Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Modulation of Adhesion Molecules Expression by Different Metalloproteases Isolated from Bothrops Snakes.

Snake venom metalloproteinases (SVMP) are involved in local inflammatory reactions observed after snakebites. Based on domain composition, they are classified as PI (pro-domain + proteolytic domain), PII (PI + disintegrin-like domains), or PIII (PII + cysteine-rich domains). Here, we studied the role of different SVMPs domains in inducing the expression of adhesion molecules at the microcirculation of the cremaster muscle of mice. We used Jararhagin (Jar)-a PIII SVMP with intense hemorrhagic activity, and Jar-C-a Jar devoid of the catalytic domain, with no hemorrhagic activity, both isolated from B. jararaca venom and BnP-1-a weakly hemorrhagic P1 SVMP from B. neuwiedi venom. Toxins (0.5 µg) or PBS (100 µL) were injected into the scrotum of mice, and 2, 4, or 24 h later, the protein and gene expression of CD54 and CD31 in the endothelium, and integrins (CD11a and CD11b), expressed in leukocytes were evaluated. Toxins induced significant increases in CD54, CD11a, and CD11b at the initial time and a time-related increase in CD31 expression. In conclusion, our results suggest that, despite differences in hemorrhagic activities and domain composition of the SVMPs used in this study, they behave similarly to the induction of expression of adhesion molecules that promote leukocyte recruitment.

Bacteroides fragilis Enterotoxin Upregulates Matrix Metalloproteinase-7 Expression through MAPK and AP-1 Activation in Intestinal Epithelial Cells, Leading to Syndecan-2 Release.

Bacteroides fragilis enterotoxin (BFT) produced by enterotoxigenic B. fragilis (ETBF) causes colonic inflammation. BFT initially contacts intestinal epithelial cells (IECs) and affects the intestinal barrier. Although molecular components of the gut epithelial barrier such as metalloproteinase-7 (MMP-7) and syndecan-2 are known to be associated with inflammation, little has been reported about MMP-7 expression and syndecan-2 shedding in response to ETBF infection. This study explores the role of BFT in MMP-7 induction and syndecan-2 release in IECs. Stimulating IECs with BFT led to the induction of MMP-7 and the activation of transcription factors such as NF-κB and AP-1. MMP-7 upregulation was not affected by NF-κB, but it was related to AP-1 activation. In BFT-exposed IECs, syndecan-2 release was observed in a time- and concentration-dependent manner. MMP-7 suppression was associated with a reduction in syndecan-2 release. In addition, suppression of ERK, one of the mitogen-activated protein kinases (MAPKs), inhibited AP-1 activity and MMP-7 expression. Furthermore, the suppression of AP-1 and ERK activity was related to the attenuation of syndecan-2 release. These results suggest that a signaling cascade comprising ERK and AP-1 activation in IECs is involved in MMP-7 upregulation and syndecan-2 release during exposure to BFT.

"Computational and Functional Characterization of a Hemorrhagic Metalloproteinase Purified from Cerastes cerastes Venom".

Structural and functional aspects of snake venoms metalloproteinases (SVMPs) have been extensively studied due to their role in envenomation. However, in the detection of certain coagulation disorders these biomolecules have been used and applied for the production of new thrombolytic drugs. CcMP-II, a SVMP-II metalloproteinase with a hemorrhagic activity, isolated from the venom of Cerastes cerastes, its sequence of 472 amino acids was identified. Predicted 3D structure showed an arrangement of CcMP-II into two distinct domains: i) a metalloproteinase domain where the zinc-binding motif is found (HXXGHNLGIDH) in addition to the sequence Cys-Ile-Met (CIM) at the Met-turn and ii) disintegrin-like domain containing RGD motif. CcMP-II inhibits platelet aggregation induced by collagen in a dose-dependent manner with an IC50 value estimated of 0.11 nM. This proteinase inhibits also aggregation of platelet stimulated by collagen even if the metal chelating agents (EDTA and 1, 10-phenontroline) are present suggesting that anti-aggregating effect is not due to its metalloproteinase domain, but to its disintegrin-like domain. Capillary pathological modifications caused by CcMP-II following intramuscular injection have as well been examined in mice. The key morphological alterations of the capillary vessels were clearly apparent from the ultrastructural study. The CcMP-II can play a key function in the pathogenesis of disorders that occurs following envenomation of Cerastes cerastes. The three-dimensional model of CcMP-II was built to explain structure-function relationships in ADAM/ADAMTs, a family of proteins having significant therapeutic potential. In order to explain structure-function relationships in ADAM / ADAMT, a family of proteins with considerable therapeutic potential, the three-dimensional model of CcMP-II was constructed.

