Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8.

In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174-239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

[The research on construction and expression of eukaryotic expression plasmid for a recombinant immunotoxin mMIP-1alpha-DT390].

OBJECTIVE: To construct a novel recombinant immunotoxin (IT) expression vector by fusing mouse macrophage inflammatory protein-1alpha gene (mMIP-1alpha) into a truncated diphtheria toxin gene (DT390), and examine the expression of mMIP-1alpha-DT390 fusion protein in NIH3T3 cells. METHODS: mMIP-1alpha cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR), and inserted in the expression plasmid SRalpha, that includes the DT390 gene, to form the recombinant vector SRa-mMIP-1alpha-DT390. The positive recombinant plasmid was identified by PCR, the restriction endonucleases digestion and DNA sequencing, and then by liposome protocol, the identified positive plasmid was transferred into NIH3T3 cells for observing the fusion protein expression by immunofluorescence, with detecting the activities of the immunotoxin in vitro through MTT. RESULTS: We have successfully constructed a recombinant immunotoxin expression vector named as SRalpha-mMIP-1alpha-DT390. The immunofluorescence photograph sourced from fluorescence immunocytochemical method showed that the fusion gene could express in the cytomembrane and cytoplasm of NIH3T3 cells. In the bioactivity detection assay, the supernatant of the transfected cell culture was observed to have the obvious cytotoxic activity to activated T-lymphocyte. CONCLUSION: The SRalpha-mMIP-1alpha-DT390 recombinant immunotoxin expression vector will provide the basis of studying the targeted cytotoxic activity to inflammatory cells, and may have some potential value for clinical application.

[Development of monoclonal latex diagnostic kit for diphtheria toxin and toxoid detection].

Latex diagnostic kit with high specificity and sensitivity of diphtheria toxin and toxoid detection has been developed on the basis of protective monoclonal antibodies to diphtheria toxin and polyacrolein microspheres. Diagnostic kit was stable during 1 year of shelf-life (time of the study).

Genetic fusion of three tandem copies of murine C3d sequences to diphtheria toxin fragment B elicits a decreased fragment B-specific antibody response.

Previous studies have demonstrated that the covalent modification of target protein and polysaccharide antigens with the activated complement product C3d results in dramatically enhanced immunogenicity of the target antigens. In this paper, we describe our attempts to enhance the immunogenicity of the non-toxic B fragment of diphtheria toxin (DT-B) by genetic fusion to polypeptides derived from the C3d coding sequence. Contrary to expectations, we found that the antibody responses elicited by immunizing mice with DT-B genetically linked to three tandem copies of C3d-derived sequences were markedly reduced relative to the antibody responses elicited by immunizing mice with DT-B alone. These results demonstrate levels of complexity in the immunomodulatory effects of the complement system that were not apparent in earlier reports on the adjuvant effects of C3d administered to mice as genetic fusions to target antigens, such as hen egg lysozyme, human immunodeficiency virus (HIV) gp120 or influenza virus hemaglutinnin. The data presented herein suggest that C3d may act as a negative regulator, in some immunological contexts, for antibody production in the mammalian host.

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.

Reconstitution of active diphtheria toxin based on a hexahistidine tagged version of the B-fragment produced to high yields in bacteria.

Diphtheria toxin consists of an A-fragment that inactivates elongation factor 2 and a B-fragment that both binds to the toxin receptor and mediates translocation of the A-fragment across cellular membranes to the cytosol. Several fragments of the toxin and an inactive version of the holotoxin have been expressed in Escherichia coli, but the B-fragment alone has proven difficult to express. Only low levels of expression have been achieved. We have designed a hexahistidine tagged version of a modified diphtheria toxin B-fragment (DT-BHis) that can be expressed to high levels in E. coli. DT-BHis contains the entire diphtheria toxin B-fragment preceded by an alanine and succeeded by a leucine, a glutamic acid and a hexahistidine tag and could be purified in a single step using nickel-coated agarose beads to 85% homogeneity. DT-BHis bound specifically to the diphtheria toxin receptor and was able to compete out the effect of the wild type diphtheria toxin. Furthermore, DT-BHis was able to form pores in cellular membranes in a manner similar to the wild type B-fragment. The high yield makes DT-BHis a suitable tool in studies of diphtheria toxin interaction with cells or liposomes since functional diphtheria toxin was easily formed upon addition of A-fragment. The reconstituted diphtheria toxin showed toxicity in the same range as the wild type.

