Jeong-Hoon Chun

The Poly-γ-d-Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

ABSTRACT The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

Poly-γ-D-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits.

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis. The two major virulence factors of B. anthracis are exotoxin and the poly-gamma-d-glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA-PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 x LD(50) challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA-PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.

Complete Genome Sequence of Bacillus anthracis H9401, an Isolate from a Korean Patient with Anthrax

Bacillus anthracis H9401 (NCCP 12889) is an isolate from a Korean patient with gastrointestinal anthrax. The whole genome of H9401 was sequenced. It is a circular chromosome containing 5,480 open reading frames (ORFs) and two plasmids, pXO1 containing 202 ORFs and pXO2 containing 110 ORFs. H9401 shows high pathogenicity and genome sequence similarity to Ames Ancestor.

Bacillus anthracis Capsule Activates Caspase-1 and Induces Interleukin-1β Release from Differentiated THP-1 and Human Monocyte-Derived Dendritic Cells

ABSTRACT The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1β (IL-1β) in a dose-dependent manner. Evaluation of IL-1β processing by Western blotting revealed that cleaved IL-1β increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1β directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1β-converting enzyme (ICE). The extracellular release of IL-1β in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1β. These results demonstrate that B. anthracis PGA elicits IL-1β production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax.

The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

Objective Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. Methods Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. Results To examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. Conclusion Neutralizing-antibody titers can be used as a surrogate marker.

Construction of Luminescence- and Fluorescence-Tagged Burkholderia pseudomallei for Pathogen Tracking in a Mouse Model

Molecular imaging is a powerful method for tracking various infectious disease-causing pathogens in host organisms. Currently, a dual molecular imaging method that can provide temporal and spatial information on infected hosts at the organism, organ, tissue, and cellular levels simultaneously has not been reported for Burkholderia pseudomallei, a high-risk pathogen that causes melioidosis. In this study, we have established an experimental method that provides spatiotemporal information on infected hosts using luminescent and fluorescent dual-labeled B. pseudomallei. Using this method, we visualized B. pseudomallei infection at the organism, organ, and tissue levels in a BALB/c mouse model by detecting its luminescence and fluorescence. The infection of B. pseudomallei at the cellular level was also visualized by its emitted fluorescence in infected macrophage cells. This method could be an extremely useful and applicable tool to study the pathogenesis of B. pseudomallei-related infectious diseases.

Performance Characterization of Two-Dimensional Paper Chromatography-based Biosensors for Biodefense, Exemplified by Detection of Bacillus anthracis Spores

Bacillus anthracis (B. anthracis), the causative agent of anthrax disease, is a Gram-positive spore-forming bacterium which can be used as a threatening bioterrorism agent. We developed enzyme-linked immunosorbent assay (ELISA)-on-a-chip biosensors for rapid, sensitive analysis of B. anthracis spores based on two-dimensional, cross-flow chromatography. In order to establish optimal assay conditions, a polyclonal antibody and four monoclonal antibodies against B. anthracis were raised and examined to characterize their analytical sensitivity as well as specificity. The biosensor results showed that a monoclonal antibody pair not only offered a relatively low detection limit for B. anthracis compared to other antibody combinations, but also displayed no cross-reactivity with other microorganisms belonging to the Bacillus genus. For detection of ELISA enzyme signal (e.g., horseradish peroxidase), chemiluminescent detection in combination with cooled charge-coupled device enhanced the sensor performance in terms of assay time, compared to that achieved by colorimetry. Under optimal conditions, the biosensor was able to detect a minimum threshold of 5×103 and 5×102 spores/mL for two different B. anthracis strains, NCCP 12860 (Sterne) and NCCP 10666 (Haman #1), respectively. Furthermore, the chemiluminometric sensor was minimally affected by the presence of potential interferents in samples such as baby powder, skim milk, and sucrose, indicating its potential utility for the analysis of bioterrorism agents directly in the field.