Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora.

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

Crystal Structure of Exotoxin A from Aeromonas Pathogenic Species.

Aeromonas exotoxin A (AE) is a bacterial virulence factor recently discovered in a clinical case of necrotising fasciitis caused by the flesh-eating Aeromonas hydrophila. Here, database mining shows that AE is present in the genome of several emerging Aeromonas pathogenic species. The X-ray crystal structure of AE was solved at 2.3 Å and presents all the hallmarks common to diphthamide-specific mono-ADP-ribosylating toxins, suggesting AE is a fourth member of this family alongside the diphtheria toxin, Pseudomonas exotoxin A and cholix. Structural homology indicates AE may use a similar mechanism of cytotoxicity that targets eukaryotic elongation factor 2 and thus inhibition of protein synthesis. The structure of AE also highlights unique features including a metal binding site, and a negatively charged cleft that could play a role in interdomain interactions and may affect toxicity. This study raises new opportunities to engineer alternative toxin-based molecules with pharmaceutical potential.

Host cell protein quantification of an optimized purification method by mass spectrometry.

Recombinant ExoProtein A (EPA), a detoxified form of Pseudomonas aeruginosa Exotoxin A, is used as a protein carrier in the vaccine field. A scaled manufacturing process, in which EPA was expressed in Escherichia coli, yielded a product that approached or exceeded our upper limit of E. coli host cell protein (HCP) content per human dose. The purification process was redeveloped to reduce HCP levels in the bulk product and HCP content was evaluated by orthogonal methods. Using a platform specific immunoassay, the HCP level from the original purification method was 1,830 ppm (0.18% w/w) while the revised purification process yielded the HCP below the detection limits of the assay. With a 2D/LC-MSE methodology the reference sample from the original process was found to contain 57 unique HCPs at a total level of 37,811 ppm (3.78% w/w). Two lots were tested after purification with the revised process and contained 730 and 598 ppm (0.07% and 0.06% w/w), respectively. To develop a high-throughput MS method, the samples were tested on a 1D/LC-MS/MS. The data sets from the two mass spectrometers correlated well. These improved HCP profiles support implementing the revised purification process for manufacturing the EPA protein carrier and 1D/LC-MS/MS for HCP analysis.

The immunotoxin activity of exotoxin A is sensitive to domain modifications.

Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically. The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance.

Two Clostridiumperfringens Type E Isolates in France.

Clostridiumperfringens type E is a less frequently isolated C.perfringens type and has not previously been reported in France. We have characterized two recent type E isolates, C.perfringens 508.17 from the intestinal content of a calf that died of enterotoxemia, and 515.17 from the stool of a 60-year-old woman, subsequent to food poisoning, which contained the plasmid pCPPB-1 with variant iota toxin and C. perfringens enterotoxin genes.

Molecular epidemiology of Clostridioides difficile and risk factors for the detection of toxin gene-positive strains.

In this study, we investigated all Clostridioides difficile strains isolated from stool samples in Nagasaki University Hospital between January 2012 and December 2014. Toxin genes (tcdA, tcdB and cdtA/cdtB) were analyzed for multiplex PCR in a total of 213 strains. In the toxin gene-positive strain, PCR ribotyping was conducted using capillary gel electrophoresis-based PCR and the Webribo database. Patients' backgrounds were analyzed by departments, disorders, antimicrobials, and clinical dates. The positive rates of tcdA, tcdB, and cdtA/cdtB genes were 62.9%, 63.4%, and 2.8%, respectively. The most frequent PCR ribotype was 047 (14.1%), followed by 014/0 (11.1%) and 002/0 (8.2%). In univariate analysis, the risk factors for the detection of toxin gene-positive strains in patients were older age (p = 0.0036), over ≥ 65 years old (p = 0.0175), the patients hospitalized at Department of Digestive Surgery (P = 0.0059), higher CRP level (P = 0.0395), and lower albumin level (p = 0.0014). In the multivariate analysis, the risk factor for detection of toxin gene-positive strains was the patients hospitalized at Department of Digestive Surgery (OR; 4.62, 95% CI; 1.18-18.0, p = 0.0274). In this study, the percentage of toxin gene-positive and cdtA/cdtB gene-positive strains was almost the same as that reported in previous studies, but the ribotype was different. In addition, we revealed that the risk factor associated with the detection of toxin gene-positive strains was the patients hospitalized at Department of digestive surgery.

Assessment of the impact of manufacturing changes on the physicochemical properties of the recombinant vaccine carrier ExoProtein A.

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.

Mono-ADP-Ribosylation Catalyzed by Arginine-Specific ADP-Ribosyltransferases.

Methods are described for determination of arginine-specific mono-ADP-ribosyltransferase activity of purified proteins and intact cells by monitoring the transfer of ADP-ribose from NAD+ to a model substrate, e.g., arginine, agmatine, and peptide (human neutrophil peptide-1 [HNP1]), and for the nonenzymatic hydrolysis of ADP-ribose-arginine to ornithine, a noncoded amino acid. In addition, preparation of purified ADP-ribosylarginine is included as a control substrate for ADP-ribosylation reactions.

Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry.

ADP-ribosylation is a posttranslational modification that involves the conjugation of monomers and polymers of the small molecule ADP-ribose onto amino acid side chains. A family of ADP-ribosyltransferases catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD+) onto a variety of amino acid side chains including aspartate, glutamate, lysine, arginine, cysteine, and serine. The monomeric form of the modification mono(ADP-ribosyl)ation (MARylation) is reversed by a number of enzymes including a family of MacroD-type macrodomain-containing mono(ADP-ribose) (MAR) hydrolases. Though it has been inferred from various chemical tests that these enzymes have specificity for MARylated aspartate and glutamate residues in vitro, the amino acid and site specificity of different family members are often not unambiguously defined. Here we describe a mass spectrometry-based assay to determine the site specificity of MAR hydrolases in vitro.

Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine.

Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.

A Size-Exclusion Chromatography Method for Analysis of Clostridium difficile Vaccine Toxins.

High-performance size-exclusion chromatography (HPSEC or SEC) is a method that can be applied to measure size distribution of proteins, including aggregates, monomers, and fragments. In the biopharmaceutical industry the quantitation of aggregates contained in biotherapeutics and protein-based vaccines is critical given the potential impact on safety, immunogenicity, and efficacy. Hence, aggregation analysis of therapeutic proteins or protein-based vaccine products is almost always a requirement of regulatory agencies. SEC, also referred to as gel-filtration chromatography, separates molecules by size through a porous resin stationary phase. Under isocratic flow small molecules are retained on the column longer than large molecules. Here we describe the use of this SEC technique to characterize aggregation levels for four different protein antigens for a Clostridium difficile vaccine.

Characterization and cytotoxic activity of apoptosis-inducing pierisin-5 protein from white cabbage butterfly.

In this study, caspase-dependent apoptosis-inducing pierisin-5 gene was identified and characterized from cabbage white butterfly, Pieris canidia. A thousand-fold increase in expression of pierisin-5 gene was observed from second to third instar larvae, gradually decreasing before pupation. Pierisin-5 was purified from the fifth-instar larvae and was found to exhibit cytotoxicity against HeLa and HepG2 human cancer cell lines. Pierisin-5 showed growth inhibition and several morphological changes such as cell shrinkage, chromatin condensation and apoptotic body formation with programmed cell death in HeLa and HepG2 cells. Moreover, DNA fragmentation was observed after gel electrophoresis analysis. Caspase substrate assay showed further cleavage of Ac-DEVD-pNA, suggesting the activation of Caspase-3. Flow cytometry analysis revealed the cell cycle arrest at G1 phase and increased the percentage of apoptotic cells in cancer cell lines treated with pierisin-5. These findings suggest that pierisin-5 could significantly induce apoptosis in cancer cell lines and is mediated by activation of caspase-3 in the mitochondrial pathway. Phylogenetic analysis using pierisin proteins from Pierid butterflies, ADP-ribosylating toxins from bacteria, human, rat, and mouse indicated the possibility of horizontal transfer of pierisin genes from bacteria to butterflies. The single copy of pierisin gene unlike other insect toxin genes also supports lateral transfer.

Vimentin mediates uptake of C3 exoenzyme.

Clostridium botulinum C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.

Surveillance snapshot of Clostridium difficile infection in hospitals across Queensland detects binary toxin producing ribotype UK 244.

In North America and Europe, the binary toxin positive Clostridium difficile strains of the ribotypes 027 and 078 have been associated with death, toxic megacolon and other adverse outcomes. Following an increase in C. difficile infections (CDIs) in Queensland, a prevalence study involving 175 hospitals was undertaken in early 2012, identifying 168 cases of CDI over a 2 month period. Patient demographics and clinical characteristics were recorded, and C. difficile isolates were ribotyped and tested for the presence of binary toxin genes. Most patients (106/168, 63.1%) were aged over 60 years. Overall, 98 (58.3%) developed symptoms after hospitalisation; 89 cases (53.0%) developed symptoms more than 48 hours after admission. Furthermore, 27 of the 62 (67.7%) patients who developed symptoms in the community ad been hospitalised within the last 3 months. Thirteen of the 168 (7.7%) cases identified had severe disease, resulting in admission to the Intensive Care Unit or death within 30 days of the onset of symptoms. The 3 most common ribotypes isolated were UK 002 (22.9%), UK 014 (13.3%) and the binary toxin-positive ribotype UK 244 (8.4%). The only other binary toxin positive ribotype isolated was UK 078 (n = 1). Of concern was the detection of the binary toxin positive ribotype UK 244, which has recently been described in other parts of Australia and New Zealand. No isolates were of the international epidemic clone of ribotype UK 027, although ribotype UK 244 is genetically related to this clone. Further studies are required to track the epidemiology of ribotype UK 244 in Australia and New Zealand.

