Improved methods for prepurification and detection of staphylococcal enterotoxin B from cell-free culture filtrate

An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.     

Suspension cultivation of Mycobacterium ulcerans for the production of mycolactones

Mycolactones are polyketide toxins produced by Mycobacterium ulcerans, the causative agent of the tropical skin disease known as Buruli ulcer. Development of novel therapeutic agents from mycolactones has been hindered by the difficulty of producing sufficient amounts of material. Here, we describe the successful adaptation of M. ulcerans to suspension cultivation and the development of a fed-batch fermentation process that was scaled up to 150 l. In addition to producing mycolactones A and B, a number of new mycolactone-related compounds were also observed.     

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.     

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.     

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5' end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.     

Development of a subunit vaccine for prevention of Clostridium difficile associated diseases: Biophysical characterization of toxoids A and B

Inactivation of bacterial toxins for use in human vaccines traditionally is achieved by treatment with formaldehyde. In contrast, the bivalent experimental vaccine for the prevention of C. difficile infections (CDI) that is currently being evaluated in clinical trials was produced using a different strategy. C. difficile toxins A and B were inactivated using site-directed mutagenesis and treatment with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS). In the present work we investigate the effect of genetic and chemical modifications on the structure of inactivated toxins (toxoids) A and B. The far-UV circular dichroism (CD) spectra of wild type toxins, mutated toxins, and EDC/NHS-inactivated toxoids reveal that the secondary structure of all proteins is very similar. The near-UV CD spectra show that aromatic residues of all proteins are in a unique asymmetric environment, indicative of well-defined tertiary structure. These results along with the fluorescence emission maxima of 335 nm observed for all proteins suggest that the tertiary structure of toxoids A and B is preserved as well. Analytical ultracentrifugation data demonstrate that all proteins are predominantly monomeric with small fractions of higher molecular weight oligomeric species present in toxoids A and B. Differential scanning calorimetry data reveal that genetic mutations induce thermal destabilization of protein structures. Subsequent treatment with EDC/NHS results either in a minimal (1 °C) increase of apparent thermostability (toxoid B) or no change at all (toxoid A). Therefore, our two-step inactivation strategy is an effective approach for the preparation of non-toxic proteins maintaining native-like structure and conformation.     

Pharmacological receptors for substance P and neurokinins

The three neurokinins identified in mammals, substance P, neurokinin A and neurokinin B, as well as their C-terminal biologically active fragments, have been used to characterize the responses of a variety of isolated organs. Three preparations selective either for substance P (the dog carotid artery), or for neurokinin A (the rabbit pulmonary artery) or for neurokinin B (the rat portal vein) are described. A neurokinin receptor classification is attempted using the neurokinins and their fragments to determine the order of potency of agonists. Three receptor subtypes have been identified: the NK-P, on which substance P (SP) is more active than neurokinin A (NKA) and neurokinin B (NKB), and the neurokinins are more active than their respective fragments; the NK-A on which NKA greater than NKB greater than SP, and some NKA fragments are more discriminative than their precursor; the NK-B on which NKB greater than NKA greater than SP, and fragments of NKB are less active than their precursor. Among the peptides studied, some potent compounds have been identified that could provide selective receptor ligands.     

Bacillus anthracis interacts with plasmin(ogen) to evade C3b-dependent innate immunity

The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.     

Comparative proteome analysis of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> with pXO1 plasmid content

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4–7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.     

Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B

Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.     

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.     

Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1

Botulinum neurotoxins (BoNTs), the causative agent of human botulism, are the most potent naturally occurring toxins known. BoNT/A1, the most studied BoNT, is also used as an important biopharmaceutical. In this study, the biological activity of BoNT/A1 is compared to that of BoNT/A2 using neuronal cell models. The data obtained indicate faster and increased intoxication of neuronal cells by BoNT/A2 than BoNT/A1, and that the mechanism underlying this increased toxicity is faster and more efficient cell entry that is independent of ganglioside binding. These results have important implications for the development of new BoNT based therapeutics and BoNT countermeasures.     

Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function

Clostridium difficile causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea. Major virulence factors of C. difficile are the glucosylating exotoxins A and B. Both toxins enter target cells in a pH- dependent manner from endosomes by forming pores. They translocate the N-terminal catalytic domains into the cytosol of host cells and inactivate Rho guanosine triphosphatases by glucosylation. The crystal structure of the catalytic domain of toxin B was solved in a complex with uridine diphosphate, glucose, and manganese ion, exhibiting a folding of type A family glycosyltransferases. Crystallization of fragments of the C-terminus of toxin A, which is characterized by polypeptide repeats, revealed a solenoid-like structure often found in bacterial cell surface proteins. These studies, which provide new insights into structure, uptake, and function of the family of clostridial glucosylating toxins, are reviewed.     

Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis

Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.     

The virulence factors of Bordetella pertussis: a matter of control

Bordetella pertussis is the causative agent of whooping cough, a contagious childhood respiratory disease. Increasing public concern over the safety of whole-cell vaccines led to decreased immunisation rates and a subsequent increase in the incidence of the disease. Research into the development of safer, more efficacious, less reactogenic vaccine preparations was concentrated on the production and purification of detoxified B. pertussis virulence factors. These virulence factors include adhesins such as filamentous haemagglutinin, fimbriae and pertactin, which allow B. pertussis to bind to ciliated epithelial cells in the upper respiratory tract. Once attachment is initiated, toxins produced by the bacterium enable colonisation to proceed by interfering with host clearance mechanisms. B. pertussis co-ordinately regulates the expression of virulence factors via the Bordetella virulence gene (bvg) locus, which encodes a response regulator responsible for signal-mediated activation and repression. This strict regulation mechanism allows the bacterium to express different gene subsets in different environmental niches within the host, according to the stage of disease progression.     

Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.     

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.     

M protein of a Streptococcus dysgalactiae human wound isolate shows multiple binding to different plasma proteins and shares epitopes with keratin and human cartilage

Besides group A (GAS), Lancefield group C beta-haemolytic streptococci (GCS) have been implicated as a causative agent in outbreaks of purulent pharyngitis. In this study we have investigated a class CI M protein of a Streptococcus dysgalactiae1:256, revealed that 26% of these sera showed serological cross-reactivity between a 68-kDa cartilage protein and the N-terminal part of MC. Only 8% of the sera of healthy patients showed this property. In additional, MC also cross-reacted with antibodies recognising epidermal keratins. The cross-reacting 68-kDa protein from cartilage was different from human serum albumin, but was recognised with anti-vimentin immune serum. The MC was cloned and the gene sequenced. By using PCR, recombinant gene fragments encoding characteristic peptide fragments of MC were expressed in Escherichia coli. The peptides were used to map the binding sites for plasma proteins and to locate the cross-reacting epitopes on the MC molecule. In consequence, sequence alignments revealed that MC shared homologous regions with vimentin and different keratins. Our data, obtained with MC, suggest that not only infections with GAS but also infections with GCS and possibly GGS (the latter species can also produce class CI M-like proteins) may be responsible for the formation of streptococcal-associated sequel diseases.     

Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state

There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.     

Purification of bacterial exotoxins. The case of botulinum, tetanus, anthrax, pertussis and cholera toxins.

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.