Toxin Variants Research Articles

An anti-Shiga toxin VHH nanobody multimer protects mice against fatal toxicosis when administered intramuscularly as repRNA.

Hemolytic uremic syndrome (HUS) is a systemic sequelae from gastrointestinal infection with Shiga toxin (Stx) producing Escherichia coli (STEC) that can result in acute kidney injury, lasting renal disease, and death. Despite a window for intervention between hemorrhagic diarrhea and onset of HUS, no specific therapies exist to prevent or treat HUS following STEC infection. Furthermore, there is no way to predict which patients with STEC will develop HUS or any rapid way to determine which Stx variant is present. To address this, we have broadened the therpay to neutralize additional toxin variants. It contains a multimer of nanobodies derived from camelid heavy chain antibody fragments (VHHs). An improved VHH-based neutralizing agent (VNA2) is delivered intramuscularly as RNA combined with LION nanoparticles rather than mRNA, that replicates on administration (repRNA), resulting in a rapidly circulating VNA that can bind systemic toxin. The RNA/VNA2-Stx administered intramuscularly prevents toxicity and death in a mouse model of acute Stx toxicity.

Pharmacological and Non-pharmacological Approaches for the Management of Neuropathic Pain in Multiple Sclerosis.

Multiple sclerosis is a chronic inflammatory disease that affects the central nervous system and can cause various types of pain including ongoing extremity pain, Lhermitte's phenomenon, trigeminal neuralgia, and mixed pain. Neuropathic pain is a major concern for individuals with multiple sclerosis as it is directly linked to myelin damage in the central nervous system and the management of neuropathic pain in multiple sclerosis is challenging as the options available have limited efficacy and can cause unpleasant side effects. The literaturesearch was conducted across two databases, PubMed, and Google Scholar. Eligible studies included clinical trials, observational studies, meta-analyses, systematic reviews, and narrative reviews. The objective of this article is to provide an overview of literature on pharmacological and non-pharmacological strategies employed in the management of neuropathic pain in multiple sclerosis. Pharmacological options include cannabinoids, muscle relaxants (tizanidine, baclofen, dantrolene), anticonvulsants (benzodiazepines, gabapentin, phenytoin, carbamazepine, lamotrigine), antidepressants (duloxetine, venlafaxine, tricyclic antidepressants), opioids (naltrexone), and botulinum toxin variants, which have evidence from various clinical trials. Non-pharmacological approaches for trigeminal neuralgia may include neurosurgical methods. Non-invasive methods, physical therapy, and psychotherapy (cognitive behavioral therapy, acceptance and commitment therapy and mindfulness-based stress reduction) may be recommended for patients with neuropathic pain in multiple sclerosis. The choice of treatment depends on the severity and type of pain as well as other factors, such as patient preferences and comorbidities. There is a pressing need for healthcare professionals and researchers to prioritize the development of better strategies for managing multiple sclerosis-induced neuropathic pain.

Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins.

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.

Isofagomine Inhibits Multiple TcdB Variants and Protects Mice from Clostridioides difficile-Induced Mortality.

Clostridioides difficile causes life-threatening diarrhea and is one of the leading causes of nosocomial infections. During infection, C. difficile releases two gut-damaging toxins, TcdA and TcdB, which are the primary determinants of disease pathogenesis and are important therapeutic targets. Once in the cytosol of mammalian cells, TcdA and TcdB use UDP-glucose to glucosylate host Rho GTPases, which leads to cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the glucocation transition state of the glucosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective C. difficile continue to be identified, and therefore, evaluation of isofagomine inhibition against multiple toxin variants is required. Here, we show that isofagomine inhibits the glucosyltransferase domain of multiple TcdB variants and protects TcdB-induced cell rounding of the most common full-length toxin variants. Furthermore, we demonstrate that isofagomine protects against C. difficile-induced mortality in two murine models of C. difficile infection. Isofagomine treatment of mouse C. difficile infection also permitted the recovery of the gastrointestinal microbiota, an important barrier to preventing recurring C. difficile infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against C. difficile-induced morbidity and mortality.

