Anthrax lethal toxin disrupts intestinal barrier function and causes systemic infections with enteric bacteria

A variety of intestinal pathogens have virulence factors that target mitogen activated protein kinase (MAPK) signaling pathways, including Bacillus anthracis. Anthrax lethal toxin (LT) has specific proteolytic activity against the upstream regulators of MAPKs, the MAPK kinases (MKKs). Using a murine model of intoxication, we show that LT causes the dose-dependent disruption of intestinal epithelial integrity, characterized by mucosal erosion, ulceration, and bleeding. This pathology correlates with an LT-dependent blockade of intestinal crypt cell proliferation, accompanied by marked apoptosis in the villus tips. C57BL/6J mice treated with intravenous LT nearly uniformly develop systemic infections with commensal enteric organisms within 72 hours of administration. LT-dependent intestinal pathology depends upon its proteolytic activity and is partially attenuated by co-administration of broad spectrum antibiotics, indicating that it is both a cause and an effect of infection. These findings indicate that targeting of MAPK signaling pathways by anthrax LT compromises the structural integrity of the mucosal layer, serving to undermine the effectiveness of the intestinal barrier. Combined with the well-described immunosuppressive effects of LT, this disruption of the intestinal barrier provides a potential mechanism for host invasion via the enteric route, a common portal of entry during the natural infection cycle of Bacillus anthracis.     

Anthrax lethal toxin paralyzes actin-based motility by blocking Hsp27 phosphorylation

Inhalation of anthrax causes fatal bacteremia, indicating a meager host immune response. We previously showed that anthrax lethal toxin (LT) paralyzes neutrophils, a major component of innate immunity. Here, we have found that LT also inhibits actin-based motility of the intracellular pathogen Listeria monocytogenes. LT inhibition of actin assembly is mediated by blockade of Hsp27 phosphorylation, and can be reproduced by treating cells with the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580. Nonphosphorylated Hsp27 inhibits Listeria actin-based motility in cell extracts, and binds to and sequesters purified actin monomers. Phosphorylation of Hsp27 reverses these effects. RNA interference knockdown of Hsp27 blocks LT inhibition of Listeria actin-based motility. Rescue with wild-type Hsp27 accelerates Listeria speed in knockdown cells, whereas introduction of Hsp27 mutants incapable of phosphorylation or dephosphorylation causes slowing down. We propose that Hsp27 facilitates actin-based motility through a phosphorylation cycle that shuttles actin monomers to regions of new actin filament assembly. Our findings provide a previously unappreciated mechanism for LT virulence, and emphasize a central role for p38 MAP kinase-mediated phosphorylation of Hsp27 in actin-based motility and innate immunity.     

Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells

Vascular endothelial dysfunction is thought to play a prominent role in systemic anthrax pathogenesis. We examined the effect of anthrax lethal toxin (LTx), a key virulence factor of Bacillus anthracis, on the expression of vascular cell adhesion molecule-1 (VCAM-1) on normal and cytokine-stimulated human lung microvascular endothelial cells. Confluent endothelial monolayers were treated with lethal factor (LF), protective antigen (PA), or both (LTx) in the presence or absence of tumor necrosis factor-alpha (TNFalpha). LTx enhanced cytokine-induced VCAM-1 expression and monocyte adhesion. LTx alone had no effect on VCAM-1 expression. LF, PA or the combination of a catalytically inactive mutant LF and PA failed to enhance cytokine-induced VCAM-1 expression. Treatment with inhibitors of mitogen-activated protein kinase kinases (MEKs) and mitogen-activated protein kinases did not reproduce the VCAM-1 enhancement effect of LTx, a known MEK metalloprotease, suggesting LTx-mediated MEK cleavage may not be a contributing factor.     

Anthrax toxin: a tripartite lethal combination

Anthrax is a severe bacterial infection that occurs when Bacillus anthracis spores gain access into the body and germinate in macrophages, causing septicemia and toxemia. Anthrax toxin is a binary A-B toxin composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA mediates the entry of either LF or EF into the cytosol of host cells. LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defences. Inhibitors targeting different steps of toxin activity have recently been developed. Anthrax toxin has also been exploited as a therapeutic agent against cancer.     

Anthrax toxin receptor 2-dependent lethal toxin killing in vivo

Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.     

p38 MAP kinase-dependent regulation of endothelial cell permeability

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.     

