complexes during transcription. Genome condensation was not observed either with increased cation concentrations at normal pH or at high pH without ammonium ions. This finding indicates that the observed genome condensation is a synergistic effect of hydroxyl and ammonium ions involving disruption of protein–RNA interactions that perhaps facilitate further charge neutralization and consequent reduction in the interstrand spacing.

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.

Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor

Clostridium difficile, a causative agent of antibiotic-associated diarrhea and its potentially lethal form, pseudomembranous colitis, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The level of toxin production appears to be critical for determining the severity of the disease, but the mechanism by which toxin synthesis is regulated is unknown. The product of a gene, txeR, that lies just upstream of the tox gene cluster was shown to be needed for tox gene expression in vivo and to activate promoter-specific transcription of the tox genes in vitro in conjunction with RNA polymerases from C. difficile, Bacillus subtilis, or Escherichia coli. TxeR was shown to function as an alternative sigma factor for RNA polymerase. Because homologs of TxeR regulate synthesis of toxins and a bacteriocin in other Clostridium species, TxeR appears to be a prototype for a novel mode of regulation of toxin genes.

Hypothalamic integration of body fluid regulation.

The progression of animal life from the paleozoic ocean to rivers and diverse econiches on the planet's surface, as well as the subsequent reinvasion of the ocean, involved many different stresses on ionic pattern, osmotic pressure, and volume of the extracellular fluid bathing body cells. The relatively constant ionic pattern of vertebrates reflects a genetic "set" of many regulatory mechanisms--particularly renal regulation. Renal regulation of ionic pattern when loss of fluid from the body is disproportionate relative to the extracellular fluid composition (e.g., gastric juice with vomiting and pancreatic secretion with diarrhea) makes manifest that a mechanism to produce a biologically relatively inactive extracellular anion HCO3- exists, whereas no comparable mechanism to produce a biologically inactive cation has evolved. Life in the ocean, which has three times the sodium concentration of extracellular fluid, involves quite different osmoregulatory stress to that in freshwater. Terrestrial life involves risk of desiccation and, in large areas of the planet, salt deficiency. Mechanisms integrated in the hypothalamus (the evolutionary ancient midbrain) control water retention and facilitate excretion of sodium, and also control the secretion of renin by the kidney. Over and above the multifactorial processes of excretion, hypothalamic sensors reacting to sodium concentration, as well as circumventricular organs sensors reacting to osmotic pressure and angiotensin II, subserve genesis of sodium hunger and thirst. These behaviors spectacularly augment the adaptive capacities of animals. Instinct (genotypic memory) and learning (phenotypic memory) are melded to give specific behavior apt to the metabolic status of the animal. The sensations, compelling emotions, and intentions generated by these vegetative systems focus the issue of the phylogenetic emergence of consciousness and whether primal awareness initially came from the interoreceptors and vegetative systems rather than the distance receptors.

The control of hematopoiesis and leukemia: from basic biology to the clinic.

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.

High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells.

Enteropathogenic Escherichia coli (EPEC), a major cause of pediatric diarrhea, adheres to epithelial cells and activates host cell signal transduction pathways. We have identified five proteins that are secreted by EPEC and show that this secretion process is critical for triggering signal transduction events in epithelial cells. Protein secretion occurs via two pathways: one secretes a 110-kDa protein and the other mediates export of the four remaining proteins. Secretion of all five proteins was regulated by temperature and the perA locus, two factors which regulate expression of other known EPEC virulence factors. Amino-terminal sequence analysis of the secreted polypeptides identified one protein (37 kDa) as the product of the eaeB gene, a genetic locus previously shown to be necessary for signal transduction. A second protein (39 kDa) showed significant homology with glyceraldehyde-3-phosphate dehydrogenase, while the other three proteins (110, 40, and 25 kDa) were unique. The secreted proteins associated with epithelial cells, and EaeB became resistant to protease digestion upon association, suggesting that intimate interactions are required for transducing signals.

Transient transfection and expression of firefly luciferase in Giardia lamblia.

We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly luciferase reporter gene. The optimal construct consisted of a GDH/luciferase fusion gene in which the first 18 codons of the GDH gene immediately preceded the luciferase gene; this fusion gene was flanked by the upstream and downstream sequences of the GDH gene. Electroporation of this construct into Giardia yielded luciferase activity that was 3000- to 50,000-fold above background. Removal of either the 5' or 3' GDH flanking sequences from this construct resulted in significantly reduced luciferase activity, and removal of both flanking sequences reduced luciferase activity to background levels. Luciferase activity was proportional to the amount of DNA electroporated and was maximal at 6 hr after electroporation.