Gastrointestinal involvement in STEC-associated hemolytic uremic syndrome: 10 years in a pediatric center

BackgroundThe gastrointestinal (GI) tract represents one of the main targets of typical hemolytic uremic syndrome (HUS) in children. In this observational study, we tried to establish (1) the main features of GI complications during STEC-HUS and (2) the relationship between Escherichia coli serotypes and Shiga toxin (Stx) variants with hepatopancreatic involvement.MethodsA total of 79 STEC-HUS patients were admitted to our pediatric nephrology department between January 2012 and June 2021. Evidence of intestinal, hepatobiliary, and pancreatic involvements was reported for each patient, alongside demographic, clinical, and laboratory features. Frequency of gastrointestinal complications across groups of patients infected by specific E. coli serotypes and Stx gene variants was evaluated.ResultsSix patients developed a bowel complication: two developed rectal prolapse, and four developed bowel perforation which resulted in death for three of them and in bowel stenosis in one patient. Acute pancreatitis was diagnosed in 13 patients. An isolated increase in pancreatic enzymes and/or liver transaminases was observed in 41 and 15 patients, respectively. Biliary sludge was detected in three, cholelithiasis in one. Forty-seven patients developed direct hyperbilirubinemia. Neither E. coli serotypes nor Shiga toxin variants correlated with hepatic or pancreatic involvement.ConclusionsDuring STEC-HUS, GI complications are common, ranging from self-limited elevation of laboratory markers to bowel perforation, a severe complication with a relevant impact on morbidity and mortality. Hepatopancreatic involvement is frequent, but usually short-lasting and self-limiting.Graphical abstractA higher resolution version of the Graphical abstract is available asSupplementary information

Moringa oleifera as a potential antimicrobial against pathogenic Clostridium perfringens isolates in farm animals.

Clostridium perfringens (CP) is an emerging anaerobic pathogen that can aggravate severe fatal infections in different hosts and livestock. This paper was designed to monitor the antibacterial efficacy of Moringa oleifera (M. oleifera) plant against different CP isolates of variant toxin genotypes comparing that with commercial antibiotics in the veterinary field. A total of 200 examined fecal, intestinal, and liver samples from cattle, sheep, and goats were investigated bacteriologically and biochemically for CP. Then, the isolates were examined by polymerase chain reaction (PCR) for toxin gene typing. Thereafter, the antimicrobial susceptibility testing as well as the antibacterial efficacy of M. oleifera were evaluated and statistically analyzed against recovered isolates. The prevalence rate of CP was 51% (102/200); of which 54.5% was from cattle, 50% from sheep, and 40% from goat. Moreover, all CP isolates were highly resistant to tetracycline and lincomycin drugs; meanwhile, they were of the least resistance against ciprofloxacin (8.3%-16.7%), cefotaxime (16.7%-25%), and gentamycin (26.7%-33.3%). For M. oleifera, high antibacterial efficacy with greater inhibition zones of the plant was recorded with its oil (20-24 mm) and ethanolic extracts (16-20 mm) against CP than the aqueous extract (≤ 10 mm). A good correlation was stated between M. oleifera oil and toxin type of CP isolates particularly type A followed by D and B types. Interestingly, the oil and ethanolic extracts of M. oleifera gave higher antibacterial efficacy than most commercial antibiotics against the recovered isolates. This study highlighted the potent antibacterial properties of M. oleifera for suppressing CP isolated from farm animals; hence, more investigations on M. oleifera are suggested to support its use as a medical herbal plant substituting antibiotics hazards and resistance problems worldwide.

Exploring the inhibitory potential of the antiarrhythmic drug amiodarone against Clostridioides difficile toxins TcdA and TcdB

ABSTRACT The intestinal pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans. The symptoms of C. difficile-associated diseases (CDADs) are directly associated with the pathogen’s toxins TcdA and TcdB, which enter host cells and inactivate Rho and/or Ras GTPases by glucosylation. Membrane cholesterol is crucial during the intoxication process of TcdA and TcdB, and likely involved during pore formation of both toxins in endosomal membranes, a key step after cellular uptake for the translocation of the glucosyltransferase domain of both toxins from endosomes into the host cell cytosol. The licensed drug amiodarone, a multichannel blocker commonly used in the treatment of cardiac dysrhythmias, is also capable of inhibiting endosomal acidification and, as shown recently, cholesterol biosynthesis. Thus, we were keen to investigate in vitro with cultured cells and human intestinal organoids, whether amiodarone preincubation protects from TcdA and/or TcdB intoxication. Amiodarone conferred protection against both toxins independently and in combination as well as against toxin variants from the clinically relevant, epidemic C. difficile strain NAP1/027. Further mechanistic studies suggested that amiodarone’s mode-of-inhibition involves also interference with the translocation pore of both toxins. Our study opens the possibility of repurposing the licensed drug amiodarone as a novel pan-variant antitoxin therapeutic in the context of CDADs.

