Friends or Foes? Emerging Impacts of Biological Toxins.

Toxins are substances produced from biological sources (e.g., animal, plants, microorganisms) that have deleterious effects on a living organism. Despite the obvious health concerns of being exposed to toxins, they are having substantial positive impacts in a number of industrial sectors. Several toxin-derived products are approved for clinical, veterinary, or agrochemical uses. This review sets out the case for toxins as 'friends' that are providing the basis of novel medicines, insecticides, and even nucleic acid sequencing technologies. We also discuss emerging toxins ('foes') that are becoming increasingly prevalent in a range of contexts through climate change and the globalisation of food supply chains and that ultimately pose a risk to health.

Delayed presence of alternatively activated macrophages during a Francisella tularensis infection.

Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive ("classical") macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive ("alternative") cells appear later and in low numbers during the development of the disease tularemia.

Targeting the "Rising DAMP" during a Francisella tularensis Infection.

Antibiotic efficacy is greatly enhanced the earlier it is administered following infection with a bacterial pathogen. However, in a clinical setting antibiotic treatment usually commences following the onset of symptoms, which in some cases (e.g., biothreat agents) may be too late. In a BALB/c murine intranasal model of infection for Francisella tularensis SCHU S4 infection, we demonstrate during a time course experiment that proinflammatory cytokines and the damage-associated molecular pattern HMGB1 were not significantly elevated above naive levels in tissue or sera until 72 h postinfection. HMGB1 was identified as a potential therapeutic target that could extend the window of opportunity for the treatment of tularemia with antibiotics. Antibodies to HMGB1 were administered in conjunction with a delayed/suboptimal levofloxacin treatment of F. tularensis We found in the intranasal model of infection that treatment with anti-HMGB1 antibody, compared to an isotype IgY control antibody, conferred a significant survival benefit and decreased bacterial loads in the spleen and liver but not the lung (primary loci of infection) 4 days into infection. We also observed an increase in the production of gamma interferon in all tested organs. These data demonstrate that treatment with anti-HMGB1 antibody is beneficial in enhancing the effectiveness of current antibiotics in treating tularemia. Strategies of this type, involving antibiotics in combination with immunomodulatory drugs, are likely to be essential for the development of a postexposure therapeutic for intracellular pathogens.

Reactive oxygen species and the MEK/ERK pathway are involved in the toxicity of clostridium perfringens α-toxin, a prototype bacterial phospholipase C.

Clostridium perfringens, the most broadly distributed pathogen in nature, produces a prototype phospholipase C, also called α-toxin, which plays a key role in the pathogenesis of gas gangrene. α-Toxin causes plasma membrane disruption at high concentrations, but the role of intracellular mediators in its toxicity at low concentrations is unknown. This work demonstrates that α-toxin causes oxidative stress and activates the MEK/ERK pathway in cultured cells and furthermore provides compelling evidence that O(2)(-.), hydrogen peroxide, and the OH(.) radical are involved in its cytotoxic and myotoxic effects. The data show that antioxidants and MEK1 inhibitors reduce the cytotoxic and myotoxic effects of α-toxin and demonstrate that edaravone, a clinically used hydroxyl radical trap, reduces the myonecrosis and the mortality caused by an experimental infection with C. perfringens in a murine model of gas gangrene. This knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis.

From "crisis to recovery": A complete insight into the mechanisms of chlorine injury in the lung.

Chlorine (Cl2) gas is a toxic industrial chemical (TIC) that poses a hazard to human health following accidental and/or intentional (e.g. terrorist) release. By using a murine model of sub-lethal Cl2 exposure we have examined the airway hyper responsiveness, cellular infiltrates, transcriptomic and proteomic responses of the lung. In the "crisis" phase at 2 h and 6 h there is a significant decreases in leukocytes within bronchoalveolar lavage fluid accompanied by an upregulation within the proteome of immune pathways ultimately resulting in neutrophil influx at 24 h. A flip towards "repair" in the transcriptome and proteome occurs at 24 h, neutrophil influx and an associated drop in the lung function persisting until 14 d post-exposure and subsequent "recovery" after 28 days. Collectively, this research provides new insights into the mechanisms of damage, early global responses and processes of repair induced in the lung following the inhalation of Cl2.

Pegylation of antimicrobial peptides maintains the active peptide conformation, model membrane interactions, and antimicrobial activity while improving lung tissue biocompatibility following airway delivery.

Antimicrobial peptides (AMPs) have therapeutic potential, particularly for localized infections such as those of the lung. Here we show that airway administration of a pegylated AMP minimizes lung tissue toxicity while nevertheless maintaining antimicrobial activity. CaLL, a potent synthetic AMP (KWKLFKKIFKRIVQRIKDFLR) comprising fragments of LL-37 and cecropin A peptides, was N-terminally pegylated (PEG-CaLL). PEG-CaLL derivatives retained significant antimicrobial activity (50% inhibitory concentrations [IC(50)s] 2- to 3-fold higher than those of CaLL) against bacterial lung pathogens even in the presence of lung lining fluid. Circular dichroism and fluorescence spectroscopy confirmed that conformational changes associated with the binding of CaLL to model microbial membranes were not disrupted by pegylation. Pegylation of CaLL reduced AMP-elicited cell toxicity as measured using in vitro lung epithelial primary cell cultures. Further, in a fully intact ex vivo isolated perfused rat lung (IPRL) model, airway-administered PEG-CaLL did not result in disruption of the pulmonary epithelial barrier, whereas CaLL caused an immediate loss of membrane integrity leading to pulmonary edema. All AMPs (CaLL, PEG-CaLL, LL-37, cecropin A) delivered to the lung by airway administration showed limited (<3%) pulmonary absorption in the IPRL with extensive AMP accumulation in lung tissue itself, a characteristic anticipated to be beneficial for the treatment of pulmonary infections. We conclude that pegylation may present a means of improving the lung biocompatibility of AMPs designed for the treatment of pulmonary infections.

RIPpore: A Novel Host-Derived Method for the Identification of Ricin Intoxication through Oxford Nanopore Direct RNA Sequencing.

Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop in the large ribosomal subunit, leading to the inhibition of protein translation and cell death. We postulated that this depurination event could be detected using Oxford Nanopore Technologies (ONT) direct RNA sequencing, detecting a change in charge in the ricin loop. In this study, A549 cells were exposed to ricin for 2-24 h in order to induce depurination. In addition, a novel software tool was developed termed RIPpore that could quantify the adenine modification of ribosomal RNA induced by ricin upon respiratory epithelial cells. We provided demonstrable evidence for the first time that this base change detected is specific to RIP activity using a neutralising antibody against ricin. We believe this represents the first detection of depurination in RNA achieved using ONT sequencers. Collectively, this work highlights the potential for ONT and direct RNA sequencing to detect and quantify depurination events caused by ribosome-inactivating proteins such as ricin. RIPpore could have utility in the evaluation of new treatments and/or in the diagnosis of exposure to ricin.

In Vitro Priming of Human T Cells by Dendritic Cells Provides a Screening Tool for Candidate Vaccines for Burkholderia pseudomallei.

Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1ß. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimulated the expression of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 from both myeloid and plasmacytoid DCs. Furthermore, B. pseudomallei-pulsed DCs cultured with naïve syngeneic T cells ex vivo, induced the activation and proliferation of the CD4+ T-cell population, which was identified by cell surface marker staining using flow cytometry. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be used to identify immunogenic components of B. pseudomallei for future therapies and vaccines.

Establishment of a Novel Oral Murine Model of Ricin Intoxication and Efficacy Assessment of Ovine Ricin Antitoxins.

Ricin, produced from the castor beans of Ricinus communis, is a cytotoxin that exerts its action by inactivating ribosomes and causing cell death. Accidental (e.g., ingestion of castor beans) and/or intentional (e.g., suicide) exposure to ricin through the oral route is an area of concern from a public health perspective and no current licensed medical interventions exist to protect from the action of the toxin. Therefore, we examined the oral toxicity of ricin in Balb/C mice and developed a robust food deprivation model of ricin oral intoxication that has enabled the assessment of potential antitoxin treatments. A lethal oral dose was identified and mice were found to succumb to the toxin within 48 h of exposure. We then examined whether a despeciated ovine F(ab')2 antibody fragment, that had previously been demonstrated to protect mice from exposure to aerosolised ricin, could also protect against oral intoxication. Mice were challenged orally with an LD99 of ricin, and 89 and 44% of mice exposed to this otherwise lethal exposure survived after receiving either the parent anti-ricin IgG or F(ab')2, respectively. Combined with our previous work, these results further highlight the benefit of ovine-derived polyclonal antibody antitoxin in providing post-exposure protection against ricin intoxication.

Erratum: Blume, C., et al. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections. Pathogens 2016, 5, 53.

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Common Host Responses in Murine Aerosol Models of Infection Caused by Highly Virulent Gram-Negative Bacteria from the Genera Burkholderia, Francisella and Yersinia.

Highly virulent bacterial pathogens cause acute infections which are exceptionally difficult to treat with conventional antibiotic therapies alone. Understanding the chain of events that are triggered during an infection of a host has the potential to lead to new therapeutic strategies. For the first time, the transcriptomic responses within the lungs of Balb/C mice have been compared during an acute infection with the intracellular pathogens Burkholderia pseudomallei, Francisella tularensis and Yersinia pestis. Temporal changes were determined using RNAseq and a bioinformatics pipeline; expression of protein was also studied from the same sample. Collectively it was found that early transcriptomic responses within the infected host were associated with the (a) slowing down of critical cellular functions, (b) production of circulatory system components, (c) lung tissue integrity, and (d) intracellular regulatory processes. One common molecule was identified, Errfi1 (ErbB receptor feedback inhibitor 1); upregulated in response to all three pathogens and a potential novel marker of acute infection. Based upon the pro-inflammatory responses observed, we sought to synchronise each infection and report that 24 h p.i. of B. pseudomallei infection closely aligned with 48 h p.i. of infection with F. tularensis and Y. pestis. Post-transcriptional modulation of RANTES expression occurred across all pathogens, suggesting that these infections directly or indirectly modulate cell trafficking through chemokine expression/detection. Collectively, this unbiased NGS approach has provided an in-depth characterisation of the host transcriptome following infection with these highly virulent pathogens ultimately aiding in the development of host-directed therapies as adjuncts or alternatives to antibiotic treatment.

Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis.

Recombinant Bacillus subtilis endospores have been used to vaccinate against tetanus and anthrax. In this work, we have developed spores that could be used to vaccinate against Clostridium perfringens alpha toxin and that could be used to protect against gas gangrene in humans and necrotic enteritis in poultry. The primary active agent in both cases is alpha toxin. A carboxy-terminal segment of the alpha toxin gene (cpa) fused to the glutathione-S-transferase (GST) gene was cloned in B. subtilis such that the encoded GST-Cpa(247-370) polypeptide had been expressed in the following three different ways: expression in the vegetative cell, expression on the surface of the spore coat (fused to the CotB spore coat protein), and a combined approach of spore coat expression coupled with expression in the vegetative cell. Mice immunized orally or nasally with three doses of recombinant spores that carried GST-Cpa(247-370) on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa(247-370)-specific immunoglobulin G (IgG) responses in their sera, a Th2 bias, and secretory IgA responses in saliva, feces, and lung samples. Neutralizing IgG antibodies to alpha toxin were detected using in vitro and in vivo assays, and a toxin challenge established protection. Mice immunized nasally or orally with recombinant spores were protected against a challenge with 12 median lethal doses of alpha toxin. Existing use of spores as competitive exclusion agents in animal feeds supports their use as a potentially economical and heat-stable vaccine for the poultry industry.