Jararhagin, a snake venom metalloproteinase, induces mechanical hyperalgesia in mice with the neuroinflammatory contribution of spinal cord microglia and astrocytes.

Jararhagin is a hyperalgesic metalloproteinase from Bothrops jararaca venom. In rodents, jararhagin induces nociceptive behaviors that correlate with an increase in peripheral cytokine levels. However, the role of the spinal cord glia in pain processing after peripheral stimulus of jararhagin has not been investigated. Aiming to explore this proposal, mice received intraplantar (i.pl.) injection of jararhagin and the following parameters were evaluated: hyperalgesia, spinal cord TNF-α, IL-1ß levels, and CX3CR1, GFAP and p-NFκB activation. The effects of intrathecal (i.t.) injection of TNF-α soluble receptor (etanercept), IL-1 receptor antagonist (IL-1Ra), and inhibitors of NFκB (PDTC), microglia (minocycline) and astrocytes (α-aminoadipate) were investigated. Jararhagin inoculation induced cytokine production (TNF-α and IL-1ß) in the spinal cord, which was reduced by treatment with PDTC (40% and 50%, respectively). Jararhagin mechanical hyperalgesia and cytokine production were inhibited by treatment with etanercept (67%), IL-1Ra (60%), PDTC (70%), minocycline (60%) and α-aminoadipate (45%). Furthermore, jararhagin induced an increase in p-NFκB, CX3CR1 and GFAP detection in the spinal cord indicating activation of NFκB, microglia and astrocytes. These results demonstrate for the first time that jararhagin-induced mechanical hyperalgesia is dependent on spinal cord activation of glial cells, consequent NFκB activation, and cytokine production in mice.

Glucosylceramide production maintains colon integrity in response to Bacteroides fragilis toxin-induced colon epithelial cell signaling.

Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro-inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E-cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT-induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide-dependent mechanism by which the colon epithelium responds to BFT.

Bacteroides fragilis Enterotoxin Induces Sulfiredoxin-1 Expression in Intestinal Epithelial Cell Lines Through a Mitogen-Activated Protein Kinases- and Nrf2-Dependent Pathway, Leading to the Suppression of Apoptosis.

Enterotoxigenic Bacteroides fragilis is a causative agent of colitis and secrets enterotoxin (BFT), leading to the disease. Sulfiredoxin (Srx)-1 serves to protect from oxidative damages. Although BFT can generate reactive oxygen species in intestinal epithelial cells (IECs), no Srx-1 expression has been reported in ETBF infection. In this study, we explored the effects of ETBF-produced BFT on Srx-1 induction in IECs. Treatment of IECs with BFT resulted in increased expression of Srx-1 in a time-dependent manner. BFT treatment also activated transcriptional signals including Nrf2, AP-1 and NF-κB, and the Srx-1 induction was dependent on the activation of Nrf2 signals. Nrf2 activation was assessed using immunoblot and Nrf2-DNA binding activity and the specificity was confirmed by supershift and competition assays. Suppression of NF-κB or AP-1 signals did not affect the upregulation of Srx-1 expression. Nrf2-dependent Srx-1 expression was associated with the activation of p38 mitogen-activated protein kinases (MAPKs) in IECs. Furthermore, suppression of Srx-1 significantly enhanced apoptosis while overexpression of Srx-1 significantly attenuated apoptosis during exposure to BFT. These results imply that a signaling cascade involving p38 and Nrf2 is essential for Srx-1 upregulation in IECs stimulated with BFT. Following this upregulation, Srx-1 may control the apoptosis in BFT-exposed IECs.

Enterotoxigenic Bacteroides fragilis infection exacerbates tumorigenesis in AOM/DSS mouse model.

The azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model is commonly used to study colitis-associated cancer. The human commensal bacterium, enterotoxigenic Bacteroides fragilis (ETBF) secretes the Bacteroides fragilis toxin (BFT) which is necessary and sufficient to cause colitis. We report that BALB/c mice infected with WT-ETBF and administered three cycles of AOM/DSS developed numerous, large-sized polyps predominantly in the colorectal region. In addition, AOM/DSS-treated BALB/c mice orally inoculated with wild-type nontoxigenic Bacteroides fragilis (WT-NTBF) overexpressing bft (rETBF) developed numerous polyps whereas mice infected with WT-NTBF overexpressing a biologically inactive bft (rNTBF) did not promote polyp formation. Unexpectedly, the combination of AOM+ETBF did not induce polyp formation whereas ETBF+DSS did induce polyp development in a subset of BALB/c mice. In conclusion, WT-ETBF promoted polyp development in AOM/DSS murine model with increased colitis in BALB/c mice. The model described herein provides an experimental platform for understanding ETBF-induced colonic tumorigenesis and studying colorectal cancer in wild-type mice.

Diversity of astacin-like metalloproteases identified by transcriptomic analysis in Peruvian Loxosceles laeta spider venom and in vitro activity characterization.

Loxosceles spiders are found in almost all countries of South America. In Peru, Loxosceles laeta species is the main responsible for the accidents caused by poisonous animals, being known as "killer spiders", due to the large number of fatal accidents observed. Astacin-like metalloproteases, named LALPs (Loxosceles astacin-like metalloproteases) are highly expressed in Loxosceles spiders venom gland. These proteases may be involved in hemorrhage and venom spreading, being relevant to the envenoming proccess. Thus, the aim of this work was to analyze Peruvian L. laeta venom gland transcripts using bioinformatics tools, focusing on LALPs. A cDNA library from Peruvian L. laeta venom glands was constructed and sequenced by MiSeq (Illumina) sequencer. After assembly, the resulting sequences were annotated, seeking out for similarity with previously described LALPs. Nine possible LALPs isoforms from Peruvian L. laeta venom were identified and the results were validated by in silico and in vitro experiments. This study contributes to a better understanding of the molecular diversity of Loxosceles venom and provide insights about the action of LALPs.

Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom.

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca2+ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. "In vitro" studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation.

Epigenetic Changes Induced by Bacteroides fragilis Toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) is a Gram-negative, obligate anaerobe member of the gut microbial community in up to 40% of healthy individuals. This bacterium is found more frequently in people with colorectal cancer (CRC) and causes tumor formation in the distal colon of multiple intestinal neoplasia (Apcmin/+ ) mice; tumor formation is dependent on ETBF-secreted Bacteroides fragilis toxin (BFT). Because of the extensive data connecting alterations in the epigenome with tumor formation, initial experiments attempting to connect BFT-induced tumor formation with methylation in colon epithelial cells (CECs) have been performed, but the effect of BFT on other epigenetic processes, such as chromatin structure, remains unexplored. Here, the changes in gene expression (transcriptome sequencing [RNA-seq]) and chromatin accessibility (assay for transposase-accessible chromatin using sequencing) induced by treatment of HT29/C1 cells with BFT for 24 and 48 h were examined. Our data show that several genes are differentially expressed after BFT treatment and that these changes relate to the interaction between bacteria and CECs. Further, sites of increased chromatin accessibility are associated with the location of enhancers in CECs and the binding sites of transcription factors in the AP-1/ATF family; they are also enriched for common differentially methylated regions (DMRs) in CRC. These data provide insight into the mechanisms by which BFT induces tumor formation and lay the groundwork for future in vivo studies to explore the impact of BFT on nuclear structure and function.