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias.

Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G4S)2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) > or =10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocyte-macrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three- to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease.

EFFECT OF DIPHTHERIA TOXIN T-DOMAIN ON ENDOSOMAL pH.

A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demonstrated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

A novel method to purify immunotoxins from free antibodies using modified recombinant toxins.

Monoclonal antibodies linked to toxin polypeptides (immunotoxins) are developed for clinical application against cancer and graft rejection. Immunotoxins prepared by many conventional methods often contain a trace amount of free antibody. Present studies describe a method to purify immunotoxins from free antibody in conjugation mixtures. Recombinant ricin A chain and a truncated form of diphtheria toxin (385 residues) containing ten consecutive histidine residues at the amino terminus were prepared. The modified toxin polypeptides retaining full biological activity were chemically linked to monoclonal antibodies (317G5 and 454C11) reactive to breast cancer cells. The high affinity of consecutive histidine residues for nickel-based resin (Ni-NTA) was exploited to purify immunotoxins from unreacted free antibodies. SDS-PAGE analysis of conjugates eluted from nickel column contained trace amounts of detectable free antibody whereas conjugates purified by other conventional methods using phenyl Sepharose or Cibacron blue Sepharose chromatography contained significant amounts of unconjugated antibody. Furthermore, the immunotoxin fraction containing predominantly two toxin molecules linked to one antibody can be separated from stoichiometric conjugates by Ni-NTA column. Cytotoxicity experiments showed that the complex of two toxin molecules linked to an antibody was more cytotoxic to tumor cells in vitro than the fraction enriched with immunotoxin containing equimolar stoichiometry.

Report on the Sixth International Meeting of the European Laboratory Working Group on Diphtheria, Brussels, Belgium.

In addition to the Eastern European resurgence of diphtheria during the last decade, there has also been an emergence of infections caused by non-toxigenic Corynebacterium diphtheriae and non-toxigenic, toxin gene bearing C. diphtheriae. Given that these strains may manifest as symptomatic infections of differing degrees of severity, their clinical and epidemiological significance need to be assessed. The persistence of toxigenic and non-toxigenic C. diphtheriae in circulation, together with genotypic and biotype variability means that innovative measures to vaccinate populations are pertinent. The most effective method of protecting the currently most vulnerable population group (adults) is to implement a booster dose of vaccine amongst the adult populations. Furthermore, in combination with an efficient surveillance system, effective antibiotic prophylaxis and an up-to-date vaccination programme, serological studies needs to be maintained to monitor the immunity status of the population.

Collaborative study for establishment of the European Pharmacopoeia BRP batch 1 for diphtheria toxin.

A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.

[PCR as an alternative method for detecting toxigenic strains of Corynebacterium diphtheriae]. / Wykorzystanie techniki PCR jako alternatywnej metody wykrywania toksynotwórczosci szczepów Corynebacterium diphtheriae.

PCR standardization was performed in order to detect a fragment and a whole tox gene which is presented in all toxigenic strains of Corynebacterium diphtheriae. PCR is one of the least time-consuming methods for detection toxigenicity of C. diphtheriae strains near EIA (enzyme immunoassay) and ICS (immunochromatographic strip) tests.

[Improved culture of Corynebacterium diphtheriae for obtaining toxin-anatoxin]. / Sovershenstvovanie kul'tivirovaniia difteriinykh korinebakterii dlia polucheniia toksina-anatoksina.

The work deals with the optimization of the cultivation of C. diphtheriae with a view to obtaining diphtheria toxin--toxoid on the basis of the study of the kinetics of microbial growth and toxin formation. The combined cultivation process consisting of 3 cycles has been experimentally developed and realized under industrial conditions. The use of this cultivation method has made it possible to obtain more balanced cultures and to standardize the biological properties of the biomass and toxin thus obtained. The characteristic features of the toxoid preparations obtained on the basis of this method are a high degree of purification, good immunogenic properties and constant fractional composition; their degree of homogeneity approximates the molecular homogeneous state.

An alternative method for purifying and detoxifying diphtheria toxin.

Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 µg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.

A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization.

Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.