Anthrax lethal toxin activates the inflammasome in sensitive rat macrophages

Anthrax lethal toxin (LT) is an important virulence factor for Bacillus anthracis. In mice, LT lyses macrophages from certain inbred strains in less than 2 h by activating the Nlrp1b inflammasome and caspase-1, while macrophages from other strains remain resistant to the toxin’s effects. We analyzed LT effects in toxin-sensitive and resistant rat macrophages to test if a similar pathway was involved in rat macrophage death. LT activates caspase-1 in rat macrophages from strains harboring LT-sensitive macrophages in a manner similar to that in toxin-sensitive murine macrophages. This activation of caspase-1 is dependent on proteasome activity, and sensitive macrophages are protected from LT’s lytic effects by lactacystin. Proteasome inhibition also delayed the death of rats in response to LT, confirming our previous data implicating the rat Nlrp1 inflammasome in animal death. Quinidine, caspase-1 inhibitors, the cathepsin B inhibitor CA-074Me, and heat shock also protected rat macrophages from LT toxicity. These data support the existence of an active functioning LT-responsive Nlrp1 inflammasome in rat macrophages. The activation of the rat Nlrp1 inflammasome is required for LT-mediated rat macrophage lysis and contributes to animal death.     

Anthrax lethal toxin increases superoxide production in murine neutrophils via differential effects on MAPK signaling pathways

The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1-4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.     

Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity

Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound ⁶⁵Zn both in solution and on protein blots. The ⁶⁵Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.     

Anthrax edema toxin cooperates with lethal toxin to impair cytokine secretion during infection of dendritic cells

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-alpha, whereas LT targets IL-10 and TNF-alpha production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-gamma secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.     

Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability

Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.     

p21-activated kinase regulates endothelial permeability through modulation of contractility

Endothelial cells lining the vasculature have close cell-cell associations that maintain separation of the blood fluid compartment from surrounding tissues. Permeability is regulated by a variety of growth factors and cytokines and plays a role in numerous physiological and pathological processes. We examined a potential role for the p21-activated kinase (PAK) in the regulation of vascular permeability. In both bovine aortic and human umbilical vein endothelial cells, PAK is phosphorylated on Ser141 during the activation downstream of Rac, and the phosphorylated subfraction translocates to endothelial cell-cell junctions in response to serum, VEGF, bFGF, TNFalpha, histamine, and thrombin. Blocking PAK activation or translocation prevents the increase in permeability across the cell monolayer in response to these factors. Permeability correlates with myosin phosphorylation, formation of actin stress fibers, and the appearance of paracellular pores. Inhibition of myosin phosphorylation blocks the increase in permeability. These data suggest that PAK is a central regulator of endothelial permeability induced by multiple growth factors and cytokines via an effect on cell contractility. PAK may therefore be a suitable drug target for the treatment of pathological conditions where vascular leak is a contributing factor, such as ischemia and inflammation.     

Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin

Anthrax lethal toxin (LT) contributes to the immune evasion strategy of Bacillus anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however, not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knockout mice, we found that depending on the mouse strain, death of bone marrow-derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1-dependent, or by a slow caspase-1-independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1-independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses.     

Homocysteine in microvascular endothelial cell barrier permeability

Redox stress activates the endothelium and upregulates matrix metalloproteinases (MMPs), which degrade the matrix and lead to blood-endothelial barrier leakage. Interestingly, elevated levels of plasma homocysteine (Hcy) are associated with vascular dementia, seizure, stroke, and Alzheimer disease. Hcy competes with the γ-aminobutyric acid (GABA)-A/B receptors and behave like an excitatory neurotransmitter. GABA stimulates the inhibitory neurotransmitter GABA-A/B receptor and decreases arterial blood pressure. However, the neural mechanisms of microvascular remodeling in hyperhomocysteinemia are unclear. This review addresses the idea that Hcy induces microvascular permeability by attenuating the GABA-A/B receptors and increasing redox stress, which activates a disintegrin and metalloproteinase that suppresses tissue inhibitors of metalloproteinase. This process causes disruption of the matrix in the blood-brain barrier. Understanding the mechanism of Hcy-mediated changes in permeability of the blood-brain barrier and extracellular matrix that can alter the neuronal environment in cerebral-vascular dementia is of great importance in developing treatments for this disease.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway

Although increased vascular permeability is known to be a major characteristic of diabetic vasculopathy, the precise mechanisms and relevance of advanced glycation end products (AGE) to hyperpermeability of vessels remains unclear. Here, we studied changes in cytoskeletal configuration and the signaling mechanism induced by AGE in human endothelial cells. AGE-BSA stimulation induced Rho activation, intercellular gap formation, prominent actin stress fiber and cell contraction without changing VE-cadherin, and subsequently transendothelial diffusion of FITC-labeled dextran. These processes induced by AGE-BSA were inhibited by either Rho-kinase inhibitor Y27632 or anti-RAGE antibody. We also showed that RhoA and RAGE spontaneously formed a complex. These findings suggest that activation of RAGE/Rho is involved in AGE-BSA-induced hyperpermeability through gap formation and actin reorganization in diabetes.

Structured summary

MINT-7301170: Rhotekin (uniprotkb:Q9BST9) physically interacts (MI:0915) with RhoA (uniprotkb:P61586) by pull down (MI:0096)MINT-7301204, MINT-7301186: RhoA (uniprotkb:P61586) physically interacts (MI:0915) with RAGE (uniprotkb:Q15109) by anti bait coimmunoprecipitation (MI:0006)