Botulinum and Tetanus Neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase.

Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (∼6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas it significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK, and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.

Dynamical properties of LFPs from mice with unilateral injection of TeNT.

Local field potential (LFP) recordings were performed from the visual cortex (V1) of a focal epilepsy mouse model. Epilepsy was induced by a unilateral injection of the synaptic blocker tetanus neurotoxin (TeNT). LFP signals were simultaneously recorded from V1 of both hemispheres of each animal under acute and chronic conditions (i.e. during and after the period of TeNT action). All data were analysed by using nonlinear time series methods. Suitable values of the lag time and embedding dimension for phase space reconstruction were estimated by employing well-known methods. The results showed that lag times are sensitive to the presence of TeNT. Interestingly, TeNT promoted an increase in the level of linear and nonlinear correlation of LFP signals. The values of the embedding dimension failed to show any dependence on the presence of the neurotoxin. However, a local nonlinear prediction method showed that the presence of TeNT increases the predictability, quantified by the normalized prediction error, of the neural recordings. From a neurophysiological point of view, the above results suggest that TeNT injected in one hemisphere strongly impacts the local electrical activity of the neural populations in the opposite hemisphere. We hypothesize that this could arise from a qualitative and quantitative alteration of the transmission properties of the callosal fibers.

Effects of iron and carbon monoxide on Lachesis muta muta venom-mediated degradation of plasmatic coagulation.

Hypofibrinogenemia is an important clinical consequence following envenomation by Lachesis muta muta, usually attenuated or prevented by administration of antivenom. The venom of L. m. muta contains both a metalloproteinase fibrinogenase and a serine protease thrombin-like enzyme, and exposure of fibrinogen to iron (Fe) and carbon monoxide (CO) has been demonstrated to decrease its catalysis by such enzymes. Using thrombelastographic analytical techniques, it was determined that this venom displayed weak procoagulant effects combined with fibrinogenolytic effects, and pretreatment of plasma with Fe and CO markedly attenuated venom-mediated effects. Additional experiments involving heparin exposure and varying calcium concentrations demonstrated that modification of fibrinogen with Fe and CO in human plasma rendered fibrinogen not recognizable to the fibrinogenolytic metalloproteinase but did not prevent polymerization by the thrombin-like serine protease. Lastly, when venom was exposed to CO in isolation and then placed in plasma, the fibrinogenase was inhibited but the thrombin-like enzyme was not inhibited. In sum, utilizing relatively facile modifications, we demonstrated with thrombelastography that Fe and/or CO addition can protect human plasmatic coagulation from fibrinogenase activity but not the effects of the thrombin-like activity of L. m. muta venom.

Hemorrhage Caused by Snake Venom Metalloproteinases: A Journey of Discovery and Understanding.

The historical development of discoveries and conceptual frames for understanding the hemorrhagic activity induced by viperid snake venoms and by hemorrhagic metalloproteinases (SVMPs) present in these venoms is reviewed. Histological and ultrastructural tools allowed the identification of the capillary network as the main site of action of SVMPs. After years of debate, biochemical developments demonstrated that all hemorrhagic toxins in viperid venoms are zinc-dependent metalloproteinases. Hemorrhagic SVMPs act by initially hydrolyzing key substrates at the basement membrane (BM) of capillaries. This degradation results in the weakening of the mechanical stability of the capillary wall, which becomes distended owing of the action of the hemodynamic biophysical forces operating in the circulation. As a consequence, the capillary wall is disrupted and extravasation occurs. SVMPs do not induce rapid toxicity to endothelial cells, and the pathological effects described in these cells in vivo result from the mechanical action of these hemodynamic forces. Experimental evidence suggests that degradation of type IV collagen, and perhaps also perlecan, is the key event in the onset of microvessel damage. It is necessary to study this phenomenon from a holistic, systemic perspective in which the action of other venom components is also taken into consideration.

Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Jararhagin-induced mechanical hyperalgesia depends on TNF-α, IL-1ß and NFκB in mice.

Jararhagin is a hemorrhagic metalloprotease from Bothrops jararaca snake venom. The hyperalgesic mechanisms of jararhagin were investigated focusing on the role of proinflammatory cytokines (TNF-α and IL-1ß) and the transcription factor NFκB. Intraplantar administration of jararhagin (1, 10, 100 and 1000 ng/paw) induced mechanical hyperalgesia, and increased TNF-α levels at 1, 3 and 5 h, and IL-1ß levels at 0.5, 1 and 3 h after its injection in the paw tissue. Pre-treatment with morphine (2, 6, 12 µg/paw) inhibited jararhagin-induced mechanical hyperagesia. The systemic or local pre-treatment with etanercept (10 mg/kg and 100 µg/paw) and IL-1ra (30 mg/kg and 100 pg/paw) inhibited jararhagin-induced mechanical hyperalgesia. Co-administration of jararhagin (0.1 ng/paw) and TNF-α (0.1 pg/paw) or jararhagin (0.1 ng/paw) and IL-1ß (1 pg/paw) enhanced the mechanical hyperalgesia. The systemic or local pre-treatment with PDTC (NFκB inhibitor; 100 mg/kg and 100 µg/paw) inhibited jararhagin-induced mechanical hyperalgesia as well as PDTC decreased the jararhagin-induced production of TNF-α and IL-1ß. Thus, these data demonstrate the involvement of pro-inflammatory cytokines TNF-α and IL-1ß and nuclear transcription factor NFκB in jararhagin-induced mechanical hyperalgesia indicating that targeting these mechanisms might contribute to reduce the pain induced by B. jararaca snake venom.

Tetanus neurotoxin utilizes two sequential membrane interactions for channel formation.

Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.

Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.

The serine protease inhibitor (SERPIN) family member corticosteroid-binding globulin (CBG) is the main carrier of glucocorticoids in plasma. Human CBG mediates the targeted release of cortisol at sites of inflammation through cleavage of its reactive center loop (RCL) by neutrophil elastase. The RCLs of SERPIN family members are targeted by diverse endogenous and exogenous proteases, including several bacterial proteases. We tested different bacteria for their ability to secrete proteases that disrupt CBG cortisol-binding activity, and characterized the responsible protease and site of CBG cleavage. Serum CBG integrity was assessed by Western blotting and cortisol-binding capacity assay. Effects of time, pH, temperature, and protease inhibitors were tested. Proteolytically active proteins from bacterial media were purified by fast protein liquid chromatography, and the active protease and CBG cleavage sites were identified by mass spectrometry. Among the bacteria tested, medium from Pseudomonas aeruginosa actively disrupted the cortisol-binding activity of CBG. This proteolytic activity was inhibited by zinc chelators and occurred most efficiently at pH 7 and elevated physiological temperature (ie, 41°C). Mass spectrometric analysis of a semi-purified fraction of P. aeruginosa media identified the virulence factor LasB as the responsible protease, and this was confirmed by assaying media from LasB-deficient P. aeruginosa. This metalloprotease cleaves the CBG RCL at a major site, distinct from that targeted by neutrophil elastase. Our results suggest that humoral responses to P. aeruginosa infection are influenced by this pathogen's ability to secrete a protease that promotes the release of the anti-inflammatory steroid, cortisol, from its plasma transport protein.