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Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development.

By leveraging ultrashort pulse laser and micro-electromechanical systems (MEMS) technologies, we are developing a miniaturized intraoral dental robotic device that clamps onto teeth, is remotely controlled, and equipped with a focusing and scanning system to perform efficient, fast, and ultra-precise laser treatments of teeth and dental restorative materials. The device will be supported by a real-time monitoring system for visualization and diagnostic analysis with appropriate digital controls. It will liberate dentists from repetitive manual operations, physical strain and proximity to the patient's oro-pharyngal area that potentially contains infectious agents. The technology will provide patients with high-accuracy, minimally invasive and pain-free treatment. Unlike conventional lasers, femtosecond lasers can ablate all materials without generating heat, thus negating the need for water irrigation, allowing for a clear field of view, and lowering cross-infection hazards. Additionally, dentists can check, analyze, and perform precise cutting of tooth structure with automatic correction, reducing human error. Performing early-stage diagnosis and intervention remotely will be possible through units installed at schools, rural health centers and aged care facilities. Not only can the combination of femtosecond lasers, robotics and MEMS provide practical solutions to dentistry's enduring issues by allowing more precise, efficient, and predictable treatment, but it will also lead to improving the overall access to oral healthcare for communities at large.

High fluence focused femtosecond laser pulses were used to perform fast, high precision and minimally damaging cavity cutting of teeth at room temperature without using any irrigation or cooling system. The optimal ablation rates were established for both enamel and dentin, and the surfaces were assessed with optical and scanning electron microscopy, Raman spectroscopy and optical profilometry. No chemical change in the composition of enamel and dentin was observed. We explored temperature variations inside the dental pulp during the laser procedure and showed the maximum increase was 5.5°C, within the acceptable limit of temperature increase during conventional dental treatments.

Existing capsular polysaccharide-based vaccines against pneumococcal disease are highly effective against vaccine-included serotypes, but they are unable to combat serotype replacement. We have developed a novel pneumococcal vaccine that confers serotype-independent protection, and could therefore constitute a "universal" vaccine formulation. This preparation is comprised of whole un-encapsulated pneumococci inactivated with gamma irradiation (γ-PN), and we have previously reported induction of cross-reactive immunity after nonadjuvanted intranasal vaccination. To further enhance vaccine immunogenicity and safety, we modified the pneumococcal vaccine strain to induce a stressed state during growth. Specifically, the substrate binding component of the psaBCA operon for manganese import was mutated to create a pneumococcal surface antigen A (psaA) defective vaccine strain. psaA mutation severely attenuated the growth of the vaccine strain in vitro without negatively affecting pneumococcal morphology, thereby enhancing vaccine safety. In addition, antibodies raised against vaccine preparations based on the modified strain [γ-PN(ΔPsaA)] showed more diversified reactivity to wild-type pneumococcal challenge strains compared to those induced by the original formulation. The modified vaccine also induced comparable protective TH 17 responses in the lung, and conferred greater protection against lethal heterologous pneumococcal challenge. Overall, the current study demonstrates successful refinement of a serotype-independent pneumococcal vaccine candidate to enhance safety and immunogenicity.

The upper respiratory tract is continuously exposed to a vast array of potentially pathogenic viruses and bacteria. Influenza A virus (IAV) has particular synergism with the commensal bacterium Streptococcus pneumoniae in this niche, and co-infection exacerbates pathogenicity and causes significant mortality. However, it is not known whether this synergism is associated with a direct interaction between the two pathogens. We have previously reported that co-administration of a whole-inactivated IAV vaccine (γ-Flu) with a whole-inactivated pneumococcal vaccine (γ-PN) enhances pneumococcal-specific responses. In this study, we show that mucosal co-administration of γ-Flu and γ-PN similarly augments IAV-specific immunity, particularly tissue-resident memory cell responses in the lung. In addition, our in vitro analysis revealed that S. pneumoniae directly interacts with both γ-Flu and with live IAV, facilitating increased uptake by macrophages as well as increased infection of epithelial cells by IAV. These observations provide an additional explanation for the synergistic pathogenicity of IAV and S. pneumoniae, as well as heralding the prospect of exploiting the phenomenon to develop better vaccine strategies for both pathogens. Co-administration of a whole-inactivated influenza virus (IAV) vaccine (γ-Flu) with a whole-inactivated Streptococcus pneumoniae vaccine (γ-PN) enhances IAV-specific immune responses due to the ability of γ-PN to directly interact with γ-Flu, thus increasing viral uptake by macrophages.

