The crystal structure of pertussis toxin.

BACKGROUND: Pertussis toxin is an exotoxin of the A-B class produced by Bordetella pertussis. The holotoxin comprises 952 residues forming six subunits (five different sequences, S1-S5). It plays an important role in the development of protective immunity to whooping cough, and is an essential component of new acellular vaccines. It is also widely used as a biochemical tool to ADP-ribosylate GTP-binding proteins in the study of signal transduction. RESULTS: The crystal structure of pertussis toxin has been determined at 2.9 A resolution. The catalytic A-subunit (S1) shares structural homology with other ADP-ribosylating bacterial toxins, although differences in the carboxy-terminal portion explain its unique activation mechanism. Despite its heterogeneous subunit composition, the structure of the cell-binding B-oligomer (S2, S3, two copies of S4, and S5) resembles the symmetrical B-pentamers of the cholera toxin and Shiga toxin families, but it interacts differently with the A-subunit. The structural similarity is all the more surprising given that there is almost no sequence homology between B-subunits of the different toxins. Two peripheral domains that are unique to the pertussis toxin B-oligomer show unexpected structural homology with a calcium-dependent eukaryotic lectin, and reveal possible receptor-binding sites. CONCLUSION: The structure provides insight into the pathogenic mechanisms of pertussis toxin and the evolution of bacterial toxins. Knowledge of the tertiary structure of the active site forms a rational basis for elimination of catalytic activity in recombinant molecules for vaccine use.

A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity.

BACKGROUND: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). RESULTS: To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). CONCLUSIONS: These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family.

Glycan mimicry as a basis for novel anti-infective drugs.

The idea of using carbohydrate-based drugs to prevent attachment of microbial pathogens to host tissues has been around for about three decades. This concept evolved from the observation that many pathogenic microbes bind to complex carbohydrate sequences on the surface of host cells. It stands to reason, therefore, that analogs of the carbohydrate sequences pathogens bind to could be used to competitively inhibit these interactions, thereby preventing microbial damage to the host. This article will summarize some of the recent advances in developing such carbohydrate-based anti-infective drugs.

The use of lectin affinity columns for selection of precursor or fully glycosylated forms of glycoprotein gD1 of herpes simplex virus type 1.

Several lectins were examined for their ability to bind to the glycoprotein gD1 polypeptide from Vero cells infected by herpes simplex virus type 1 (HVS-1), strain KOS. At least four distinct forms of gD1 (1, 2, 3 and 4), ranging in size from 59K to 52K, were resolved by SDS-10% polyacrylamide gel electrophoresis. Wheat germ agglutinin (WGA) did not bind to any of these forms, suggesting that if any sialic residues are present in the carbohydrate moieties of gD1, they are not available for binding to WGA. The entire population of forms 1 and 2 (approximately 59K) bound to castor bean-120 (CB-120) lectin affinity columns, suggesting the presence of terminal galactose residues on the mature and more fully glycosylated carbohydrate moieties of gD1. The forms 3 and 4, representing precursor gD1 molecules, did not bind. The majority of forms 2 and 4, and a portion of form 3 bound to lentil lectin, suggesting the presence of fucose and alpha-linked mannosyl residues on these molecules. A gD1-specific, high molecular weight species (120-125K) was detected in the lentil lectin-binding fraction but not in the fraction bound to CB-120 lectin or in the original infected-cell extract. The results indicated that lectin affinity chromatography, using lentil and CB-120 lectins, is useful as an initial step for the selection and purification of the individual glycosylated forms of gD1.

Towards integrated hygiene and food safety management systems: the Hygieneomic approach.

Integrated hygiene and food safety management systems in food production can give rise to exceptional improvements in food safety performance, but require high level commitment and full functional involvement. A new approach, named hygieneomics, has been developed to assist management in their introduction of hygiene and food safety systems. For an effective introduction, the management systems must be designed to fit with the current generational state of an organisation. There are, broadly speaking, four generational states of an organisation in their approach to food safety. They comprise: (i) rules setting; (ii) ensuring compliance; (iii) individual commitment; (iv) interdependent action. In order to set up an effective integrated hygiene and food safety management system a number of key managerial requirements are necessary. The most important ones are: (a) management systems must integrate the activities of key functions from research and development through to supply chain and all functions need to be involved; (b) there is a critical role for the senior executive, in communicating policy and standards; (c) responsibilities must be clearly defined, and it should be clear that food safety is a line management responsibility not to be delegated to technical or quality personnel; (d) a thorough and effective multi-level audit approach is necessary; (e) key activities in the system are HACCP and risk management, but it is stressed that these are ongoing management activities, not once-off paper generating exercises; and (f) executive management board level review is necessary of audit results, measurements, status and business benefits.

Therapeutic potential of egg yolk antibodies for treating Clostridium difficile infection.

