Foodborne Toxigenic Agents Investigated in Central Italy: An Overview of a Three-Year Experience (2018-2020).

Foodborne diseases (FBDs) represent a worldwide public health issue, given their spreadability and the difficulty of tracing the sources of contamination. This report summarises the incidence of foodborne pathogens and toxins found in food, environmental and clinical samples collected in relation to diagnosed or suspected FBD cases and submitted between 2018 and 2020 to the Food Microbiology Unit of the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (IZSLT). Data collected from 70 FBD investigations were analysed: 24.3% of them started with an FBD diagnosis, whereas a further 41.4% involved clinical diagnoses based on general symptomatology. In total, 5.6% of the 340 food samples analysed were positive for the presence of a bacterial pathogen, its toxins or both. Among the positive samples, more than half involved meat-derived products. Our data reveal the probable impact of the COVID-19 pandemic on the number of FBD investigations conducted. In spite of the serious impact of FBDs on human health and the economy, the investigation of many foodborne outbreaks fails to identify the source of infection. This indicates a need for the competent authorities to continue to develop and implement a more fully integrated health network.

Cell-Penetrating Antimicrobial Peptides Derived from an Atypical Staphylococcal δ-Toxin.

Revisiting underutilized classes of antibiotics is a pragmatic approach to the identification of alternative therapies for antimicrobial-resistant pathogens. To this end, we designed and screened a set of seven staphylococcal δ-toxin-inspired peptides (STIPs) for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, a pathogen-specific protease was leveraged to generate shorter peptides from these δ-toxin derivatives to expand the screen of putative antimicrobial peptides (AMPs) and to counterscreen against AMP inactivation. Remarkably, a 17-amino acid peptide based on the atypical δ-toxin sequence of Staphylococcus auricularis was discovered to possess an ability to kill MRSA and related pathogens. An alanine scan and series of rational substitutions improved AMP activity, and phenotypic assays characterized the STIPs' ability to rapidly interact with and permeabilize the staphylococcal membrane without causing lysis on a commensurate timescale. Instead of rapid lysis, both l- and d-enantiomers of STIP3-29, an AMP with low micromolar activity, were observed to penetrate and accumulate within cells. Finally, we observed that STIP3-29 was capable of controlling MRSA infection in a three-dimensional skin infection model. Overall, the results suggest that this unconventional source of AMPs can provide promising candidates for further development as therapeutic agents. IMPORTANCE The continued emergence and global distribution of infections caused by antimicrobial-resistant pathogens fuel our perpetual need for new or alternative therapies. Here, we present the discovery and initial characterization of bacterial cell-penetrating AMPs that were based on a family of virulence factors. In contrast to the multitude of AMPs that are sourced from animals, these potential therapeutic molecules have not undergone extensive selection for their antimicrobial properties and have proven to be amenable to activity-optimizing modifications. The staphylococcal toxin-inspired peptides described here represent a source of AMPs that can kill common opportunistic pathogens, such as MRSA, and have the potential to be improved for application in medicine.

Bacteria and bacterial anticancer agents as a promising alternative for cancer therapeutics.

Cancer is the leading cause of deaths worldwide, though significant advances have occurred in its diagnosis and treatment. The development of resistance against chemotherapeutic agents, their side effects, and non-specific toxicity urge to screen for the novel anticancer agent. Hence, the development of novel anticancer agents with a new mechanism of action has become a major scientific challenge. Bacteria and bacterially produced bioactive compounds have recently emerged as a promising alternative for cancer therapeutics. Bacterial anticancer agents such as antibiotics, bacteriocins, non-ribosomal peptides, polyketides, toxins, etc. These are adopted different mechanisms of actions such as apoptosis, necrosis, reduced angiogenesis, inhibition of translation and splicing, and obstructing essential signaling pathways to kill cancer cells. Also, live tumor-targeting bacteria provided a unique therapeutic alternative for cancer treatment. This review summarizes the anticancer properties and mechanism of actions of the anticancer agents of bacterial origin and antitumor bacteria along with their possible future applications in cancer therapeutics.

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins.

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

Adsorption of Tolaasins, the Toxins Behind Mushroom Bacterial Blotch, by Microbacterium spp. is Insufficient for Its Detoxification.

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of bacterial blotch on several kinds of cultivated mushrooms. Our previous study reported on tolaasin detoxification by Microbacterium sp. K3-5 as a potential biocontrol of the disease. In this study, the tolaasin-detoxifying activities of various type strains of Microbacterium spp. were evaluated through chemical and biological assays. The bacterial cells of all tested strains of Microbacterium spp. showed tolaasin I-elimination from liquid phase. However, the toxin activities of tolaasins were still retained on the tolaasin-treated bacterial cells of all Microbacterium strains except M. foliorum NBRC 103072T. Furthermore, intact tolaasin I was recovered from the tolaasin-treated bacterial cells of all tested strains except M. foliorum NBRC 103072T. Our data reveal that Microbacterium spp. can be characterized as effective tolaasin I-eliminating bacteria through cell adsorption, but that this adsorption alone is insufficient for actual tolaasin detoxification. The biological degradation process must be needed to carry out the detoxification.