Serotypes, Pathotypes, Shiga Toxin Variants and Antimicrobial Resistance in Diarrheagenic Escherichia coli Isolated from Rectal Swabs and Sheep Carcasses in an Abattoir in Mexico

Sheep represent one of the main reservoirs of diarrheagenic Escherichia coli; this microorganism is an etiological agent of food-borne diseases; therefore, this work aimed to identify and characterize the principal pathotypes of diarrheagenic E. coli (DEC) obtained through rectal swabs and carcasses samples from sheep slaughtered in an abattoir at the central region of Mexico. The isolates were subjected to bacteriological identification, serotyping; phylogenetic classification; detection for virulence factors, and antimicrobial sensibility. A total of 90 E. coli isolates were obtained. It was observed through 49 E. coli isolates (54%), 8 of them from carcasses, and 43 from feces was DEC. DEC serotypes with health public relevance were found: O76:H19 (n = 5), O146:H21 (n = 3), O91:H10 (n = 1), O6:NM (n = 1), and O8:NM (n = 1). Regarding the presence of Shiga toxin-producing E.coli (STEC), 43/90 (47.7%) isolates have the stx1 w/o stx2 genes, and therefore were assigned as STEC non-O157; only one isolate expressed stx1 and eae genes and was classified as t-STEC (typical STEC). Additionally, 3/90 (3.3%) harbored only the eae gene and were classified as enteropathogenic E. coli (EPEC), the stp gene was found in 2/90 isolates (2.2%) and were classified as enterotoxigenic E. coli (ETEC); 1/90 (1.1%) isolates harboring the ipaH were classified as enteroinvasive E. coli EIEC. Regarding stx1 genes subtypes, stx1c only was found in 60.5% (26/43), followed by stx1a-stx1c 20.9% (9/43) and stx1a-stx1d 2.3% (1/43). The presence of both, stx1 and stx2 genes was found in 7/43 isolates (16.3%) from rectal swabs; the combination stx1c-stx2g was detected in 3/43 isolates (6.9%), while 4 (9.4%) isolates showed different patterns (stx1a-stx1c-stx2g; stx1c-stx2b-stx2g; stx1c-stx2b and stx1a-stx1c-stx2b-stx2g). STEC isolates showed the major diversity of phylogenetic groups, although phylogroup B1 was predominant in 90.6% (39/43) while there was only one isolate (2.3%) in each remaining phylogroup (A, B2, C, and F). All EPEC, ETEC, and EIEC isolates were clustered in phylogroup B1. We observed that 27.9% (12/43) of STEC isolates carried at least one antibiotic resistance: nine isolates expressed the tetB gene, one isolate the tetA gene, two isolates the sul2 gene, one isolate the sul1 and one isolate the sul1-tetB genes. These results highlight the importance of diarrheagenic E. coli as a potential risk for public health during the slaughtering process.

Group Selection as a Basis for Screening Mutagenized Libraries of Public Goods (Bacillus thuringiensis Cry Toxins).

The pesticidal toxins of Bacillus thuringiensis (Bt) supply the active proteins for genetically modified insect-resistant crops. There is therefore keen interest in finding new toxins, or improving known toxins, in order to increase the mortality of various targets. The production and screening of large libraries of mutagenized toxins are among the means of identifying improved toxins. Since Cry toxins are public goods, and do not confer advantages to producers in competition, conventional directed evolution approaches cannot be used here. Instead, thousands of individual mutants have to be sequenced and assayed individually, a costly and time-consuming process. In this study, we tested a group selection-based approach that could be used to screen an uncharacterized pool of Cry toxin mutants. This involved selecting for infectivity between subpopulations of Bt clones within metapopulations of infected insects in three rounds of passage. We also tested whether additional mutagenesis from exposure to ethyl methanesulfonate could increase infectivity or supply additional Cry toxin diversity during passage. Sequencing of pools of mutants at the end of selection showed that we could effectively screen out Cry toxin variants that had reduced toxicity with our group selection approach. The addition of extra mutagenesis during passage decreased the efficiency of selection for infectivity and did not produce any additional novel toxin diversity. Toxins with loss-of-function mutations tend to dominate mutagenized libraries, and so a process for screening out these mutants without time-consuming sequencing and characterization steps could be beneficial when applied to larger libraries. IMPORTANCE Insecticidal toxins from the bacterium Bacillus thuringiensis are widely exploited in genetically modified plants. This application creates a demand for novel insecticidal toxins that can be used to better manage resistant pests or control new or recalcitrant target species. An important means of producing novel toxins is via high-throughput mutagenesis and screening of existing toxins, a lengthy and resource-intensive process. This study describes the development and testing of an efficient means of screening a test library of mutagenized insecticidal toxins. Here, we showed that it is possible to screen out loss-of-function mutations with low infectivity within a pool without the need to characterize and sequence each mutant individually. This has the potential to improve the efficiency of processes used to identify novel proteins.