Gamma-irradiation, particularly an irradiation dose of 50 kGy, has been utilised widely to sterilise highly pathogenic agents such as Ebola, Marburg Virus, and Avian Influenza H5N1. We have reported previously that intranasal vaccination with a gamma-irradiated Influenza A virus vaccine (γ-Flu) results in cross-protective immunity. Considering the possible inclusion of highly pathogenic Influenza strains in future clinical development of γ-Flu, an irradiation dose of 50 kGy may be used to enhance vaccine safety beyond the internationally accepted Sterility Assurance Level (SAL). Thus, we investigated the effect of irradiation conditions, including high irradiation doses, on the immunogenicity of γ-Flu. Our data confirm that irradiation at low temperatures (using dry-ice) is associated with reduced damage to viral structure compared with irradiation at room temperature. In addition, a single intranasal vaccination with γ-Flu irradiated on dry-ice with either 25 or 50 kGy induced seroconversion and provided complete protection against lethal Influenza A challenge. Considering that low temperature is expected to reduce the protein damage associated with exposure to high irradiation doses, we titrated the vaccine dose to verify the efficacy of 50 kGy γ-Flu. Our data demonstrate that exposure to 50 kGy on dry-ice is associated with limited effect on vaccine immunogenicity, apparent only when using very low vaccine doses. Overall, our data highlight the immunogenicity of influenza virus irradiated at 50 kGy for induction of high titre antibody and cytotoxic T-cell responses. This suggests these conditions are suitable for development of γ-Flu vaccines based on highly pathogenic Influenza A viruses.

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by impaired antibody production, which is in many instances secondary to defective T-cell function (T-CVID). We have previously identified a subset of patients with T-CVID characterized by defective T-cell receptor (TCR)-dependent protein tyrosine phosphorylation. In these patients, ZAP-70 fails to be recruited to the TCR as the result of impaired CD3ζ phosphorylation, which is, however, not dependent on defective Lck expression or activity. Here we show that neither Fyn nor CD45 is affected in these patients. On the other hand, T-CVID T cells show dramatic defects in the Vav/Rac pathway controlling F-actin dynamics. A significant deficiency in Vav protein was indeed observed; in 3 of 4 patients with T-CVID, it was associated with reduced VAV1 mRNA levels. The impairment in Vav expression correlated with defective F-actin reorganization in response to TCR/CD28 coengagement. Furthermore, TCR/CD28-dependent up-regulation of lipid rafts at the cell surface, which requires F-actin dynamics, was impaired in these patients. The actin cytoskeleton defect could be reversed by reconstitution of Vav1 expression in the patients' T cells. Results demonstrate an essential role of Vav in human T cells and strongly suggest Vav insufficiency in T-CVID. (Blood. 2005;106:626-634)

Cholera toxin (Ctx) from Vibrio cholerae and its closely related homologue, heat-labile enterotoxin (Etx) from Escherichia coli have become superb tools for illuminating pathways of cellular trafficking and immune cell function. These bacterial protein toxins should be viewed as conglomerates of highly evolved, multi-functional elements equipped to engage the trafficking and signalling machineries of cells. Ctx and Etx are members of a larger family of A-B toxins of bacterial (and plant) origin that are comprised of structurally and functionally distinct enzymatically active A and receptor-binding B sub-units or domains. Intoxication of mammalian cells by Ctx and Etx involves B pentamer-mediated receptor binding and entry into a vesicular pathway, followed by translocation of the enzymatic A1 domain of the A sub-unit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase and a sequence of events culminating in life-threatening diarrhoeal disease. Importantly, Ctx and Etx also have the capacity to induce a wide spectrum of remarkable immunological processes. With respect to the latter, it has been found that these toxins activate signalling pathways that modulate the immune system. This review explores the complexities of the cellular interactions that are engaged by these bacterial protein toxins, and highlights some of the new insights to have recently emerged.

Epstein-Barr virus (EBV) is associated with a number of important human cancers, including nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin's lymphoma. These tumors express a viral nuclear antigen, EBV nuclear antigen 1 (EBNA1), which cannot be presented to T cells in a major histocompatibility complex class I context, and the viral latent membrane proteins (LMPs). Although the LMPs are expressed in these tumors, no effective immune response is made. We report here that exposure to the cholera-like enterotoxin B subunit (EtxB) in EBV-infected lymphoblastoid cell lines (LCLs) enhances their susceptibility to killing by LMP-specific CD8 + cytotoxic T lymphocytes (CTLs) in a HLA class I-restricted manner. CTL killing of LCLs is dramatically increased through both transporter-associated protein-dependent and -independent epitopes after EtxB treatment. The use of mutant B subunits revealed that the enhanced susceptibility of LCLs to CTL killing is dependent on the B subunit's interaction with GM 1 but not its signaling properties. These important findings could underpin the development of novel approaches to treating EBV-associated malignancies and may offer a general approach to increasing the presentation of other tumor and viral antigens.

Current immunization strategies, using peptide or protein antigens, generally fail to elicit cytotoxic-T-lymphocyte responses, since these antigens are unable to access intracellular compartments where loading of major histocompatibility complex class I (MHC-I) molecules occurs. In an attempt to circumvent this, we investigated whether the GM1 receptor-binding B subunit ofEscherichia coliheat-labile toxin (EtxB) could be used to deliver class I epitopes. When a class I epitope was conjugated to EtxB, it was delivered into the MHC-I presentation pathway in a GM1-binding-dependent fashion and resulted in the appearance of MHC-I-epitope complexes at the cell surface. Importantly, we show that the efficiency of EtxB-mediated epitope delivery could be strikingly enhanced by incorporating, adjacent to the class I epitope, a 10-amino-acid segment from the C terminus of the DNA polymerase (Pol) of herpes simplex virus. The replacement of this 10-amino-acid segment by a heterologous sequence or the introduction of specific amino acid substitutions within this segment either abolished or markedly reduced the efficiency of class I epitope delivery. If the epitope was extended at its C terminus, EtxB-mediated delivery into the class I presentation pathway was found to be completely dependent on proteasome activity. Thus, by combining the GM1-targeting function of EtxB with the 10-amino-acid Pol segment, highly efficient delivery of exogenous epitopes into the endogenous pathway of class I antigen processing and presentation can be achieved.

Following ligand binding the TCR segregates to plasma membrane microdomains, termed lipid rafts, characterized by a highly ordered lipid structure favoring partitioning of glycosyl phosphatidyl inositol-linked costimulatory receptors and acylated signaling molecules. Here we show that the inducible association of the TCR and key signaling proteins with lipid rafts is dependent on the actin cytoskeleton through a mechanism involving raft coalescence. Although lipid rafts are required for full activation of the TCR-dependent tyrosine phosphorylation cascade and sustained signaling, triggering of TCR-proximal events, including Fyn activation and a first wave of Vav phosphorylation, is independent of lipid rafts, while a second wave of raft-dependent Vav phosphorylation occurs after raft coalescence, as also supported by the finding that Vav is phosphorylated in response to lipid raft clustering by GM1 aggregation. The constitutive association found between Vav and the CD3zeta chain suggests a model whereby the TCR-associated signaling machinery initiates raft aggregation by promoting F-actin reorganization, which permits full activation of the tyrosine phosphorylation cascade, further reorganization of the actin cytoskeleton and sustained signaling, leading to cell activation.

Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.

Heat-labile enterotoxin from enterotoxinogenicEscherichia coliis not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8+T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8+T cells but not of CD4+T cells triggers the specific up-regulation of the transcription factorc-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-mycantisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IκBα turnover, suggesting that elevated Myc expression may be dependent on NF-κB. When CD8+T cells were pretreated with inhibitors of IκBα turnover and NF-κB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relativeEscherichia coliheat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 μg of rEtxB or above with 10 μg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 μg) with a trace (0.5 μg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 μg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

Cholera toxin and E. coli heat-labile enterotoxin are structurally homologous proteins comprised of an enzymatically active A-subunit and five B-subunits that bind with high affinity to GM1-ganglioside receptors found on the surface of mammalian cells. The B-subunits have long been thought of simply as trafficking vehicles that trigger entry and subsequent delivery of the 'toxic' A-subunit into cells. Indeed, such is the capacity of the B-subunits to enter cells, that they have been developed as generic carriers for attachment and delivery of a variety of peptides into mammalian cells. However, the B-subunits also appear to possess discrete 'signalling functions', that induce both transcription factor and cell activation. These are thought to be directly responsible for the potent immunomodulatory properties of the B-subunits, and have resulted in their use as adjuvants and as agents to suppress inflammatory immune disorders. The relationship between the signalling properties of the B-subunits and their capacity to act as trafficking vehicles has remained unclear. In an effort to understand the structural requirements for these two functions, a set of mutant B-subunits, with amino acid substitutions at position His-57, have been generated and studied. Importantly, such mutant B-subunits retain an ability to bind with high affinity to GM1 and to traffic into cells, but have entirely lost their capacity to activate immune cell populations. Thus, while binding via GM1 appears to be sufficient to trigger cellular uptake it is not sufficient to activate signal transduction. The His-57 region is therefore speculated to be actively engaged in triggering signalling events, possibly via cognate interaction with other cell surface molecules.

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and likely endoplasmic reticulum (ER) (Lencer et al., J. Cell Biol. 131, 951-962 (1995)). We have recently found that the toxin's apical membrane receptor ganglioside GM1 acts specifically in this signal transduction pathway, likely by coupling CT with caveolae or caveolae-related membrane domains (lipid rafts) (Wolf et al., J. Cell Biol. 141, 917-927 (1998)). Work in progress shows that 1) cholesterol depletion uncouples the CT-GM1 receptor complex from signal transduction, a characteristic of lipid rafts; 2) the GM1 acyl chains rather than the carbohydrate head groups appear to account for the structural basis of ganglioside specificity in toxin trafficking; and 3) intestinal epithelial cells obtained from normal adult humans exhibit lipid rafts which differentiate between CT-GM1 and LTIIb-GD1a complexes and which contain caveolin 1.

In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein–protein interactions and may be developed to target specific cellular compartments.