The role of membrane-bound metal ions in toxicity of a human cancer cell-active pore-forming toxin Cry41Aa from Bacillus thuringiensis.

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action.

Genetically-engineered plants yield an orally immunogenic PirA-like toxin from Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the main etiological agent of human gastroenteritis by seafood consumption and some strains from this species causing the Acute Hepatopancreatic Necrosis Disease in shrimp have been recently reported. The PirA-like toxin from V. parahaemolyticus (ToxA) has been recently reported as an attractive antigen implicated in subunit vaccine development. Since plants are attractive hosts for the production and delivery of vaccines in the present study plants expressing ToxA were developed to account with a low cost platform for the production and oral delivery of ToxA. Tobacco plants were genetically engineered by Agrobacterium-mediated transformation to stably integrate the ToxA-coding gene into the nuclear genome. Transgenic lines were rescued in kanamycin-containing medium and analyzed by ELISA to determine ToxA yields observing levels up to 9 µg g-1 FW leaf tissues. Western blot analysis confirmed the presence of the ToxA protein in plant extracts. Immunogenicity assessment of the plant-made ToxA was performed in mice, comprising a 4-dose oral immunization scheme; revealing the induction of anti-ToxA humoral responses (IgG in serum and IgA in feces). This study opens the path for the development of low cost plant-based vaccines against Vibrio parahaemolyticus.

Virulence mechanisms of plant-pathogenic Streptomyces species: an updated review.

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces.

Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease.

We found that Escherichia coli MazF toxin, an ACA-sequence-specific endoribonuclease, was sensitive to N6-methyladenosine (m6A), representing the first m6A-sensitive RNA cleavage enzyme. The methyl-sensitivity of MazF allowed simple analyses of both m6A demethylase and methyltransferase activity. Furthermore, the approach could be used for inhibitor screening.

Cyanobacterial Toxins of the Laurentian Great Lakes, Their Toxicological Effects, and Numerical Limits in Drinking Water.

Cyanobacteria are ubiquitous phototrophic bacteria that inhabit diverse environments across the planet. Seasonally, they dominate many eutrophic lakes impacted by excess nitrogen (N) and phosphorus (P) forming dense accumulations of biomass known as cyanobacterial harmful algal blooms or cyanoHABs. Their dominance in eutrophic lakes is attributed to a variety of unique adaptations including N and P concentrating mechanisms, N2 fixation, colony formation that inhibits predation, vertical movement via gas vesicles, and the production of toxic or otherwise bioactive molecules. While some of these molecules have been explored for their medicinal benefits, others are potent toxins harmful to humans, animals, and other wildlife known as cyanotoxins. In humans these cyanotoxins affect various tissues, including the liver, central and peripheral nervous system, kidneys, and reproductive organs among others. They induce acute effects at low doses in the parts-per-billion range and some are tumor promoters linked to chronic diseases such as liver and colorectal cancer. The occurrence of cyanoHABs and cyanotoxins in lakes presents challenges for maintaining safe recreational aquatic environments and the production of potable drinking water. CyanoHABs are a growing problem in the North American (Laurentian) Great Lakes basin. This review summarizes information on the occurrence of cyanoHABs in the Great Lakes, toxicological effects of cyanotoxins, and appropriate numerical limits on cyanotoxins in finished drinking water.

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 µg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.

Dissecting the contribution of Staphylococcus aureus α-phenol-soluble modulins to biofilm amyloid structure.

The opportunistic pathogen Staphylococcus aureus is recognized as one of the most frequent causes of biofilm-associated infections. The recently discovered phenol soluble modulins (PSMs) are small α-helical amphipathic peptides that act as the main molecular effectors of staphylococcal biofilm maturation, promoting the formation of an extracellular fibril structure with amyloid-like properties. Here, we combine computational, biophysical and in cell analysis to address the specific contribution of individual PSMs to biofilm structure. We demonstrate that despite their highly similar sequence and structure, contrary to what it was previously thought, not all PSMs participate in amyloid fibril formation. A balance of hydrophobic/hydrophilic forces and helical propensity seems to define the aggregation propensity of PSMs and control their assembly and function. This knowledge would allow to target specifically the amyloid properties of these peptides. In this way, we show that Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, prevents the assembly of amyloidogenic PSMs and disentangles their preformed amyloid fibrils.

Longitudinal whole genome analysis of pre and post drug treatment Mycobacterium tuberculosis isolates reveals progressive steps to drug resistance.

Tuberculosis (TB) is one of the leading causes of death due to an infectious disease in the world. Understanding the mechanisms of drug resistance has become pivotal in the detection and treatment of newly emerging resistant TB cases. We have analyzed three pairs of Mycobacterium tuberculosis strains pre- and post-drug treatment to identify mutations involved in the progression of resistance to the drugs rifampicin and isoniazid. In the rifampicin resistant strain, we confirmed a mutation in rpoB (S450L) that is known to confer resistance to rifampicin. We discovered a novel L101R mutation in the katG gene of an isoniazid resistant strain, which may directly contribute to isoniazid resistance due to the proximity of the mutation to the katG isoniazid-activating site. Another isoniazid resistant strain had a rare mutation in the start codon of katG. We also identified a number of mutations in each longitudinal pair, such as toxin-antitoxin mutations that may influence the progression towards resistance or may play a role in compensatory fitness. These findings improve our knowledge of drug resistance progression during therapy and provide a methodology to monitor longitudinal strains using whole genome sequencing, polymorphism comparison, and functional annotation.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantitation of Microcystins in Blue-Green Algal Dietary Supplements.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous detection and quantitation of seven microcystin congeners (1-7) and nodularin-R (8) in blue-green algal dietary supplements. Single-laboratory method validation data were collected in four supplement matrices (capsule, liquid, powder, and tablet) fortified at toxin concentrations from 0.25-2.00 µg/g (ppm). Average recoveries and relative standard deviations (RSD) using matrix-corrected solvent calibration curves were 101% (6% RSD) for all congeners and supplements investigated. Limits of detection (0.006-0.028 µg/g) and quantitation (0.018-0.084 µg/g) were sufficient to confirm the presence of microcystin contamination at the Oregon-mandated guidance concentration of 1.0 µg of microcystin-LReq/g. Quantitated concentrations of microcystin contamination in market-available Aphanizomenon flos-aquae blue-green algal supplements ranged from 0.18-1.87 µg of microcystin-LReq/g for detected congeners microcystin-LR, microcystin-LA, and microcystin-LY (3-5). Microcystin-RR, -YR, -LW, and -LF and nodularin-R (1, 2, and 6-8) were not detected in the supplements examined.

Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity.

In a time in which mucosal vaccines development has been delayed by the lack of safe and effective mucosal adjuvants, the combination of adjuvants has started to be explored as a strategy to obtain potent vaccine formulations. This study describes a novel adjuvant combination as an effective approach for a nasal vaccine - the association of the mast cell activator compound 48/80 with chitosan based nanoparticles. It was hypothesized that mucoadhesive nanoparticles would promote the cellular uptake and prolong the antigen residence time on nasal cavity. Simultaneously, mast cell activation would promote a local microenvironment favorable to the development of an immune response. To test this hypothesis, two different C48/80 loaded nanoparticles (NPs) were prepared: Chitosan-C48/80 NP (Chi-C48/80 NP) and Chitosan/Alginate-C48/80 NP (Chi/Alg-C48/80 NP). The potential as a vaccine adjuvant of the two delivery systems was evaluated and directly compared. Both formulations had a mean size near 500nm and a positive charge; however, Chi-C48/80 NP was a more effective adjuvant delivery system when compared with Chi/Alg-C48/80 NP or C48/80 alone. Chi-C48/80 NP activated mast cells at a greater extent, were better internalized by antigen presenting cells than Chi/Alg-C48/80 NP and successfully enhanced the nasal residence time of a model antigen. Superiority of Chi-C48/80 NP as adjuvant was also observed in vivo. Therefore, nasal immunization of mice with Bacillus anthracis protective antigen (PA) adsorbed on Chi-C48/80 NP elicited high levels of serum anti-PA neutralizing antibodies and a more balanced Th1/Th2 profile than C48/80 in solution or Chi/Alg-C48/80 NP. The incorporation of C48/80 within Chi NP also promoted a mucosal immunity greater than all the other adjuvanted groups tested, showing that the combination of a mast cell activator and chitosan NP could be a promising strategy for nasal immunization.

A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions.

Cytotoxic and pathogenic properties of Klebsiella oxytoca isolated from laboratory animals.

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.

Phytotoxins produced by plant pathogenic Streptomyces species.

Streptomyces is a large genus consisting of soil-dwelling, filamentous bacteria that are best known for their capability of producing a vast array of medically and agriculturally useful secondary metabolites. In addition, a small number of Streptomyces spp. are capable of colonizing and infecting the underground portions of living plants and causing economically important crop diseases such as potato common scab (CS). Research into the mechanisms of Streptomyces plant pathogenicity has led to the identification and characterization of several phytotoxic secondary metabolites that are known or suspected of contributing to diseases in various plants. The best characterized are the thaxtomin phytotoxins, which play a critical role in the development of CS, acid scab and soil rot of sweet potato. In addition, the best-characterized CS-causing pathogen, Streptomyces scabies, produces a molecule that is predicted to resemble the Pseudomonas syringae coronatine phytotoxin and which contributes to seedling disease symptom development. Other Streptomyces phytotoxic secondary metabolites that have been identified include concanamycins, FD-891 and borrelidin. Furthermore, there is evidence that additional, unknown metabolites may participate in Streptomyces plant pathogenicity. Such revelations have implications for the rational development of better management procedures for controlling CS and other Streptomyces plant diseases.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Small molecule inhibitors of Bacillus anthracis protective antigen proteolytic activation and oligomerization.

Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis , are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1-10 µM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells and, surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA's cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins.