Jason L. Larabee

Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

Glycogen Synthase Kinase 3 Activation Is Important for Anthrax Edema Toxin-Induced Dendritic Cell Maturation and Anthrax Toxin Receptor 2 Expression in Macrophages

ABSTRACT Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3β. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocyte-derived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.

Increased cAMP in Monocytes Augments Notch Signaling Mechanisms by Elevating RBP-J and Transducin-like Enhancer of Split (TLE)

Background: Many bacterial pathogens, such as Bacillus anthracis, increase cAMP in monocytes, leading to disruption of immune responses. Results: In human monocytes, cAMP up-regulates transducin-like enhancer of split (TLE) and activates Notch signaling. Conclusion: Our findings demonstrate novel signaling mechanisms used by cAMP to enhance Notch signaling. Significance: This work delineates how cAMP modifies a signaling pathway critical to innate immune responses during infection. In cells of the innate immune system, pathological increases in intracellular cAMP attenuate immune responses and contribute to infections by bacteria such as Bacillus anthracis. In this work, cAMP from B. anthracis edema toxin (ET) is found to activate the Notch signaling pathway in both mouse macrophages and human monocytes. ET as well as a cell-permeable activator of PKA induce Notch target genes (HES1, HEY1, IL2RA, and IL7R) and are able to significantly enhance the induction of these Notch target genes by a Toll-like receptor ligand. Elevated cAMP also resulted in increased levels of Groucho/transducin-like enhancer of Split (TLE) and led to increased amounts of a transcriptional repressor complex consisting of TLE and the Notch target Hes1. To address the mechanism used by ET to activate Notch signaling, components of Notch signaling were examined, and results revealed that ET increased levels of recombinant recognition sequence binding protein at the Jκ site (RBP-J), a DNA binding protein and principal transcriptional regulator of Notch signaling. Overexpression studies indicated that RBP-J was sufficient to activate Notch signaling and potentiate LPS-induced Notch signaling. Further examination of the mechanism used by ET to activate Notch signaling revealed that C/EBP β, a transcription factor activated by cAMP, helped activate Notch signaling and up-regulated RBP-J. These studies demonstrate that cAMP activates Notch signaling and increases the expression of TLE, which could be an important mechanism utilized by cAMP to suppress immune responses.

Exposure of neutralizing epitopes in the carboxyl-terminal domain of TcdB is altered by a proximal hypervariable region.

Background: TcdB from 027 strains is not as sensitive to antibody neutralization as TcdB from 012 strains, although neutralizing epitopes are present on both forms. Results: Sequence alterations in strain-specific TcdB influence intramolecular protein-protein interactions and exposure of neutralizing epitopes. Conclusion: Our work uncovers a mechanism used by TcdB to shield neutralizing epitopes. Significance: This study demonstrates how strain-specific TcdB can avoid antibody targeting. The sequence, activity, and antigenicity of TcdB varies between different strains of Clostridium difficile. As a result, ribotype-specific forms of TcdB exhibit different toxicities and are not strongly cross-neutralized. Using a combination of biochemical and immunological approaches, we compared two important variants of TcdB (TcdB012 and TcdB027) to identify the mechanisms through which sequence differences alter epitopes and activity of the toxin. These analyses led to the discovery of a critical variation in the 1753–1851 (B2′) region of TcdB, which affects the exposure of neutralizing epitopes in the toxin. Sequence comparisons found that the B2′ region exhibits only 77% identity and is the most variable sequence between the two forms of TcdB. A combination of biochemical, analytical, and mutagenesis experiments revealed that the B2′ region promotes protein-protein interactions. These interactions appear to shield neutralizing epitopes that would otherwise be exposed in the toxin, an event found to be less prominent in TcdB012 due to sequence differences in the 1773–1780 and 1791–1798 regions of the B2′ domain. When the carboxyl-terminal domains of TcdB012 and TcdB027 are swapped, neutralization experiments suggest that the amino terminus of TcdB interacts with the B2′ region and impacts the exposure of neutralizing epitopes in the carboxyl terminus. Collectively, these data suggest that variations in the B2′ region affect protein-protein interactions within TcdB and that these interactions influence the exposure of neutralizing epitopes.

Serum Profiling of Rat Dermal Exposure to JP-8 Fuel Reveals an Acute-Phase Response

ABSTRACT Dermal exposure to JP-8 petroleum jet fuel leads to toxicological responses in humans and rodents. Serum profiling is a molecular analysis of changes in the levels of serum proteins and other molecules in response to changes in physiology. This present study utilizes serum profiling approaches to examine biomolecular changes in the sera of rats exposed to dermal applications of JP-8 (jet propulsion fuel-8). Using gel electrophoresis and electrospray ionization (ESI) mass spectrometry (MS), levels of serum proteins as well as low-mass constituents were found to change after dermal exposures to JP-8. The serum protein levels altered included the acute-phase response proteins haptoglobin, ceruloplasmin, α1-inhibitor III, and apolipoprotein A-IV. Haptoglobin levels increased after a 1-day JP-8 dermal exposure and continued to increase through 7 days of exposure. Ceruloplasmin levels increased after 5 days of exposure. Serum α1-inhibitor III was reduced after a 1-day exposure and the depletion continued after 7 days of exposure. Apolipoprotein A-IV increased after a 1-day exposure and then returned to basal levels after 3- and 5-day exposures of JP-8. Levels of the acute-phase protein α2-macroglobulin were found to not vary over these time course studies. Using ESI-MS analysis directly on the sera from rats exposed to dermal JP-8, low-mass sera constituents were found to correlate with control (acetone) or JP-8 exposure.