Cholera toxin (Ctx) and E. coli heat-labile enterotoxin (Etx) are structurally and functionally similar AB5 toxins with over 80% sequence identity. When their action in polarized human epithelial (T84) cells was monitored by measuring toxin-induced Cl- ion secretion, Ctx was found to be the more potent of the two toxins. Here, we examine the structural basis for this difference in toxicity by engineering a set of mutant and hybrid toxins and testing their activity in T84 cells. This revealed that the differential toxicity of Ctx and Etx was (i) not due to differences in the A-subunit's C-terminal KDEL targeting motif (which is RDEL in Etx), as a KDEL to RDEL substitution had no effect on cholera toxin activity; (ii) not attributable to the enzymatically active A1-fragment, as hybrid toxins in which the A1-fragment in Ctx was substituted for that of Etx (and vice versa) did not alter relative toxicity; and (iii) not due to the B-subunit, as the replacement of the B-subunit in Ctx for that of Etx caused no alteration in toxicity, thus excluding the possibility that the broader receptor specificity of EtxB is responsible for reduced activity. Remarkably, the difference in toxicity could be mapped to a 10-amino acid segment of the A2-fragment that penetrates the central pore of the B-subunit pentamer. A comparison of the in vitro stability of two hybrid toxins, differing only in this 10-amino acid segment, revealed that the Ctx A2-segment conferred a greater stability to the interaction between the A- and B-subunits than the corresponding segment from Etx A2. This suggests that the reason for the relative potency of Ctx compared with Etx stems from the increased ability of the A2-fragment of Ctx to maintain holotoxin stability during uptake and transport into intestinal epithelia.

Cholera toxin and its close relative, Escherichia coli heat-labile enterotoxin, are potent immunogens and mucosal adjuvants. The recent findings that their B subunits can promote tolerance highlights the complexity of their interactions with the immune system. Here, Neil Williams and colleagues review the mechanisms by which these molecules modulate leukocyte populations and seek to explain the paradox.

Bioreactor selection is important for maximising the productivity of recombinant organisms. In this paper a comparison is made between growth and recombinant protein synthesis in three types of bioreactor containing a marine Vibrio capable of heterologous expression and secretion of the non-toxic B-subunit pentamer of Escherichia coli heat-labile enterotoxin, EtxB. The heterologous gene was located on the plasmid pMMB68. Resistance to carbenicillin was used to select for plasmid-containing cells. In batch and continuous culture. Volumetric productivities were highest when cells were grown in the presence of carbenicillin. Without antibiotic selection, the highest volumetric productivity (9.4 mg EtxB-1 h-1) was observed in hollow-fibre bioreactors, and the production phase could be maintained for over 50 h. The highest specific productivity under these conditions was found in batch culture, but the maximal production phase was only of 5 h duration. In hollow-fibre reactors the type of fibre used significantly affected productivity, both with regards to the maintenance of reactor integrity and by allowing passage of the recombinant toxoid through the selectively permeable membrane. Where contamination of the product with carbenicillin is to be avoided, these bioreactors are superior to batch or continuous culture.

Cholera and Escherichia coliheat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed “nicking”). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl− secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 μA/cm2). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the “basolateral” secretory responses to “apical” levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T. R., 1994, Protein Expression Purif. 5, 198–204). However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB–pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB–pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60. Addition of 0.3 m M EDTA to the growth medium blocked proteolysis of the secreted EtxB–pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB–pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol. This modified method for expression and purification of EtxB–pol should be of general utility for the preparation of other EtxB-based fusion proteins.

The B subunits of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) assemble in vivo into exceptionally stable homopentameric complexes, which maintain their quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. Recently, we showed that the simultaneous protonation of two of the COOH-terminal carboxylates in pentameric EtxB was required to cause its disassembly at pH values below 2.0 (Ruddock, L., Ruston, S. P., Kelly, S. M., Price, N. C., Freedman, R. B., and Hirst, T. R.(1995) J. Biol. Chem. 270, 29953-29958). Here, we investigate the influence of environmental parameters on the kinetics of reassembly of acid-generated EtxB monomers in vitro. Such monomers were found to undergo a further acid-mediated conformational change, with an activation energy of 76 +/- 2 J.mol-1.K-1, consistent with isomerization of the cis-proline residue at position 93, and which prevented subsequent EtxB reassembly. By using rapid neutralization of acid-generated monomers, a high proportion of the B-subunits adopted an assembly-competent conformation, which resulted in up to 75% of the protein reassembling into a stable pentameric complex, indistinguishable from native EtxB pentamers. The rate-limiting step in reassembly, over a concentration range of 50-200 microg/ml, was shown to be due to an intramolecular event, which exhibited a pH dependence with a pKa of 7.0. Modification of EtxB with amine-specific probes revealed that the protonation state of the NH2-terminal alanine residue was responsible for the pH dependence of reassembly. The implications of these findings for the biogenesis of Escherichia coli enterotoxin and related enterotoxins in vivo, are considered.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

The non-covalently associated B-subunit moieties of AB(5) toxins, such as cholera toxin and related diarrheagenic enterotoxins, exhibit exceptional pH stability and remain pentameric at pH values as low as 2.0. Here, we investigate the structural basis of a pH-dependent conformational change which occurs within the B-5 structure of Escherichia coli heat-labile enterotoxin (EtxB) at around pH 5.0. The use of far-UV CD and fluorescence spectroscopy showed that EtxB pentamers undergo a fully reversible pH-dependent conformational change with a pK(a) of 4.9 +/- 0.1 (R(2) = 0.999) or 5.13 +/- 0.01 (R(2) = 0.999), respectively, This renders the pentamer susceptible to SDS-mediated disassembly and decreases its thermal stability by 18 degrees C. A comparison of the pH-dependence of the structural change in EtxB(5), with that of a mutant containing a Ser substitution at His 57, revealed that the pK(a) of the conformational change was shifted from ca. 5.1 to 4.4. This finding suggests that protonation of the imidazole side chain of His 57 might facilitate disruption of a spatially adjacent salt bridge, located between Glu 51 and Lys 91 in each B-subunit, thus triggering the conformational change in the pentameric structure. The pH-dependent conformational change was found to be inhibited when B-subunits bound to monosialoganglioside, G(M1); and to have no effect on the stability of interaction between A- and B-subunits within the AB(5) complex. This suggests that the conformational change is unlikely to have a direct involvement in toxicity. Conservation of the pH-dependent conformational change in the AB(5) toxin family, combined with the potential exposure of the hydrophobic core of beta-barrel in the monomeric units, leads to the proposal that the conformational change may be the common feature that ensures the secretion of these proteins from the Vibrionaceae.

Gangliosides are glycosphingolipids found ubiquitously on the surface of mammalian cells. They contain a ceramide tail that is inserted into the membrane and exposed carbohydrate and sialic acid moieties. The non-toxic B subunit oligomer (EtxB) of Escherichia coli heat-labile enterotoxin (Etx) is a potent immunogen in vivo and has profound modulatory effects on EtxB-primed lymphocytes in vitro, properties which are dependent on its ability to bind to GM1 ganglioside receptors. Here, it is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin. Addition of EtxB to such cultures led to the complete depletion of CD8(+) T cells compared with enhanced activation of CD4(+) cells [as measured by expression of CD25 (IL-2Ralpha)]. By contrast, addition of a mutant EtxB, EtxB(G33D), which does not bind to GM1, failed to trigger CD8(+) T cell depletion. When EtxB was added to isolated non-immune CD8(+) lymphocytes rapid (12-18 h) alterations in nuclear morphology and the appearance of sub-G0/G1 levels of DNA were induced; properties which are characteristic of cells undergoing apoptosis. EtxB(G33D) failed to trigger apoptosis, indicating that the induction of the apoptotic signal was dependent on the binding of GM1. These findings provide an insight into the potent immunogenicity and immunomodulatory properties of E. coli enterotoxins as well as heralding a novel method for the selective induction of apoptosis in mature CD8(+) T lymphocytes.

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is highly stable, maintaining its quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. In this paper the structural stability of EtxB has been studied as a function of pH by electrophoretic, immunochemical, and spectroscopic techniques. Disassembly of the cyclic pentameric structure of human EtxB occurs only below pH 2. As determined by changes in intrinsic fluorescence this process follows first-order kinetics, with the rate constant for disassembly being proportional to the square of the H ion concentration, and with an activation energy of 155 kJ mol. A C-terminal deletion mutant, hEtxB214, similarly shows first-order kinetics for disassembly but with a higher pH threshold, resulting in disassembly being seen at pH 3.4 and below. These findings are consistent with the rate-limiting step for disassembly of human EtxB being the simultaneous disruption of two interfaces by protonation of two C-terminal carboxylates. By comparison, disassembly of the B-subunit of cholera toxin (CtxB), a protein which shows 80% sequence identity with EtxB, exhibits a much lower stability to acid conditions; with disassembly of CtxB occurring below pH 3.9, with an activation energy of 81 kJ mol. Reasons for the observed differences in acid stability are discussed, and the implications of these findings to the development of oral vaccines using EtxB and CtxB are considered.

Cholera toxin and the related Escherichia coli heat-labile enterotoxin are hexameric proteins comprising one A-subunit and five B-subunits. In this paper we report the generation and characterization of a monoclonal antibody, designated LDS47, that recognizes and precipitates in vivo assembly intermediates of the B-subunit (EtxB) of E. coli heat-labile enterotoxin. The monoclonal antibody is unable to precipitate native B-subunit pentamers, thus making LDS47 a useful probe for studying the early stages of enterotoxin biogenesis. The use of LDS47 to monitor the in vivo turnover of newly synthesized B-subunits in the periplasm of E. coli demonstrated that (i) the turnover of unassembled B-subunits followed an apparent first order process and (ii) it occurred concomitantly with the assembly of native B-pentamers (k = 0.317 +/- 0.170 min-1; t1/2 = 2.2 min). No other proteins were co-precipitated with the newly synthesized B-subunits; a finding that implies that unassembled B-subunits do not stably associate with other periplasmic proteins prior to their assembly into a macromolecular complex. The use of overlapping synthetic peptides corresponding to the entire EtxB polypeptide demonstrated that the epitope recognized by LDS47 is located within the amino-terminal decapeptide of the B-subunit. From the x-ray structural analysis of the toxin (Sixma, T., Kalk, K., van Zanten, B., Dauter, Z., Kingma, J., Witholt, B., and Hol, W. G. J. (1993) J. Mol. Biol. 230, 890-918), this region appears to resemble a curved finger that clasps the adjacent B-subunit. Thus, this region might be expected to be exposed in the unfolded or unassembled subunit, but to become partially buried upon assembly and thus inaccessible to recognition by the monoclonal antibody.

The immunoregulatory role of H-2 and intra-H-2 alleles on antibody responses to recombinant preparations of B-subunits of Escherichia coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB) is reported. Oral delivery of rEtxB to congenic mice of several different H-2 haplotypes resulted in H-2 dependent serum IgG responses (H-2(d) > H-2(b) = H-2(q) > H-2(a) > H-2(k)) and a similar spectrum of intestinal IgA responses in those strains tested Responses to rEtxB and rCtxB were found to be differentially modulated by the H-2 locus, with significant differential effects in H-2(b) and H-2(d) congenic strains (H-2(d) > H-2(b) for rEtxB; H-2(b) > H-2(d) for rCtxB), Additionally, it was found that when rEtxB was fed to mice previously primed (orally) with either rEtxB or rCtxB only those mice primed with rEtxB exhibited a booster response. A second booster immunisation with rEtxB in rCtxB-primed mice produced an H-2 dependent spectrum of responses characteristic of those elicited by rEtxB, with the antibodies predominantly directed against rEtxB and not rCtxB. These results indicate that the differential response to rEtxB and rCtxB is set at the T- and B-cell level. Also, immunoregulation of antibody responses to rEtxB by intra-H-2 I-E in mice transgenic for the entire IE(a)(k) gene was investigated. No significant difference between responses in transgene-positive and -negative mice was found, suggesting that antigen presentation does not involve I-E, but occurs in the context of I-A. The implications of these results for the design of vaccines against enterotoxigenic E. coli and cholera diarrhoea are discussed.

With the elucidation of the properties and modes of action of a wide range of bacterial protein toxins has come the realisation that with appropriate adaptation, these molecules can be used to deliver other moieties to the immune system and thereby enhance immunogenicity. Given the diversity of bacterial toxins as a group of proteins, this objective can be contemplated from several perspectives. Many toxins possess the ability to bind to receptors at the surface of mammalian cells. Depending upon the fate of the toxin-receptor complex, this may provide a route for enhancing the uptake of antigen into endocytic pathways which ultimately result in presentation to the immune system. The ability of staphylococcal enterotoxins to interact with MHC class II may allow targeting of attached sequences specifically to those cells capable of processing and presenting antigen. As it becomes clearer which vesicular trafficking pathways are followed by those bacterial toxins which are taken up by cells, it may be possible to manipulate events such that attached materials are released specifically in those cellular compartments where antigen processing and/or the loading of MHC class II molecules with peptides occurs with greatest efficiency. Since several toxins have the ability to translocate from acidic endosomal or lysosomal compartments to the cell cytoplasm, this or the ability to traffic to the endoplasmic reticulum, may offer a mechanism with which to stimulate MHC class I-restricted responses against defined moieties borne by the toxin.

Mimetic peptides capable of selectively disrupting protein-protein interactions represent potential therapeutic agents for inhibition of viral and cellular enzymes. This approach was first suggested by the observation that the peptide YAGAVVNDL, corresponding to the carboxyl-terminal 9 amino acids of the small subunit of ribonucleotide reductase of herpes simplex virus, specifically inhibited the viral enzyme in vitro. Evaluation and use of this peptide as a potential antiviral agent has, however, been thwarted by its failure to inhibit virus replication in vivo, presumably because the peptide is too large to enter eukaryotic cells unaided. Here, we show that the nontoxic B subunit of Escherichia coli heat-labile enterotoxin can be used as a recombinant carrier for the receptor-mediated delivery of YAGAVVNDL into virally infected cells. The resultant fusion protein specifically inhibited herpes simplex virus type 1 replication and ribonucleotide reductase activity in quiescent Vero cells. Preincubation of the fusion protein with soluble GM1 ganglioside abolished this antiviral effect, indicating that receptor-mediated binding to the target cell is necessary for its activity. This provides direct evidence of the usefulness of carrier-mediated delivery to evaluate the intracellular efficacy of a putative antiviral peptide.

Heat-labile enterotoxins (Etx) are plasmid-encoded, multimeric proteins produced by certain diarrheagenic strains of Escherichia coli. The nontoxic, receptor-binding B subunit (EtxB) of such toxins may be useful as a component of vaccines against enterotoxigenic E. coli, or as a carrier for the delivery of heterologous epitopes to the mucosal immune system. Here we describe a simple method for the purification of EtxB from a marine vibrio harboring a broad-host range controlled expression vector containing the etxB gene. Induction of EtxB resulted in its specific secretion to the medium, to a concentration of greater than 25 mg/liter of culture. The techniques of ultrafiltration and hydrophobic interaction chromatography were used to purify EtxB to homogeneity from the medium of this organism (with a yield of 60.7%). EtxB-epitope fusion proteins were also successfully expressed and secreted in this marine vibrio, suggesting that this system may be of general use in the preparation of EtxB-based vaccines.

Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15-20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies.

