Alexandra Rucavado

Snake venom metalloproteinases: Their role in the pathogenesis of local tissue damage

The biochemical characteristics of hemorrhagic metalloproteinases isolated from snake venoms are reviewed, together with their role in the pathogenesis of the local tissue damage characteristic of crotaline and viperine snake envenomations. Venom metalloproteinases differ in their domain structure. Some enzymes comprise only the metalloproteinase domain, others have disintegrin-like and high cysteine domains and others present, besides these domains, an additional lectin-like subunit. All of them are zinc-dependent enzymes with highly similar zinc binding environments. Some metalloproteinases induce hemorrhage by directly affecting mostly capillary blood vessels. It is suggested that hemorrhagic enzymes cleave, in a highly selective fashion, key peptide bonds of basement membrane components, thereby affecting the interaction between basement membrane and endothelial cells. As a consequence, these cells undergo a series of morphological and functional alterations in vivo, probably associated with biophysical hemodynamic factors such as tangential fluid shear stress. Eventually, gaps are formed in endothelial cells through which extravasation occurs. In addition to hemorrhage, venom metalloproteinases induce skeletal muscle damage, myonecrosis, which seems to be secondary to the ischemia that ensues in muscle tissue as a consequence of bleeding and reduced perfusion. Microvessel disruption by metalloproteinases also impairs skeletal muscle regeneration, being therefore responsible of fibrosis and permanent tissue loss after snakebites. Moreover, venom metalloproteinases participate in the degradation of extracellular matrix components and play a relevant role in the prominent local inflammatory response that characterizes snakebite envenomations, since they induce edema, activate endogenous matrix metalloproteinases (MMPs) and are capable of releasing TNF-alpha from its membrane-bound precursor. Owing to their protagonic role in the pathogenesis of local tissue damage, snake venom metalloproteinases constitute relevant targets for natural and synthetic inhibitors which may complement antivenoms in the neutralization of these effects.

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

Background To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections.

Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases.

Hemorrhage is one of the most significant effects in envenomings induced by viperid snakebites. Damage to the microvasculature, induced by snake venom metalloproteinases (SVMPs), is the main event responsible for this effect. The precise mechanism by which SVMPs disrupt the microvasculature has remained elusive, although recent developments provide valuable clues to deciphering the details of this pathological effect. The main targets of hemorrhagic SVMPs are components of basement membrane (BM) and surrounding extracellular matrix (ECM), which provide mechanical stability to capillaries. P-III SVMPs, comprising disintegrin-like and cysteine-rich domains in addition to the catalytic domain, are more potent hemorrhagic toxins than P-I SVMPs, constituted only by the metalloproteinase domain. This is likely due to the presence of exosites in the additional domains, which contribute to the binding of SVMPs to relevant targets in the microvasculature. Recent in vivo studies have shown that P-III SVMPs are preferentially located in microvessels. On the other hand, the structural determinants responsible for the different hemorrhagic potential of P-I SVMPs remain largely unknown, although backbone flexibility in a loop located near the active site is likely to play a role. Moreover, hemorrhagic and non-hemorrhagic SVMPs differ in their capacity to hydrolyze in vivo key BM proteins, such as type IV collagen and perlecan, as well as other ECM proteins, like types VI and XV collagens, which play a critical role by connecting BM components to perivascular fibrillar collagens. The evidence gathered support a two-step model for the pathogenesis of SVMP-induced hemorrhage: initially, hemorrhagic SVMPs bind to and hydrolyze components of the BM and associated extracellular matrix proteins that play a key role in the mechanical stability of BM. In conditions of normal blood flow in the tissues, such cleavage results in the weakening, distension and eventual disruption of capillary wall due to the action of biophysical forces operating in vivo.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue‐damaging activities

BaP1 is a 22.7‐kD P‐I‐type zinc‐dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue‐damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117–Cys 197, Cys 159–Cys 181, Cys 157–Cys 164). It has the consensus sequence H142E143XXH146XXGXXH152, as well as the sequence C164I165M166, which characterize the “metzincin” superfamily of metalloproteinases. The active‐site cleft separates a major subdomain (residues 1–152), comprising four α‐helices and a five‐stranded β‐sheet, from the minor subdomain, which is formed by a single α‐helix and several loops. The catalytic zinc ion is coordinated by the Nε2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active‐site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P‐I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active‐site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

