Hsin-Hsien Huang

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

Background Photocatalysis of titanium dioxide (TiO2) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO2 substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. Methodology/Principal Findings Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. Conclusion/Significance Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host.

Antiplatelet Activities of Anthrax Lethal Toxin Are Associated with Suppressed p42/44 and p38 Mitogen-Activated Protein Kinase Pathways in the Platelets

Anthrax lethal toxin (LT) is the major virulence factor produced by Bacillus anthracis, but the mechanism by which it induces high mortality remains unclear. We found that LT treatment could induce severe hemorrhage in mice and significantly suppress human whole-blood clotting and platelet aggregation in vitro. In addition, LT could inhibit agonist-induced platelet surface P-selectin expression, resulting in the inhibition of platelet-endothelial cell engagements. Data from Western blot analysis indicated that LT treatment resulted in the suppression of p42/44 and p38 mitogen-activated protein kinase pathways in platelets. Combined treatments with LT and antiplatelet agents such as aspirin and the RGD-containing disintegrin rhodostomin significantly increased mortality in mice. Our data suggest that platelets are a pathogenic target for anthrax LT.

DNA Vaccination Using the Fragment C of Botulinum Neurotoxin Type A Provided Protective Immunity in Mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.

Cytotoxic effects of anthrax lethal toxin on macrophage-like cell line J774A.1

The cytotoxic effects of anthrax lethal toxin purified from an avirulent strain were examined on mouse macrophage-like J774A.1 cells. Cell death induced by high concentration of purified lethal toxin had the characteristics of necrosis. At lower concentrations, the toxin caused no morphological change and most of the cells were viable. Interestingly, apoptotic cells were observed when the cells were preincubated with a serine/threonine phosphatase inhibitor, calyculin A, and then exposed to a toxin concentration of 0.1 μg/ml. This is the first report that lethal toxin of the anthrax bacillus can induce both necrosis and apoptosis and that protein phosphatases are implicated in the regulation of bacterial toxin-induced apoptosis.

Calyculin A sensitive protein phosphatase is required for Bacillus anthracis lethal toxin induced cytotoxicity.

Previous studies have shown that the Bacillus anthracis lethal toxin can induce both necrosis and apoptosis in mouse macrophage-like J774A.1 cells depending on both the toxin concentration and the phosphatase activity. In this study several protein kinase or phosphatase inhibitors were employed to evaluate the hypothesis that the lethal toxin induces cell death via protein phosphorylation processes. Pretreatment with a serine/threonine phosphatase inhibitor Calyculin A (300 nM) could inhibit about 78% of cell death induced by the lethal toxin, whereas inhibitors of kinases, such as H7, HA, Sphingosine, and Genestein, but other inhibitors of phosphatases, such as Okadaic acid, Tautomycin, and Cyclosporin A, did not. In addition, recent reports have demonstrated that the MEK1 protein may serve as a proteolytic target within its N-terminus for lethal factor cleavage. In this study, Calyculin A is shown to enhance the phosphorylation of the MEK1 protein. This prevents the cleavage of the MEK1 by lethal factor. These results suggest that a putative Calyculin A-sensitive protein phosphatase is involved in anthrax toxin induced cytotoxicity and that the blocking effect of Calyculin A on lethal factor cytotoxicity may be mediated through the MEK signaling pathway.

Sublethal Doses of Anthrax Lethal Toxin on the Suppression of Macrophage Phagocytosis

Background Lethal toxin (LT), the major virulence factor produced by Bacillus anthracis, has been shown to suppress the immune system, which is beneficial to the establishment of B. anthracis infections. It has been suggested that the suppression of MEK/MAPK signaling pathways of leukocytes contributes to LT-mediated immunosuppressive effects. However, the involvement of MAPK independent pathways has not been clearly elucidated; nor has the crucial role played by LT in the early stages of infection. Determining whether LT exerts any pathological effects before being enriched to an MEK inhibitory level is an important next step in the furtherance of this field. Methodology/Principal Findings Using a cell culture model, we determined that low doses of LT inhibited phagocytosis of macrophages, without influencing MAPK pathways. Consistent low doses of LT significantly suppressed bacterial clearance and enhanced the mortality of mice with bacteremia, without suppressing the MEK1 of splenic and peripheral blood mononuclear cells. Conclusion/Significance These results suggest that LT suppresses the phagocytes in a dose range lower than that required to suppress MEK1 in the early stages of infection.

Suppressive effects of anthrax lethal toxin on megakaryopoiesis.

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34+ cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.