Dissecting the contributions of membrane affinity and bivalency of the spider venom protein DkTx to its sustained mode of TRPV1 activation

The spider venom protein, double-knot toxin (DkTx), partitions into the cellular membrane and binds bivalently to the pain-sensing ion channel, TRPV1, triggering long-lasting channel activation. In contrast, its monovalent single knots membrane partition poorly and invoke rapidly reversible TRPV1 activation. To discern the contributions of the bivalency and membrane affinity of DkTx to its sustained mode of action, here, we developed diverse toxin variants including those containing truncated linkers between individual knots, precluding bivalent binding. Additionally, by appending the single-knot domains to the Kv2.1 channel-targeting toxin, SGTx, we created monovalent double-knot proteins that demonstrated higher membrane affinity and more sustained TRPV1 activation than the single-knots. We also produced hyper-membrane affinity-possessing tetra-knot proteins, (DkTx)2 and DkTx-(SGTx)2, that demonstrated longer-lasting TRPV1 activation than DkTx, establishing the central role of the membrane affinity of DkTx in endowing it with its sustained TRPV1 activation properties. These results suggest that high membrane affinity-possessing TRPV1 agonists can potentially serve as long-acting analgesics.

The Role of Glycoconjugates as Receptors for Insecticidal Proteins.

Bacillus thuringiensis (Bt) proteins are an environmentally safe and effective alternative to chemical pesticides and have been used as biopesticides, with great commercial success, for over 50 years. Global agricultural production is predicted to require a 70% increase until 2050 to provide for an increasing population. In addition to agriculture, Bt proteins are utilised to control human vectors of disease - namely mosquitoes - which account for >700,000 deaths annually. The evolution of resistance to Bt pesticial toxins threatens the progression of sustainable agriculture. Whilst Bt protein toxins are heavily utilised, the exact mechanisms behind receptor binding and toxicity are unknown. It is critical to gain a better understanding of these mechanisms in order to engineer novel toxin variants and to predict, and prevent, future resistance evolution. This review focuses on the role of carbohydrate binding in the toxicity of the most utilised group of Bt pesticidal proteins - three domain Cry (3D-Cry) toxins.

First cross-border outbreak of foodborne botulism in the European Union associated with the consumption of commercial dried roach (Rutilus rutilus).

Botulism outbreaks due to commercial products are extremely rare in the European Union. Here we report on the first international outbreak of foodborne botulism caused by commercial salt-cured, dried roach (Rutilus rutilus). Between November and December 2016, an outbreak of six foodborne botulism type E cases from five unrelated households was documented in Germany and Spain. The outbreak involved persons of Russian and Kazakh backgrounds, all consumed unheated salt-cured, dried roach-a snack particularly favored in Easter-European countries. The implicated food batches had been distributed by an international wholesaler and were recalled from Europe-wide outlets of a supermarket chain and other independent retailers. Of interest, and very unlike to other foodborne disease outbreaks which usually involves a single strain or virus variant, different Clostridium botulinum strains and toxin variants could be identified even from a single patient's sample. Foodborne botulism is a rare but potentially life-threatening disease and almost exclusively involves home-made or artisan products and thus, outbreaks are limited to individual or few cases. As a consequence, international outbreaks are the absolute exception and this is the first one within the European Union. Additional cases were likely prevented by a broad product recall, underscoring the importance of timely public health action. Challenges and difficulties on the diagnostic and epidemiological level encountered in the outbreak are highlighted.

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

New Knowledge on Distribution and Abundance of Toxic Microalgal Species and Related Toxins in the Northwestern Black Sea.

Numerous potentially toxic plankton species commonly occur in the Black Sea, and phycotoxins have been reported. However, the taxonomy, phycotoxin profiles, and distribution of harmful microalgae in the basin are still understudied. An integrated microscopic (light microscopy) and molecular (18S rRNA gene metabarcoding and qPCR) approach complemented with toxin analysis was applied at 41 stations in the northwestern part of the Black Sea for better taxonomic coverage and toxin profiling in natural populations. The combined dataset included 20 potentially toxic species, some of which (Dinophysis acuminata, Dinophysis acuta, Gonyaulax spinifera, and Karlodinium veneficum) were detected in over 95% of the stations. In parallel, pectenotoxins (PTX-2 as a major toxin) were registered in all samples, and yessotoxins were present at most of the sampling points. PTX-1 and PTX-13, as well as some YTX variants, were recorded for the first time in the basin. A positive correlation was found between the cell abundance of Dinophysis acuta and pectenotoxins, and between Lingulodinium polyedra and Protoceratium reticulatum and yessotoxins. Toxic microalgae and toxin variant abundance and spatial distribution was associated with environmental parameters. Despite the low levels of the identified phycotoxins and their low oral toxicity, chronic toxic exposure could represent an ecosystem and human health hazard.

Genetic Divergence of Vibrio vulnificus Clinical Isolates with Mild to Severe Outcomes.

ABSTRACTThe marine bacterium Vibrio vulnificus infects humans via food or water contamination, leading to serious manifestations, including gastroenteritis, wound infections, and septic shock. Previous studies suggest phylogenetic Lineage 1 isolates with the vcgC allele of the vcg gene cause human infections, whereas Lineage 2 isolates with the vcgE allele are less pathogenic. Mouse studies suggest that some variants of the primary toxin could drive more serious infections. A collection of 109 V. vulnificus United States human clinical isolates from 2001 to 2019 with paired clinical outcome data were assembled. The isolates underwent whole-genome sequencing, multilocus-sequence phylogenetic analysis, and toxinotype analysis of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. In contrast to prior reports, clinical isolates were equally distributed between lineages. We found no correlation between phylogenetic lineage or MARTX toxinotype and disease severity. Infections caused by isolates in Lineage 1 demonstrated a borderline statistically significant higher mortality. Lineage 1 isolates had a trend toward a higher proportion of M-type MARTX toxins compared with Lineage 2, although this was not statistically significant.

Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 μM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 μM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 μg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Introduction: the secret lives of microbial mobile genetic elements.

Introduction: the secret lives of microbial mobile genetic elements.

Reduced Toxicity of Centruroides vittatus (Say, 1821) May Result from Lowered Sodium β Toxin Gene Expression and Toxin Protein Production.

Body tissue and venom glands from an eastern population of the scorpion Centruroides vittatus (Say, 1821) were homogenized and molecular constituents removed to characterize putative sodium β toxin gene diversity, RT-qPCR, transcriptomic, and proteomic variation. We cloned sodium β toxins from genomic DNA, conducted RT-qPCR experiments with seven sodium β toxin variants, performed venom gland tissue RNA-seq, and isolated venom proteins for mass spectrophotometry. We identified >70 putative novel sodium β toxin genes, 111 toxin gene transcripts, 24 different toxin proteins, and quantified sodium β toxin gene expression variation among individuals and between sexes. Our analyses contribute to the growing evidence that venom toxicity among scorpion taxa and their populations may be associated with toxin gene diversity, specific toxin transcripts variation, and subsequent protein production. Here, slight transcript variation among toxin gene variants may contribute to the major toxin protein variation in individual scorpion venom composition.

Acellular Pertussis Vaccine from Antigens of Freshly Isolated and Vaccine Strains of Bordetella pertussis with Different Genotypic Characteristics

Relevance. The development of effective and safe vaccines for pertussis prevention remains an urgent public health challenge.Aim. To study the protective activity and safety of acellular pertussis vaccine (AcPV) containing a complex of protective antigens from freshly isolated and vaccine strains of Bordetella pertussis.Materials and methods. Freshly isolated (No. 287, and No. 317) and vaccine (No. 305 and No. 475) B. pertussis strains with «non-vaccine» and «vaccine» allelic variants of the pertussis toxin (PT) subunit A gene, the PT promoter gene, the pertactin gene, the fimbria 2 gene, and the fimbria 3 gene strains were used for the production of AcPV.Results. All the studied variants of AcPV were harmless in the test of changes in the body weight of mice and sensitivity to histamine. The protective activity of AcPV3 (strains No. 287, No. 317 and No. 305) and AcPV1 (strains No. 287, No. 305 and No. 475) was higher than that of AcPV2 (strains No. 317, No. 305, and No. 475). IgG antibody titers to PT were also higher in mice immunized with AcPV1 and AcPV3.Conclusion. The higher protective activity of AcPV3 and AcPV1 may be associated with the genotype of strain No. 287, which has a ptxP3 PT promoter and is characterized by an increased level of PT production and high virulence. The most promising for further preclinical and clinical studies is AcPV3, which contains 2/3 of the antigens of the dominant «non-vaccine» genotype and 1/3 of the «vaccine» genotype, corresponding to the genes of PT, pertactin and fimbria to the currently circulating B. pertussis strains.