Recently, a gene (dsbA) involved in the biogenesis of secreted oligomeric enterotoxins in Vibrio cholerae was described, which encodes an exported protein possessing a -Cys-Pro-His-Cys- motif similar to that found in the active sites of eukaryotic and prokaryotic thiol-disulfide oxidoreductases (Yu, J., Webb, H., and Hirst, T. R (1992) Mol. Microbiol. 6, 1949-1958). Here, we report the cloning of the dsbA gene of V. cholerae and the demonstration that the encoded periphlasmic enzyme has disulfide isomerase-like activity. Oligonucleotide-directed mutagenesis of either of the 2 Cys residues to Ala in the putative active site of DsbA abolished both its isomerase activity and its capacity to promote enterotoxin biogenesis. We conclude that the Cys residues constitute the active site domain of DsbA and are essential for its activity in vivo and in vitro.

Cholera toxin and the related heat-labile enterotoxin (LT) produced by Escherichia coli consist of a holotoxin of one A subunit and five B subunits (AB5). Here we investigate the domains of the A subunit (EtxA) of E. coli LT which influence the events of B-subunit (EtxB) oligomerization and the formation of a stable AB5 holotoxin complex. We show that the C-terminal 14 amino acids of the A subunit comprise two functional domains that differentially affect oligomerization and holotoxin stability. Deletion of the last 14 amino acids (-14) from the A subunit resulted in a molecule that was significantly impaired in its capacity to promote the assembly of a mutant B subunit, EtxB191.5. In contrast, deletion of the last four amino acids (-4) from the A subunit gave a molecule that retained such a capacity. This suggests that C-terminal residues within the -14 to -4 region of the A subunit are important for promoting the oligomerization of EtxB. In addition, we demonstrate that the truncated A subunit lacking the last 4 amino acids was unable to form a stable AB5 holotoxin complex even though it promoted B-subunit oligomerization. This suggests that the last 4 residues of the A subunit function as an "anchoring" sequence responsible for maintaining the stability of A/B subunit interaction during holotoxin assembly. These data represent an important example of how intermolecular interactions between polypeptides in vivo can modulate the folding and assembly of a macromolecular complex.

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.

Minimal alterations at the carboxyl terminus of the B subunit (EtxB) of heat-labile enterotoxin from Escherichia coli were found to have a marked effect on the assembly and release of this polypeptide into the periplasm. Nine mutant EtxB polypeptides were obtained by genetic manipulation of the 3'-end of the etxB gene using Bal31 nuclease digestion and codon substitution. A correlation was observed between the magnitude of the changes introduced at the carboxyl terminus and the extent to which the mutant polypeptides were defective in assembly and release. Some of the mutant B subunits, exemplified by those in which the last 2 amino acids had been deleted or in which the last 4 residues had been replaced by three different ones, were found to be only partially defective, with a proportion being associated with the periplasmic face of the cytoplasmic membrane and the remainder being exported to the periplasm. The portion associated with membranes was detected as monomers on sodium dodecyl sulfate-polyacrylamide gels, whereas the portion exported to the periplasm were detected as assembled oligomers. We conclude that the last few amino acids at the carboxyl terminus of EtxB exert a profound influence on the assembly and release of the B subunit from the cytoplasmic membrane during export in E. coli.

This chapter focuses on the remarkable life of Cholera toxin (Ctx) from Vibrio cholerae, and subsequently the closely related heat-labile enterotoxins (Etx) from certain toxigenic strains of Escherichia col , From the genetics of toxin expression, assembly, and secretion in the bacteria, to their interaction, uptake, and action in disease, all of this is discussed. Purification and structural analysis of cholera toxin (Ctx) from sterile culture filtrates of V. cholerae was first attempted in 1966, but it was not until the successful crystallization and X-ray analyses of Ctx almost 30 years later that the toxin arrangement of six non-covalently associated subunits was confirmed. Ctx is now well-characterized as a hexameric protein toxin comprised of a single A-subunit arranged on a toroidal ring of five B-subunits. While much is now known about the biogenesis of these toxins, the precise molecular basis by which the toxins are translocated across the bacterial outer membrane remains a challenging area for future investigation. The increasing insights being gained into the uptake and trafficking of the molecules in mammalian cells is also shedding light on aspects of eukaryotic cell biology. These toxins will therefore remain excellent models for investigating important cellular processes in both prokaryotic and eukaryotic systems.

Escherichia coli heat-labile enterotoxin (Etx) is composed of a single catalytically active A subunit (EtxA) and a pentameric B subunit (EtxB) that mediates receptor interaction and uptake of the toxin by target cells (34). In addition to the well-documented deleterious effects of the toxin, a wealth of data is accumulating suggesting that Etx can be used to modulate immune responses in a variety of beneficial ways (45). In this regard, Etx is a highly potent adjuvant and has been used in a large number of experimental, mucosal vaccines. Importantly, the toxicity of the holotoxin does not appear to be a prerequisite for this activity, and detoxified and B-subunit preparations of Etx retain the ability to act as adjuvants (8, 14, 29), at least when intranasal routes are used. Furthermore, recombinant preparations of EtxB act as immunomodulatory proteins capable of suppressing Th1-associated inflammatory processes in vivo (45). These findings have led to investigations of the ability of EtxB and the related cholera toxin B subunit, CtxB, to prevent and treat autoimmune processes in animal models of disease (23, 35, 36, 46). In an attempt to understand the in vivo immunomodulatory properties of the cholera-like enterotoxins, a number of studies have documented the effects of Etx and Ctx and their subunits on immune cell function. Both CtxB and EtxB induce the activation of murine B cells in vitro, leading to enhancement of major histocompatibility complex class II and costimulatory molecule expression (12, 27). EtxB stimulates monocyte interleukin-10 (IL-10) production, while it inhibits IL-12 production (41); these effects are also mediated by Ctx but not by CtxB (3, 6). In contrast to these stimulatory properties, both EtxB and CtxB induce apoptosis of murine CD8+ T cells (25, 26, 32, 40, 48). Additional studies indicate that injection of CtxB into mice results in depletion of CD8+ T cells from Peyer's patches, suggesting that these effects may also occur in vivo (9). The immunomodulatory activity of EtxB is dependent on binding to cell surface receptors, as evidenced by a failure of a non-receptor-binding mutant, EtxB(G33D), to induce any of the events described above. The principal receptor for both EtxB and CtxB is the ubiquitous ganglioside GM1 [Gal(β1-3)GalNAc(β1-4)NeuAc(α2-3)Gal(β1-4)Glc(β1-1)ceramide] (45). Both B subunits also bind ganglioside GD1b, while EtxB interacts with a number of additional receptors and galactoproteins for which CtxB has no affinity. Ganglioside GM1 is a principal constituent of glycolipid membrane rafts (4, 16, 21). We have postulated that receptor interaction with EtxB alters raft-associated intracellular signaling pathways and consequently leads to the activation, differentiation, or death of target cells. In this regard, it has recently been reported that EtxB-mediated apoptosis of CD8+ T cells results from receptor-dependent activation of NF-κB and c-Myc (31, 33). These events cause activation of caspases within the cells and proceed in a Fas- and p55 tumor necrosis factor receptor-independent pathway. Conversely, the activation of B cells by EtxB occurs as a result of phosphoinositide 3-kinase- and protein kinase C-dependent Erk mitogen-activated protein kinase activation (2). Data from a number of groups have suggested that inhibition of caspase activity during apoptosis may be insufficient to prevent cell death. Inhibition of caspases following Fas signaling leads to the induction of necrotic, rather than apoptotic, cell death (24, 43). Similarly, tumor necrosis factor-induced necrosis of L929 cells is enhanced in the presence of caspase inhibitors (42). Furthermore, T-cell death induced by anti-CD2 antibodies or staurosporine does not require caspase activity (7). Data from previous studies indicated that caspase activity was required for the induction of DNA fragmentation during EtxB-induced apoptosis of CD8+ T cells (31, 32). However, it is not clear whether the activation of caspases via NF-κB is the only signal triggered by EtxB in CD8+ T cells and whether the fate of the cells is dependent upon this.

The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.

PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB). Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies. Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay. Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic. Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA. The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic. Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects. These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA. Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.

Receptor-binding by the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) induces apoptosis of peripheral CD8 + , but not CD4 + T-cells. Given that peripheral CD8 + and CD4 + T cells arise from a common developmental pathway in the thymus, we investigated the effects of EtxB on different thymocyte populations. We show that the acquisition of sensitivity to EtxB-mediated cell death arises following transition of CD4 + CD8 + double positive cells into the CD4 − CD8 + pathway. Maturation of T cells into CD4 − CD8 + single positive cells is associated with upregulated expression of receptors for EtxB.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule capable of treating and preventing autoimmune disease. These properties result from its ability to bind to glycolipid receptors, principally G(M1) ganglioside, and modulate immune cell function. EtxB receptor binding causes B cell activation, modulates monocyte cytokine secretion and triggers apoptosis of CD8+ T cells. These wide-ranging effects suggest that B subunit receptor interaction triggers signaling events affecting cellular differentiation. We have investigated the processes by which EtxB induces CD8+ T cell apoptosis. We show that receptor interaction by EtxB activates caspase-3 in CD8+ but not in CD4+ T cells. Inhibition of caspase-3 blocks the apoptotic process. EtxB induces the activation of NF-kappaB in both CD8+ and CD4+ T cells. The findings that (i) SN50, a peptide inhibitor of NF-kappaB nuclear translocation, prevents caspase-3 activation and subsequent apoptosis, and (ii) CD8+CD4- thymocytes from transgenic mice expressing a dominant-negative form of the IkappaBalpha protein were markedly less susceptible to EtxB-induced apoptosis than cells from wild-type mice, indicate that NF-kappaB is important in the induction of the apoptotic pathway. Further investigations revealed that while caspase-8 activity is detected concomitant to caspase-3, caspase-9 activation, following mitochondrial cytochrome c release, is detectable later on. These observations are consistent with death receptor-mediated signaling, however, experiments using lpr/lpr and p55 TNFR -/- mice rule out the involvement of Fas and the p55 TNF receptor, respectively. The data therefore indicate that EtxB-mediated apoptosis occurs via a novel pathway involving NF-kappaB.