Current and future developments in the treatment of virus-induced hypercytokinemia.

Emerging pathogenic viruses such as Ebola and Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV) can cause acute infections through the evasion of the host's antiviral immune responses and by inducing the upregulation of inflammatory cytokines. This immune dysregulation, termed a cytokine storm or hypercytokinemia, is potentially fatal and is a significant underlying factor in increased mortality of infected patients. The prevalence of global outbreaks in recent years has offered opportunities to study the progression of various viral infections and have provided an improved understanding of hypercytokinemia associated with these diseases. However, despite this increased knowledge and the study of the infections caused by a range of emerging viruses, the therapeutic options still remain limited. This review aims to explore alternative experimental strategies for treating hypercytokinemia induced by the Ebola, avian influenza and Dengue viruses; outlining their modes of action, summarizing their preclinical assessments and potential clinical applications.

Production, Characterisation and Testing of an Ovine Antitoxin against Ricin; Efficacy, Potency and Mechanisms of Action.

Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab')2 polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab')2 antitoxin as a treatment for ricin intoxication.

Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections.

The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24-72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel(®) reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option.

Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity.

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.

Mechanisms of Disease: Host-Pathogen Interactions between Burkholderia Species and Lung Epithelial Cells.

Members of the Burkholderia species can cause a range of severe, often fatal, respiratory diseases. A variety of in vitro models of infection have been developed in an attempt to elucidate the mechanism by which Burkholderia spp. gain entry to and interact with the body. The majority of studies have tended to focus on the interaction of bacteria with phagocytic cells with a paucity of information available with regard to the lung epithelium. However, the lung epithelium is becoming more widely recognized as an important player in innate immunity and the early response to infections. Here we review the complex relationship between Burkholderia species and epithelial cells with an emphasis on the most pathogenic species, Burkholderia pseudomallei and Burkholderia mallei. The current gaps in knowledge in our understanding are highlighted along with the epithelial host-pathogen interactions that offer potential opportunities for therapeutic intervention.

Immune profiling of the progression of a BALB/c mouse aerosol infection by Burkholderia pseudomallei and the therapeutic implications of targeting HMGB1.

INTRODUCTION: The role of damage-associated molecular pattern HMGB1 signalling in a murine BALB/c model of severe respiratory melioidosis (Burkholderia pseudomallei infection) was explored in this study. METHODS: Time course experiments were performed. RESULTS: It was established that HMGB1 was released in concert with increasing weight of organs and increasing concentration of liver enzymes in the blood a short time after cytokine release. Differences in the cytokine response between organs were observed, where the lungs contained higher concentrations of chemokines and interleukin 17, while the spleen produced more interferon-gamma, which is essential in the host defence against B. pseudomallei. This is evidence as to why the disease is seemingly more severe in the respiratory form. The effect of depleting HMGB1 using an antibody was also evaluated. It was found that this treatment significantly reduced bacterial load in the liver, spleen, and, to a greater degree, in the lungs. Cytokine quantification indicated that this reduction in bacterial load is likely due to the treatment reducing the release of a variety of pro-inflammatory cytokines. CONCLUSION: It is concluded that anti-HMGB1 treatment would be effective alongside other therapeutics, where it would reduce the characteristic over-inflammation associated with late stage infection.

Targeting the "cytokine storm" for therapeutic benefit.

Inflammation is the body's first line of defense against infection or injury, responding to challenges by activating innate and adaptive responses. Microbes have evolved a diverse range of strategies to avoid triggering inflammatory responses. However, some pathogens, such as the influenza virus and the Gram-negative bacterium Francisella tularensis, do trigger life-threatening "cytokine storms" in the host which can result in significant pathology and ultimately death. For these diseases, it has been proposed that downregulating inflammatory immune responses may improve outcome. We review some of the current candidates for treatment of cytokine storms which may prove useful in the clinic in the future and compare them to more traditional therapeutic candidates that target the pathogen rather than the host response.