Herein we present evidence for the therapeutic potential of colonization factor (CF)-specific egg yolk antibodies (IgY) for potentially treating acute and recurring Clostridium difficile infection (CDI) in humans. The study involved cloning, expressing as 6×His-tagged proteins in Escherichia coli, and Ni-affinity purifying three previously identified CFs (FliC, FliD and Cwp84) from C. difficile. The recombinant CF antigens were then used to immunize Leghorn chickens and CF-specific IgY antibodies were prepared from their eggs. The specificity and titre of the resulting C. difficile CF-specific IgY antibodies were assessed by ELISA and Western immunoblotting techniques. The antibodies were also screened for their ability to inhibit C. difficile adherence to human colon-derived T84 cells, and, based on these findings, one of them (FliD-specific IgY) was evaluated for its potential to prevent C. difficile-mediated morbidity and mortality in Syrian hamsters. The results revealed that purified FliD-specific IgY significantly protected hamsters from C. difficile strain 630 infection relative to control animals treated with carbonate buffer alone or IgY produced from unimmunized chicken eggs. The results suggest that egg yolk preparations obtained from chickens immunized with recombinant C. difficile CFs may represent another safe and cost-effective treatment option in humans suffering from acute or recurring CDI.

Carbohydrate receptor mimicry as a basis for antibacterial therapy.

In the world of microbial pathogens, successful colonization of mucosal surfaces frequently involves initial binding interactions between proteinaceous attachment factors, called adhesins, lectins or hemagglutinins, expressed on the surface of the microorganism and complex carbohydrate sequences, or receptors, on host epithelial cell membranes. Many bacterial exotoxins also employ a strategy of lectin-like binding to host cell carbohydrate receptors in order to cause their pathophysiological effects. It is these specific lectin-carbohydrate interactions that often determine the species or tissue tropism for a particular pathogen. Conceptually, therefore, it should be possible to use analogs of carbohydrate receptors to competitively inhibit microbial colonization of host tissues and this hypothesis is the basis for carbohydrate-based anti-infective drugs. This review will summarize some of the past and present thinking related to the feasibility, problems and potential benefits of such novel carbohydrate-based agents.

Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining.

We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili. The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques.

Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose.

We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigations into the nature of the interaction and activity of PT in host tissues.

Interactions between Campylobacter jejuni and lipids.

We previously showed that motility plays several key roles in Campylobacter jejuni pathogenesis, including increasing the efficiency of C. jejuni attachment to host epithelial cells. To further characterize C. jejuni attachment, we first examined the role of carbohydrates. Experiments with Chinese hamster ovary (CHO) cell mutants with defined defects in complex carbohydrate biosynthesis revealed that oligosaccharide sequences probably play a subordinate role in C. jejuni attachment to eukaryotic cells. Simple sugars such as mannose, fucose, glucose, N-acetylglucosamine, maltose, and galactose also did not significantly alter the binding of C. jejuni to CHO cells. Thin-layer chromatography overlay analysis with lipids extracted from CHO cells suggested that C. jejuni binds to lipids. Lipid binding was further investigated using a receptor-based enzyme-linked immunosorbent assay. Hydrophobic interactions were determined to play a minor role in binding, since tetramethylurea, a strong inhibitor of hydrophobic interactions, did not significantly decrease binding between C. jejuni and lipids. The interaction was dissected further by comparing the binding of C. jejuni to lipids and their derivatives. The results showed that binding was greatest to the entire lipid structure and decreased in affinity when portions of the lipid were removed. Thin-layer chromatography overlay analysis showed that lipids with unsaturated fatty acids were bound with the highest affinity. Our results suggest that C. jejuni may interact with lipids in host cell membranes. However, lipids only partially inhibited C. jejuni binding to CHO cells, suggesting that multiple interactions occur between the bacteria and host cells.

Comparison of the lectin-like activity of pertussis toxin with two plant lectins that have differential specificities for alpha (2-6) and alpha (2-3)-linked sialic acid.

In this report we have compared the lectin-like properties of Pertussis toxin with two plant lectins which are known to possess different specificities towards terminal Neu5Ac Gal linkages on glycoconjugates. The hemagglutinin from elderberry bark (Sambucus nigra) has a binding specificity for terminal Neu5Ac alpha (2-6) Gal sequences and was found to bind a series of glycoconjugates with a similar specificity as Pertussis toxin. The binding specificity of Pertussis toxin was different from that of the leukoagglutinin from the seeds of Maackia amurensis which preferentially binds terminal Neu5Ac alpha (2-3) Gal sequences. These observations confirm the specificity of Pertussis toxin for Neu5Ac alpha (2-6) Gal glycoconjugate sequences.

Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli.

A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolic repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce beta-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3',5-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.

Lymphocyte receptors for pertussis toxin.

We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

Effect of carbon source on localized adherence of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) strains attach to epithelial cells as discrete clusters of bacteria which are localized at a few sites on the cell surface. Previously, it was shown that this localized-adherence (LA) phenotype is induced by specific growth conditions. We found that wild-type EPEC attached to HEp-2 cells in an LA pattern when the bacteria were grown in Dulbecco's modified Eagle medium (DMEM) containing glucose as the carbon source. In contrast, bacteria incubated in DMEM containing galactose did not adhere to epithelial cells. The latter results were similar to those observed when JPN15, an LA-negative strain, was grown under conditions which promoted bacterial binding. The differences in attachment of wild-type EPEC were independent of the stage of log-phase growth of the cultures and of the number of CFU incubated with the HEp-2 monolayers. Expression of the adherence phenotype by organisms grown in glucose was associated with increased expression of intimin and bundle-forming pilin. In contrast, bacteria grown in medium containing galactose expressed these proteins at levels similar to those observed when JPN15 was grown in medium containing glucose.

Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells.

Enteropathogenic Escherichia coli (EPEC) O111:H2, O119:H6, or O142:H6 caused rapid detachment of Chinese hamster ovary (CHO) cell monolayers within 2 to 4 h of cocultivation. CHO cell detachment was not promoted by nonenteropathogenic E. coli (O125:H4, O126:H27, O157:H7, and O26:H11) and could not be attributed to EPEC production of enterohemolysin or Shiga-like toxins. In contrast, EPEC strains did not promote rapid detachment of Lec1, Lec2, or Lec8 CHO cell monolayers. These CHO cell Lec mutants all express abbreviated glycan sequences on membrane glycoproteins and glycolipids. Although EPEC strains failed to alter the adherent properties of Lec2 cells lacking only terminal sialic acid groups, EPEC adherence to the Lec2 mutant was indistinguishable from that observed with wild-type CHO cells. There was also no significant difference in EPEC-induced actin accumulation or invasion of Lec2 cells. In contrast, EPEC localized adherence to Lec1 and Lec8 mutants, lacking sialyllactosamine (Lec1) or sialic acid and galactose (Lec8) sequences, was reduced by 84 and 93%, respectively. Our results suggest that lactosamine sequences [beta Gal(1-4 or 1-3)beta GlcNAc] not containing sialic acid are sufficient for EPEC adherence, actin accumulation, and invasion of CHO cells. Sialic acid groups, however, may be necessary for EPEC-mediated CHO cell detachment.

Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin.

Synthetic peptides corresponding to selected sequences in the S2 and S3 subunits of pertussis toxin were prepared and evaluated for their ability to inhibit the binding of biotinylated pertussis toxin and three biotinylated sialic acid specific plant lectins to fetuin and asialofetuin. The screening results indicated that two regions in the S2 subunit corresponding to amino acids 78-98 and 123-154 inhibited pertussis toxin binding to fetuin at submillimolar concentrations, while S3 sequences corresponding to amino acids 87-108 and 134-154 inhibited pertussis toxin-biotin binding to asialofetuin albeit with lower affinity. These results confirm earlier findings, which suggest that the S2 subunit is responsible for binding sialylated glycoconjugates. This was further confirmed by the ability of S2 peptides to inhibit the binding of the lectins from Maackia amurensis and wheat germ to fetuin. Two additional peptides from the S2 subunit of pertussis toxin corresponding to sequences 9-23 and 1-23 were found to contain within their sequences a 6-amino acid fragment which has strong homology with a sequence in wheat germ agglutinin that has been shown to be a component of the sialic acid binding site as determined by x-ray crystallography. One of these sequences from S2 (9-23) was biotinylated and evaluated for its ability to bind to carbohydrate. Through a series of experiments using fetuin, asialofetuin, asialoagalactofetuin, and simple saccharides, the biotinylated peptide was shown to bind with high affinity to sialic acid-containing glycoconjugates indicating that these sequences within the S2 subunit of pertussis toxin also play an important role in binding sialic acid.

Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin.

Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.

The 70-kilodalton pertussis toxin-binding protein in Jurkat cells.

125I-ASD photoaffinity-labeling derivatives of pertussis toxin (125I-ASD-PT) or lipopolysaccharide (125I-ASD-LPS) labeled similar 70-kDa proteins in Jurkat cells, a cell line derived from human CD4+ T lymphocytes. Labeling of this 70-kDa protein by 125I-ASD-PT was inhibited by underivatized PT but not by underivatized LPS. However, an immunoglobulin M monoclonal antibody with specificity for the p73 LPS receptor in murine splenocytes (S. W. Bright, T.-Y. Chen, L. M. Flebbe, M.-G. Lei, and D. C. Morrison, J. Immunol. 145:1-7, 1990) inhibited 125I-ASD-PT labeling of the 70-kDa species in Jurkat cells. Our results suggested that PT may bind to the same 70-kDa protein as LPS does in Jurkat cells but that PT and LPS bind to different sites on this receptor candidate. 125I-ASD-PT photoaffinity labeling of the 70-kDa protein was also inhibited by underivatized glycoproteins to which PT has been shown to bind, and this inhibition correlated with the relative binding affinities of the glycoproteins for PT. 125I-ASD derivatives of two sialic acid-specific plant lectins, Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin, with oligosaccharide binding specificities similar to those of PT also labeled a 70-kDa protein in Jurkat cells. This suggests that the 70-kDa PT receptor candidate in Jurkat cells likely contains sialooligosaccharide sequences to which PT, M. amurensis leukoagglutinin, and S. nigra agglutinin bind. The cross-reacting epitope recognized by monoclonal antibody 5D3 in this 70-kDa species might overlap the PT- and LPS-binding sites.