Erythropoiesis suppression is associated with anthrax lethal toxin-mediated pathogenic progression.

Anthrax is a disease caused by the bacterium Bacillus anthracis, which results in high mortality in animals and humans. Although some of the mechanisms are already known such as asphyxia, extensive knowledge of molecular pathogenesis of this disease is deficient and remains to be further investigated. Lethal toxin (LT) is a major virulence factor of B. anthracis and a specific inhibitor/protease of mitogen-activated protein kinase kinases (MAPKKs). Anthrax LT causes lethality and induces certain anthrax-like symptoms, such as anemia and hypoxia, in experimental mice. Mitogen-activated protein kinases (MAPKs) are the downstream pathways of MAPKKs, and are important for erythropoiesis. This prompted us to hypothesize that anemia and hypoxia may in part be exacerbated by erythropoietic dysfunction. As revealed by colony-forming cell assays in this study, LT challenges significantly reduced mouse erythroid progenitor cells. In addition, in a proteolytic activity-dependent manner, LT suppressed cell survival and differentiation of cord blood CD34+-derived erythroblasts in vitro. Suppression of cell numbers and the percentage of erythroblasts in the bone marrow were detected in LT-challenged C57BL/6J mice. In contrast, erythropoiesis was provoked through treatments of erythropoietin, significantly ameliorating the anemia and reducing the mortality of LT-treated mice. These data suggested that suppressed erythropoiesis is part of the pathophysiology of LT-mediated intoxication. Because specific treatments to overcome LT-mediated pathogenesis are still lacking, these efforts may help the development of effective treatments against anthrax.

Activated protein C ameliorates Bacillus anthracis lethal toxin-induced lethal pathogenesis in rats

BackgroundLethal toxin (LT) is a major virulence factor of Bacillus anthracis. Sprague Dawley rats manifest pronounced lung edema and shock after LT treatments, resulting in high mortality. The heart failure that is induced by LT has been suggested to be a principal mechanism of lung edema and mortality in rodents. Since LT-induced death occurs more rapidly in rats than in mice, suggesting that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy may contribute to circulatory failure and lung injury. However, the effect of LT on coagulation-induced lung dysfunction is unclear.MethodsTo investigate the involvement of coagulopathy in LT-mediated pathogenesis, the mortality, lung histology and coagulant levels of LT-treated rats were examined. The effects of activated protein C (aPC) on LT-mediated pathogenesis were also evaluated.ResultsFibrin depositions were detected in the lungs of LT-treated rats, indicating that coagulation was activated. Increased levels of plasma D-dimer and thrombomodulin, and the ameliorative effect of aPC further suggested that the activation of coagulation-fibrinolysis pathways plays a role in LT-mediated pathogenesis in rats. Reduced mortality was associated with decreased plasma levels of D-dimer and thrombomodulin following aPC treatments in rats with LT-mediated pathogenesis.ConclusionsThese findings suggest that the activation of coagulation in lung tissue contributes to mortality in LT-mediated pathogenesis in rats. In addition, anticoagulant aPC may help to develop a feasible therapeutic strategy.

Acquired coagulant factor VIII deficiency induced by Bacillus anthracis lethal toxin in mice

Mice treated with anthrax lethal toxin (LT) exhibit hemorrhage caused by unknown mechanisms. Moreover, LT treatment in mice induced liver damage. In this study, we hypothesized that a suppressed coagulation function may be associated with liver damage, because the liver is the major producing source of coagulation factors. The hepatic expression of coagulant factors and the survival rates were analyzed after cultured cells or mice were exposed to LT. In agreement with our hypothesis, LT induces cytotoxicity against hepatic cells in vitro. In addition, suppressed expression of coagulation factor VIII (FVIII) in the liver is associated with a prolonged plasma clotting time in LT-treated mice, suggesting a suppressive role of LT in coagulation. Accordingly, we further hypothesized that a loss-of-function approach involving treatments of an anticoagulant should exacerbate LT-induced abnormalities, whereas a gain-of-function approach involving injections of recombinant FVIII to complement the coagulation deficiency should ameliorate the pathogenesis. As expected, a sublethal dose of LT caused mortality in the mice that were non-lethally pretreated with an anticoagulant (warfarin). By contrast, treatments of recombinant FVIII reduced the mortality from a lethal dose of LT in mice. Our results indicated that LT-induced deficiency of FVIII is involved in LT-mediated pathogenesis. Using recombinant FVIII to correct the coagulant defect may enable developing a new strategy to treat anthrax.