The nitrogenase regulatory enzyme dinitrogenase reductase ADP-ribosyltransferase (DraT) is activated by direct interaction with the signal transduction protein GlnB.

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P(II) proteins. In A. brasilense, two P(II) proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P(II) proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P(II) effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P(II) proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K(m) value for NAD(+). Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.

Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells.

The mammalian mono-ADP-ribosyltransferases are a family of enzymes related to bacterial toxins that can catalyse both intracellular and extracellular mono-ADP-ribosylation of target proteins involved in different cellular processes, such as cell migration, signalling and inflammation. Here, we report the molecular cloning and functional characterisation of a novel glycosylphosphatidylinositol (GPI)-anchored mono-ADP-ribosyltransferase isoform from Chinese hamster ovary (CHO) cells (cARTC2.1) that has both NAD-glycohydrolase and arginine-specific ADP-ribosyltransferase activities. cARTC2.1 has the R-S-EXE active-site motif that is typical of arginine-specific ADP-ribosyltransferases, with Glu209 as the predicted catalytic amino acid. When over-expressed in CHO cells, the E209G single point mutant of cARTC2.1 cannot hydrolyse NAD(+), although it retains low arginine-specific ADP-ribosyltransferase activity. This ADP-ribosyltransferase activity was abolished only with an additional mutation in the R-S-EXE active-site motif, with both of the glutamate residues of the EKE sequence of cARTC2.1 mutated to glycine (E207/209G). These glutamate-mutated proteins localise to the plasma membrane, as does wild-type cARTC2.1. Thus, the partial or total loss of enzymatic activity of cARTC2.1 that arises from these mutations does not affect its cellular localisation. Importantly, an endogenous ADP-ribosyltransferase is indeed expressed and active in a subset of CHO cells, while a similar activity cannot be detected in ovarian cancer cells. With respect to this endogenous ecto-ART activity, we have identified two cell populations: ART-positive and ART-negative CHO cells. The subset of ART-positive cells, which represented 5% of the total cells, is tightly maintained in the CHO cell population.

Fusogenics: a recombinant immunotoxin-based screening platform to select internalizing tumor-specific antibody fragments.

Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer.

Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli.

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.

A laboratory study of survival of selected microorganisms after heat treatment of biowaste used in biogas plants.

The aim of the study was to assess the effect of pasteurisation, as set by the European regulation EC 1774/2002, on selected pathogens and indicator organisms. Unpasteurised substrate (biowaste), including animal by-products from a full-scale biogas plant was heat treated under laboratory conditions at 70 degrees C and 55 degrees C for 30 min and 60 min. Heat treatment at 55 degrees C for 60 min was not sufficient to achieve a hygienically acceptable product. Heat treatment at 70 degrees C for 30 min and 60 min was effective in reducing pathogenic bacteria, Ascaris suum eggs, Swine vesicular disease virus and indicator organisms. However, this level of pasteurisation will still not reduce the quantity of Clostridia spores, or completely inactivate heat-resistant viruses such as Porcine parvovirus or Salmonella phage 28B. The results still give cause for some concern regarding the use of digested residue from biogasplants in agriculture.

Purification and molecular cloning of a DNA ADP-ribosylating protein, CARP-1, from the edible clam Meretrix lamarckii.

The cabbage butterflies Pieris rapae and Pieris brassicae have unique enzymes, named pierisin-1 and -2, respectively, that catalyze the ADP-ribosylation of guanine residues of DNA, which has been linked with induction of apoptosis and mutation in mammalian cell lines. In the present study, we identified ADP-ribosylation activity targeting DNA in six kinds of edible clam. Similar to our observations with pierisin-1 and -2, crude extracts from the clams Meretrix lamarckii, Ruditapes philippinarum, and Corbicula japonica incubated with calf thymus DNA and beta-NAD resulted in production of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine. The DNA ADP-ribosylating protein in the hard clam M. lamarckii, designated as CARP-1, was purified by column chromatography, and its cDNA was cloned. The cDNA encodes a 182-aa protein with a calculated molecular mass of 20,332. The protein synthesized in vitro from the cDNA in a reticulocyte lysate exhibited the same ADP-ribosylating activity as that of purified CARP-1. Neither the nucleotide nor the deduced amino acid sequence of CARP-1 showed homology with pierisin-1 or -2. However, a glutamic acid residue (E128) at the putative NAD-binding site, conserved in all ADP-ribosyltransferases, was found in CARP-1, and replacement of aspartic acid for this glutamic acid resulted in loss of almost all ADP-ribosylating activity. CARP-1 in the culture medium showed no cytotoxicity against HeLa and TMK-1 cells; however, introduction of this protein by electroporation induced apoptosis in these cells. The finding of clam ADP-ribosylating protein targeting guanine residues in DNA could offer new insights into the biological significance of ADP-ribosylation of DNA.