Adenomatous polyposis coli protein associates with C/EBP β and increases Bacillus anthracis edema toxin stimulated gene expression in macrophages

The production of cAMP from Bacillus anthracis edema toxin (ET) activates gene expression in macrophages through a complex array of signaling pathways, most of which remain poorly defined. In this study, the tumor suppressor protein adenomatous polyposis coli (APC) was found to be important for the up-regulation of previously defined ET-stimulated genes (Vegfa, Ptgs2, Arg2, Cxcl2, Sdc1, and Cebpb). A reduction in the expression of these genes after ET exposure was observed when APC was disrupted in macrophages using siRNA or in bone marrow-derived macrophages obtained from C57BL/6J-ApcMin mice, which are heterozygous for a truncated form of APC. In line with this observation, ET increased the expression of APC at the transcriptional level, leading to increased amounts of APC in the nucleus. The mechanism utilized by APC to increase ET-induced gene expression was determined to depend on the ability of APC to interact with C/EBP β, which is a transcription factor activated by cAMP. Coimmunoprecipitation experiments found that APC associated with C/EBP β and that levels of this complex increase after ET exposure. A further connection was uncovered when silencing APC was determined to reduce the ET-induced phosphorylation of C/EBP β at Thr-188. This ET-mediated phosphorylation of C/EBP β was blocked by glycogen synthase kinase 3 (GSK-3) inhibitors, suggesting that GSK-3 is involved in the activation of C/EBP β and supporting the idea of APC helping direct interactions between GSK-3 and C/EBP β. These results indicate that ET stimulates gene expression by promoting the formation of an inducible protein complex consisting of APC and C/EBP β.

Toxin Inhibition of Antimicrobial Factors Induced by Bacillus anthracis Peptidoglycan in Human Blood

ABSTRACT Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LTs capacity to suppress this response.

Systemic molecular and cellular changes induced in rats upon inhalation of JP-8 petroleum fuel vapor

Limited information is available regarding systemic changes in mammals associated with exposures to petroleum/hydrocarbon fuels. In this study, systemic toxicity of JP-8 jet fuel was observed in a rat inhalation model at different JP-8 fuel vapor concentrations (250, 500, or 1000 mg/m3, for 91 days). Gel electrophoresis and mass spectrometry sequencing identified the α-2 microglobulin protein to be elevated in rat kidney in a JP-8 dose-dependent manner. Western blot analysis of kidney and lung tissue extracts revealed JP-8 dependent elevation of inducible heat shock protein 70 (HSP70). Tissue changes were observed histologically (hematoxylin and eosin staining) in liver, kidney, lung, bone marrow, and heart, and more prevalently at medium or high JP-8 vapor phase exposures (500–1000 mg/m3) than at low vapor phase exposure (250 mg/m3) or non-JP-8 controls. JP-8 fuel-induced liver alterations included dilated sinusoids, cytoplasmic clumping, and fat cell deposition. Changes to the kidneys included reduced numbers of nuclei, and cytoplasmic dumping in the lumen of proximal convoluted tubules. JP-8 dependent lung alterations were edema and dilated alveolar capillaries, which allowed clumping of red blood cells (RBCs). Changes in the bone marrow in response to JP-8 included reduction of fat cells and fat globules, and cellular proliferation (RBCs, white blood cells-WBCs, and megakaryocytes). Heart tissue from JP-8 exposed animals contained increased numbers of inflammatory and fibroblast cells, as well as myofibril scarring. cDNA array analysis of heart tissue revealed a JP-8 dependent increase in atrial natriuretic peptide precursor mRNA and a decrease in voltage-gated potassium (K+) ion channel mRNA.

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 μl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IκBα (inhibitor of NF-κB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IκBα were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).

Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB

ABSTRACT Clostridium difficile infection (CDI) is a major cause of hospital-associated, antibiotic-induced diarrhea, which is largely mediated by the production of two large multidomain clostridial toxins, TcdA and TcdB. Both toxins coordinate the action of specific domains to bind receptors, enter cells, and deliver a catalytic fragment into the cytosol. This results in GTPase inactivation, actin disassembly, and cytotoxicity. TcdB in particular has been shown to encode a region covering amino acids 1753 to 1851 that affects epitope exposure and cytotoxicity. Surprisingly, studies here show that several peptides derived from this region, which share the consensus sequence 1769NVFKGNTISDK1779, protect cells from the action of TcdB. One peptide, PepB2, forms multiple interactions with the carboxy-terminal region of TcdB, destabilizes TcdB structure, and disrupts cell binding. We further show that these effects require PepB2 to form a higher-order polymeric complex, a process that requires the central GN amino acid pair. These data suggest that TcdB1769–1779 interacts with repeat sequences in the proximal carboxy-terminal domain of TcdB (i.e., the CROP domain) to alter the conformation of TcdB. Furthermore, these studies provide insights into TcdB structure and functions that can be exploited to inactivate this critical virulence factor and ameliorate the course of CDI. IMPORTANCE Clostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment. IMPORTANCE Clostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment.