VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme.     

Anthrax toxins

Bacillus anthracis, the etiological agent of anthrax, secretes three polypeptides that assemble into toxic complexes on the cell surfaces of the host it infects. One of these polypeptides, protective antigen (PA), binds to the integrin-like domains of ubiquitously expressed membrane proteins of mammalian cells. PA is then cleaved by membrane endoproteases of the furin family. Cleaved PA molecules assemble into heptamers, which can then associate with the two other secreted polypeptides: edema factor (EF) and/or lethal factor (LF). The heptamers of PA are relocalized to lipid rafts where they are quickly endocytosed and routed to an acidic compartment. The low pH triggers a conformational change in the heptamers, resulting in the formation of cation-specific channels and the translocation of EF/LF. EF is a calcium- and calmodulin-dependent adenylate cyclase that dramatically raises the intracellular concentration of cyclic adenosine monophosphate (cAMP). LF is a zinc-dependent endoprotease that cleaves the amino terminus of mitogen-activated protein kinase kinases (Meks). Cleaved Meks cannot bind to their substrates and have reduced kinase activity, resulting in alterations of the signaling pathways they govern. The structures of PA, PA heptamer, EF, and LF have been solved and much is now known about the molecular details of the intoxication mechanism. The in vivo action of the toxins, on the other hand, is still poorly understood and hotly debated. A better understanding of the toxins will help in the design of much-needed anti-toxin drugs and the development of new toxin-based medical applications.Abbreviations CMG2 Capillary morphogenesis protein 2 - DTA Diphtheria toxin A chain - EF Edema factor - EFn N-terminal fragment of EF - ETx Edema toxin - GR Glucocorticoid receptors - GSK3 Glycogen synthase kinase 3 - I domain Integrin-like domain - iNOS Inducible nitric oxide synthase - LF Lethal factor - LFn N-terminal fragment of LF - LTx Lethal toxin - MAPK Mitogen-activated protein kinase - Mek MAPK kinases - PA Protective antigen - PA₂₀ 20-kDa N-terminal fragment of PA - PA₆₃ 63-kDa C-terminal fragment of PA - TEM8 Tumor endothelial marker 8     

The effects of anthrax lethal factor on the macrophage proteome: potential activity on nitric oxide synthases

Anthrax lethal factor (LeTx) is a critical virulence factor in toxin-challenged cells, as lethal factor (LF) cleaves mitogen-activated protein kinase kinases (MKKs), inhibiting their activity. The physiological importance of this cleavage for macrophage cytolysis remains unclear, because similar proteolysis has been also observed in LeTx-resistant macrophages. Here, we analyzed in vitro proteomic profiles of Raw264.7 lysates treated with LF. In our experiments, neuronal NO synthase (nNOS) was found to be a fragment, suggesting that LF may act on nNOS cleavage. A similar cleavage of nNOS was shown in LeTx-challenged HEK293 cells expressing nNOS by a transient transfection. However, the cleavage site on nNOS is a unique leader sequence among the NOS family and this LF-mediated cleavage was not observed in iNOS, a major NOS isoform for anti-bactericidal NO production, even though NO level in LeTx-challenged cells was dramatically reduced. Our findings suggest that LF is directly capable of cleaving cellular protein(s) other than MKKs, and that these actions potentiate to promote the cytotoxic mechanisms of anthrax.     

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.     

Signalling pathways in the pathogenesis of Cryptococcus

Efficient communication with the environment is critical for all living organisms. Fungi utilize complex signalling systems to sense their environments and control proliferation, development and in some cases virulence. Well-studied signalling pathways include the protein kinase A/cyclic AMP (cAMP), protein kinase C (PKC)/mitogen-activated protein kinase (MAPK), lipid signalling cascades, and the calcium–calcineurin signalling pathway. The human pathogenic basidiomycetous fungus Cryptococcus neoformans deploys sensitive signalling systems to survive in the human host, leading to life-threatening meningoencephalitis. Known virulence traits of this fungus, including the antioxidant melanin production, the antiphagocytic polysaccharide capsule and the ability to grow at 37°C, are orchestrated by complex signalling networks, whose understanding is crucial to better treat, diagnose and prevent cryptococcosis.     

Stop the killer: how to inhibit the anthrax lethal factor metalloprotease

Inhalation of anthrax spores rapidly develops into a deadly bacteraemia and toxaemia. Anthrax toxins include the lethal factor (LF), a mitogen-activated protein kinase (MAPK)-kinase-specific metalloprotease, which acts in the cell cytosol and plays a major part in anthrax pathogenesis. Recently, screening methods have led to the discovery of LF inhibitors that are membrane permeable. This will pave the way for design of novel anthrax therapeutics that are capable of inhibiting the metalloprotease activity of LF in vivo.     

Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.     

Purification and biophysical characterization of the core protease domain of anthrax lethal factor

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn²⁺ binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF_672-776) that harbours the enzyme’s core protease domain. The biophysical characterization and backbone assignments (¹H, ¹³C, ¹⁵N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn²⁺, suitable for high resolution structural analysis by NMR.     

Exploring the evolutionary path of plant MAPK networks

The evolutionarily conserved mitogen-activated protein kinase (MAPK) signaling network comprises connected protein kinases arranged in MAPK modules. In this Opinion article, we analyze MAPK signaling components in evolutionarily representative species of the plant lineage and in Naegleria gruberi, a member of an early diverging eukaryotic clade. In Naegleria, there are two closely related MAPK kinases (MKKs) and a single conventional MAPK, whereas in several species of algae, there are two distinct MKKs and multiple MAPKs belonging to different groups. This suggests that the formation of multiple MAPK modules began early during plant evolution. The expansion of MAPK signaling components through gene duplications and the evolution of interaction motifs could have contributed to the highly connected complex MAPK signaling network that we know in Arabidopsis.     

Function of the Yersinia effector YopJ

The Yersinia virulence factor YopJ inhibits the host immune response and induces apoptosis by blocking multiple signaling pathways, including the MAPK and NFkappaB pathways in the infected cell. YopJ is a cysteine protease that cleaves a reversible post-translational modification in the form of ubiquitin or a ubiquitin-like protein. Homologues of YopJ are expressed in animal and plant pathogens, as well as a plant symbiont, suggesting a universal mechanism of regulating or modulating a variety of signaling pathways. The ability of YopJ to block the innate immune response, its activity as a ubiquitin-like protein protease and its activity with respect to mammalian signalling pathways are discussed in this review.     

Anthrax oedema toxin induces anthrax toxin receptor expression in monocyte-derived cells

Bacillus anthracis, the causative agent of anthrax, secretes two bipartite toxins that help the bacterium evade the immune system and contribute directly to pathogenesis. Both toxin catalytic moieties, lethal factor (LF) and oedema factor (OF), are internalized into the host-cell cytosol by a third factor, protective antigen (PA), which binds to cellular anthrax toxin receptors (ANTXRs). Oedema factor is an adenylate cyclase that impairs host defences by raising cellular cAMP levels. Here we demonstrate that oedema toxin (PA + OF) induces an increase in ANTXR expression levels in macrophages and dendritic cells resulting in an increased rate of toxin internalization. Furthermore, we show that increases in ANTXR mRNA levels depends on the ability of OF to increase cAMP levels, is mediated through protein kinase A-directed signalling and is monocyte-lineage-specific. To our knowledge, this is the first report of a bacterial toxin inducing host target cells to increase toxin receptor expression.     

The Acetyltransferase Activity of the Bacterial Toxin YopJ of Yersinia Is Activated by Eukaryotic Host Cell Inositol Hexakisphosphate

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP₆). We extend the applicability of our results to show that IP₆ also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP₆-induced conformational change in AvrA suggests that IP₆ acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP₆; once injected into mammalian cells by the pathogen these toxins bind host cell IP₆, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity.     

The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.     

The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.     

Cytotoxicity of Anthrax Lethal Toxin to Human Acute Myeloid Leukemia Cells Is Nonapoptotic and Dependent on Extracellular Signal-Regulated Kinase 1/2 Activity

In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx). LeTx consists of protective antigen (PrAg) and lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the MEK1/2-extracellular signal-regulated kinase (ERK1/2) branch of the MAPK pathway. The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LeTx and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LeTx-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LeTx-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.     

cAMP blocks MAPK activation and sclerotial development via Rap-1 in a PKA-independent manner in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen-activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)-independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap-1 or Ras is involved in cAMP action. The Rap-1 inhibitor, GGTI-298, restored MAPK activation in the presence of cAMP, further suggesting that Rap-1 is responsible for cAMP-dependent MAPK inhibition. Importantly, inhibition of Rap-1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA-independent cAMP signalling pathway. Cross-talk between these two pathways is mediated by Rap-1.