Trends in Snakebite Envenomation Therapy: Scientific, Technological and Public Health Considerations

The therapy of snakebite envenomation has been based on the parenteral administration of animal-derived antivenoms. Despite the success of this treatment at reducing the impact of snakebite mortality and morbidity, mostly due to their capacity to neutralize systemically-acting toxins, antivenoms are of relatively low efficacy in the prevention of venom-induced local tissue damage, which often leads to permanent disability. The issue of safety also remains a concern, particularly for some antivenoms which induce a relatively high incidence of adverse reactions. Consequently, there is a need to improve the therapy of snakebite envenomations on the following lines: (a) the technologies to produce antivenoms require improvements aimed at obtaining more refined preparations of higher efficacy and safety, while being affordable for the public health systems of developing countries. (b) The growing knowledge on the biochemistry and toxicology of snake venoms should pave the way for the identification of natural and synthetic inhibitors of venom toxins, particularly of those involved in local tissue pathology. Such inhibitors might become a highly effective therapeutic tool for the abrogation of venom-induced local tissue damage. (c) A better knowledge of the inflammatory events secondary to venom actions may open the possibility of modulating such response, in order to prevent further tissue damage and to promote successful tissue repair and regeneration. A global partnership, involving many participants and combining scientific, technological and public health actions, is required to achieve a leap forward in the treatment of snakebite envenomations world-wide.

Experimental pathology of local tissue damage induced by Bothrops asper snake venom

Envenomations by Bothrops asper are often associated with complex and severe local pathological manifestations, including edema, blistering, dermonecrosis, myonecrosis and hemorrhage. The pathogenesis of these alterations has been investigated at the experimental level. These effects are mostly the consequence of the direct action of zinc-dependent metalloproteinases (SVMPs) and myotoxic phospholipases A(2) (PLA(2)s). SVMPs induce hemorrhage, blistering, dermonecrosis and general extracellular matrix degradation, whereas PLA(2)s induce myonecrosis and also affect lymphatic vessels. In addition, the prominent vascular alterations leading to hemorrhage and edema may contribute to ischemia and further tissue necrosis. The mechanisms of action of SVMPs and PLA(2)s are discussed in detail in this review. Venom-induced tissue damage plays also a role in promoting bacterial infection. A prominent inflammatory reaction develops as a consequence of these local pathological alterations, with the synthesis and release of abundant mediators, resulting in edema and pain. However, whether inflammatory cells and mediators contribute to further tissue damage is not clear at present. Muscle tissue regeneration after venom-induced pathological effects is often impaired, thus resulting in permanent tissue loss and dysfunction. SVMP-induced microvessel damage is likely to be responsible of this poor regenerative outcome. Antivenoms are only partially effective in the neutralization of B. asper-induced local effects, and the search for novel toxin inhibitors represents a potential avenue for improving the treatment of this serious aspect of snakebite envenomation.

Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper

Envenomations by the snake Bothrops asper are characterized by prominent local tissue damage (i.e. myonecrosis), blistering, hemorrhage and edema. Various phospholipases A2 and metalloproteinases that induce local pathological alterations have been purified from this venom. Since these toxins induce a conspicuous inflammatory response, it has been hypothesized that inflammatory mediators may contribute to the local pathological alterations described. This study evaluated the local production of cytokines and matrix metalloproteinases (MMPs) as a consequence of intramuscular injections of an Asp-49 myotoxic phospholipase A2 (myotoxin III (MT-III)) and a P-I type hemorrhagic metalloproteinase (BaP1) isolated from B. asper venom. Both enzymes induced prominent tissue alterations and conspicuous increments in interleukin (IL)-1beta, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-alpha by resident peritoneal macrophages in vitro. Despite the conspicuous expression of latent forms of MMPs in muscle, evidenced by zymography, there were no increments in activated MMP-2 and only a small increase in activated MMP-9, as detected by a functional enzymatic assay. This suggests that MMP activity was regulated by a highly controlled activation of latent forms and, probably, by a concomitant synthesis of MMP inhibitors. Since no hemorrhage nor dermonecrosis were observed after injection of MT-III, despite a prominent increase in MMP expression, and since inflammatory exudate did not enhance hemorrhage induced by BaP1, it is suggested that endogenous MMPs released in the tissue are not responsible for the dermonecrosis and hemorrhage characteristic of B. asper envenomation. Moreover, pretreatment of mice with the peptidomimetic MMP inhibitor batimastat did not reduce myotoxic nor edema-forming activities of MT-III, suggesting that MMPs do not play a prominent role in the pathogenesis of these effects in this experimental model. It is concluded that MT-III and BaP1 induce a local inflammatory response associated with the synthesis of IL-1beta, IL-6 and MMPs. MMPs do not seem to play a prominent role in the acute local pathological alterations induced by these toxins in this experimental model.

Experimental pathophysiology of systemic alterations induced by Bothrops asper snake venom.

Moderate and severe envenomations by the snake Bothrops asper provoke systemic alterations, such as systemic bleeding, coagulopathy, hypovolemia, hemodynamic instability and shock, and acute renal failure. Systemic hemorrhage is a typical finding of these envenomations, and is primarily caused by the action of P-III snake venom metalloproteinases (SVMPs). This venom also contains a thrombin-like serine proteinase and a prothrombin-activating P-III SVMP, both of which cause defibrin(ogen)ation. Thrombocytopenia, predominantly induced by a C-type lectin-like protein, and platelet hypoaggregation, caused by the two defibrin(ogen)ating enzymes, also contribute to hemostatic disturbances, which potentiate the systemic bleeding induced by hemorrhagic SVMPs. Cardiovascular disturbances leading to shock are due to the combined effects of hemorrhagic toxins, other venom components that increase vascular permeability, the action of hypotensive agents in the venom and of endogenous mediators, and the potential cardiotoxic effect of some toxins. Renal alterations are likely to be caused by direct cytotoxicity of venom components in the kidney, and by renal ischemia resultant from hypovolemia and hypoperfusion. Lethality induced by B. asper venom is the consequence of several combined effects among which the action of P-III SVMPs is especially relevant.

Snake Venomics of the Lesser Antillean Pit Vipers Bothrops caribbaeus and Bothrops lanceolatus: Correlation with Toxicological Activities and Immunoreactivity of a Heterologous Antivenom†

The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.

Effectiveness of batimastat, a synthetic inhibitor of matrix metalloproteinases, in neutralizing local tissue damage induced by BaP1, a hemorrhagic metalloproteinase from the venom of the snake bothrops asper.

Batimastat (BB-94), a synthetic hydroxamate peptidomimetic matrix metalloproteinase inhibitor, was tested for its ability to inhibit proteolytic and toxic effects induced by BaP1, a 24-kDa hemorrhagic metalloproteinase isolated from the venom of Bothrops asper, the medically most important snake species in Central America and southern Mexico. Batimastat inhibited proteolytic activity on biotinylated casein, with anIC(50) of 80 nM. In addition, batimastat was effective in inhibiting hemorrhagic, dermonecrotic, and edema-forming activities of this metalloproteinase if incubated with the enzyme prior to the assays. When the inhibitor was administered i.m. at the site of the toxin injection without preincubation, rapidly after metalloproteinase administration, it totally abrogated the hemorrhagic and dermonecrotic effects of BaP1. Inhibition was less effective as the time lapse between toxin and batimastat injection increased, due to the extremely rapid development of BaP1-induced local tissue damage in this experimental model. On the other hand, batimastat was ineffective if administered by the i.p. route immediately after toxin injection. It is concluded that batimastat, and probably other synthetic metalloproteinase inhibitors, may become useful therapeutic tools aimed at the in situ inhibition of venom metalloproteinases, when injected at the site of the bite rapidly after envenomation.