Jungchan Park

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.

3,4-Disubstituted oxazolidin-2-ones as constrained ceramide analogs with anticancer activities.

Heterocyclic analogs of ceramide as 3-alkanoyl or benzoyl-4-(1-hydroxy-2-enyl)-oxazolidin-2-ones were designed by binding of primary alcohol and amide in sphinogosine backbone as a carbamate. They were synthesized by addition of acyl halide to the common ring 4-(1-t-butyldimethylsilyloxyhexadec-2-enyl)-oxazolidin-2-one which was elaborated from chiral aziridine-2-carboxylate including stereoselective reduction and ring opening reactions as key steps. Other analogs with different carbon frame at C4 position which is corresponding to the sphingoid backbone were prepared from 3-cyclopentanecarbonyl-4-(1-t-butyldimethylsilyloxybut-2-enyl)-oxazolidin-2-one and straight and cyclic alkenes by cross metathesis. All compounds were tested as antileukemic drugs against human leukemia HL-60 cells. Many of them including propionyl, cyclopentanoyl and p-nitrobenzoyl-4-(1-hydroxyhexadec-2-enyl)-oxazolidin-2-ones showed better antileukemic activities than natural C2-ceramide with good correlation between cell death and DNA fragmentation. There is a drastic change of the activities by the carbon chain lengths at C4 position. Cytotoxicity was induced by caspase activation without significant accumulation of endogenous ceramide concentration or any perturbation of ceramide metabolism.

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.

The Vibrio cholerae VarS/VarA two-component system controls the expression of virulence proteins through ToxT regulation.

Although the conditions for inducing virulence protein expression in vitro are different, both classical and El Tor biotypes of Vibrio cholerae have been reported to regulate the expression of virulence proteins such as cholera toxin (CT) and toxin-coregulated pili (Tcp) through the ToxR/S/T system. The transcription activator ToxR responds to environmental stimuli such as pH and temperature and activates the second transcriptional regulator ToxT, which upregulates expression of virulence proteins. In addition to the ToxR/S/T signalling system, V. cholerae has been proposed to utilize another two-component system VarS/VarA to modulate expression of virulence genes. Previous study has shown that VarA of the VarS/VarA system is involved in the regulation of virulence proteins in the classical V. cholerae O395 strain; however, no further analysis was performed concerning VarS. In this study, we constructed varS mutants derived from the classical O395 and El Tor C6706 strains and demonstrated that VarS is also involved in the expression of the virulence proteins CT and Tcp from the V. cholerae classical and El Tor strains. This expression is through regulation of ToxT expression in response to environmental changes due to different toxin-inducing conditions.

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 Is a Novel Toll-Like Receptor 2 Agonist

ABSTRACT Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1β (IL-1β) (M. H. Cho et al., Infect Immun 78:387–392, 2010, http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4−/− but not TLR2−/− BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88−/− BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.

Identification and validation of calmodulin as a binding protein of an anti-proliferative small molecule 3,4-dihydroisoquinolinium salt.

IHY‐153 (2‐(2,5‐difluorobenzyl)‐3,4‐dihydro‐5‐(10‐hydroxydecyl)‐6‐methoxy‐1‐undecylisoquinolinium bromide) was recently discovered as a small molecule that potently inhibits proliferation of tumor cells by inducing cell‐cycle arrest at G0‐G1 phase. To investigate the basis of anti‐proliferative activity of IHY‐153, cellular binding proteins of biotinyl‐IHY‐153 were screened using T7 phage displayed human cDNA libraries. Calmodulin‐expressing phage specifically bound to immobilized IHY‐153 in a Ca2+‐dependent manner. The interaction between IHY‐153 and Ca2+/CaM was validated through phage competition binding assays, surface plasmon resonance analysis, and molecular modeling. IHY‐153 induced sustained phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2 and subsequently increased p21WAF1 expression in colon cancer cells. These results demonstrate that IHY‐153, a novel small molecule, targets Ca2+/CaM and indicate that this compound functions as an anti‐proliferative agent by influencing Ca2+/CaM‐dependent signal transduction.

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.

Muramyl dipeptide potentiates a Bacillus anthracis poly-γ-d-glutamic acid capsule surrogate that induces maturation and activation of mouse dendritic cells

HighlightsMDP enhances PGA‐induced maturation of mouse DCs.MDP enhances production of inflammatory mediators by PGA.TLR2, NOD2, MAP kinase, and NF‐&kgr;B signaling pathways are involved in PGA/MDP‐induced cytokine production.MDP increases lymphocyte activation induced by PGA‐matured DCs. &NA; Poly‐&ggr;‐d‐glutamic acid (PGA) of anthrax is an important pathogenic factor due to its anti‐phagocytic activity. Additionally, PGA has the ability to activate mouse macrophages for the secretion of cytokines through Toll‐like receptor (TLR) 2. Peptidoglycan (PGN), a major bacterial cell‐wall component, induces inflammatory responses in the host. We assessed whether PGA can induce maturation and cytokine expression in immature mouse dendritic cells (DCs) in the existence of muramyl dipeptide (MDP), the minimum motif of PGN with immunostimulatory activity. Stimulation of immature DCs with PGA or MDP alone augmented expression of costimulatory molecules and MHC class II proteins, which are all cell surface markers indicative of maturation. The observed effects were further enhanced by costimulation of PGA and MDP. PGA alone was sufficient to induce expression of TNF‐&agr;, IL‐6, MCP‐1, and MIP1‐&agr;, whereas MDP alone did not under the same conditions. Treatment with MDP enhanced PGA‐induced expression of the tested inflammatory mediators; however, the synergistic effect found for PGA and MDP was not observed in TLR2‐ or nucleotide‐binding oligomerization domain (NOD) 2–knockout DCs. Additionally, MDP augmented PGA‐induced MAP kinases and NF‐&kgr;B activation, which is crucial for expression of cytokines. Furthermore, MAP kinase and NF‐&kgr;B inhibitors attenuated MDP enhancement of PGA‐induced cytokine production. In addition, co‐culture of splenocytes and PGA/MDP‐matured DCs induced higher expression of IL‐2 and IFN‐&ggr; compared to that of splenocytes and PGA‐matured DCs. Collectively, our results suggest that PGA and MDP cooperatively induce inflammatory responses in mouse DCs through TLR2 and NOD2 via MAP kinase and NF‐&kgr;B pathways, subsequently leading to lymphocyte activation.