Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

Development of a time-resolved immunofluorometric assay for quantitation of mucosal and systemic antibody responses.

We developed a solid phase immunoassay that measured mucosal and systemic antibody responses from mice inoculated with either a staphylococcal enterotoxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiviral origin. The assay used time-resolved fluorescence (TRF) with affinity-purified goat anti-mouse IgA and IgG conjugated to samarium and europium chelates, respectively. By employing these fluorogenic conjugates with different spectral emissions, IgA and IgG specific for SEB or VLP were readily detected in serum and saliva from mice inoculated intranasally. The TRF assay detected antigen-specific IgA in saliva 10 min after the addition of enhancement solution, while a conventional alkaline phosphatase-based assay for salivary IgA required 18 h after substrate addition. The TRF assay also provided a significantly higher signal-to-noise ratio and exhibited greater sensitivity. TRF assays detected both IgA and IgG in the same well, thereby reducing sample and reagent requirements.

A non-radioactive receptor assay for snake venom postsynaptic neurotoxins.

A non-radioactive assay was developed for detecting the binding of postsynaptic neurotoxins to acetylcholine receptor (AchR) from Torpedo californica. Enzyme linked immunosorbent assay (ELISA) wells coated with long or short chain neurotoxins specifically bound to purified AchR while crotamine or two different cardiotoxins did not. Bound receptor was detected by antibody against AchR. Specificity was determined by dose-response experiments and competition studies using carbamylcholine chloride, acetylcholine chloride, or Naja naja atra cobrotoxin mixed with receptor.

Acetylcholine receptor binding characteristics of snake and cone snail venom postsynaptic neurotoxins: further studies with a non-radioactive assay.

The binding of postsynaptic neurotoxins from snake and marine cone snail (Conus sp.) venoms to nicotinic acetylcholine receptor (AchR) was investigated with an ELISA-based, non-radioactive assay. Three snake postsynaptic toxins from the long-chain group (Naja naja kaouthia cobratoxin, Naja oxiana neurotoxin I, Bungarus multicinctus alpha-bungarotoxin) and short-chain group (Naja naja atra cobrotoxin, Naja oxiana neurotoxin II, and Laticauda semifasciata erabutoxin b) were studied. Both types of snake postsynaptic toxins showed a dose-response with constant AchR (50 micrograms/ml) and varying toxin concentrations (50-0.035 micrograms/ml). The minimum detection limits of the assay for snake toxins ranged from 310 to 1240 ng/ml (40-160 pmole/ml), depending on the toxin. Unlike any of the short-chain toxins, long-chain toxins consistently bound less receptor and reached maximum absorbance levels with toxin concentrations of 10-50 micrograms/ml. Competition for AchR binding between cone snail postsynaptic neurotoxins (conotoxins GI, MI, SI) and alpha-bungarotoxin or cobrotoxin resulted in a dose-response. The postsynaptic conotoxins were uniformly better competitors for AchR binding with alpha-bungarotoxin than with cobrotoxin. Heat stability studies with neurotoxin I, erabutoxin b, or cobrotoxin revealed a loss in AchR binding activity with increasing temperature. alpha-Bungarotoxin heated at 90 degrees C had increased AchR binding activity by 105%, relative to 25 degrees C samples, but lost the majority of its binding activity after 100 degrees C. The enhanced binding of heated alpha-bungarotoxin to AchR was specific, as evidenced by a competitive dose-response with unheated alpha-bungarotoxin, but heated toxin lacked any biological activity in the mouse lethal assay. When conotoxins GI or MI were heated at 100 degrees C, there was no detectable loss in AchR binding activity, and only a slight decrease in mouse lethality.

Detection of Clostridium perfringens alpha toxin using a capture antibody ELISA.

Clostridium perfringens phospholipase C (PLC), commonly known as alpha toxin, is the lethal, dermonecrotic toxin produced by all strains and is considered a major virulence factor in clostridial myonecrosis. We developed a capture antibody ELISA that accurately and specifically quantitates alpha toxin produced by C. perfringens. Another PLC, derived from Bacillus cereus, and culture filtrates from various bacterial species including Clostridium bifermentans and Clostridium novyi were not cross-reactive in this ELISA. Standard curves generated with homogenous C. perfringens alpha toxin revealed detection limits of 19 ng/ml. The ELISA was more sensitive in detecting alpha toxin than techniques such as PLC enzymatic activity and mouse lethality assays.

Clostridium perfringens iota toxin: synergism between two proteins.

The iota toxin of Clostridium perfringens type E is a guinea pig dermonecrotic, mouse lethal toxin which cross-reacts with the iota-like toxin of Clostridium spiroforme. Antiserum raised against C. spiroforme or C. perfringens type E neutralizes the toxin from both species. By using C. spiroforme antiserum and crossed immunoelectrophoresis, we have found that there are two cross-reacting proteins, designated iota a (ia) and iota b (ib) in the culture filtrate of C. perfringens type E. Both proteins of C. perfringens were separated by preparative isoelectric focusing and had very little toxic activity when tested alone. However, when they were recombined there were 8- and 25-fold increases in bioactivity as determined by mouse lethal and guinea pig dermonecrotic assays, respectively. These results demonstrate that the iota toxin of C. perfringens requires two immunologically and biochemically different proteins for maximum activity.

Immunoreactivity, epitope mapping and protection studies with anti-conotoxin GI sera and various conotoxins.

Goat or rabbit anti-conotoxin GI sera recognized native or dithiothreitol-reduced alpha-conotoxins GI, MI and SI in an enzyme-linked immunosorbent assay (ELISA). Native mu-conotoxin GIIIA or omega-conotoxin GVIA did not react with either anti-conotoxin GI serum in the assay. The goat anti-conotoxin GI serum neutralized 2.5 LD50S of alpha-conotoxins GI or MI in mouse lethal assays, while the rabbit antiserum had little protective capabilities. Epitope mapping of synthesized conotoxin peptide fragments revealed that both anti-conotoxin GI sera recognized linear sequences from five different alpha-conotoxins: GI, GIA, GII, MI and SI. The CCNPAC sequence was optimally recognized by both antisera.

Antibody-mediated neutralization and binding-reversal studies on alpha-neurotoxins from Micrurus nigrocinctus nigrocinctus (coral snake) venom.

An ELISA based, non-radioactive acetylcholine receptor (AchR) binding assay was used to detect the alpha-neurotoxins present in Micrurus nigrocinctus nigrocinctus venom. Sera from horses hyperimmunized against M. nigrocinctus venom contain antibodies which inhibit the binding of M. n. nigrocinctus alpha-neurotoxins to AchR and reverse the binding of toxins already complexed with the receptor. This result supports the importance of using antivenom therapeutically in M. n. nigrocinctus envenomations even after the onset of neurological symptoms. M. nigrocinctus antivenoms cross-reacted in an ELISA with several elapid alpha-neurotoxins and inhibited the binding of Bungarus multicinctus alpha-bungarotoxin and Naja naja oxiana neurotoxin II to AchR in vitro, suggesting the presence of short-chain and long-chain alpha-neurotoxins in M. nigrocinctus venom. In vivo neutralization experiments with M. nigrocinctus antivenom demonstrate that M. nigrocinctus venom contains short-chain alpha-neurotoxin(s) which share common neutralizing epitope(s) with Naja naja oxiana neurotoxin II.

Antibacterial effects of different snake venoms: purification and characterization of antibacterial proteins from Pseudechis australis (Australian king brown or mulga snake) venom.

Venoms from 30 different snake species were tested in a disc diffusion assay for antibacterial effects against gram-positive and gram-negative bacteria. A number of venoms gave a zone of inhibition against both groups of bacteria, including Aeromonas hydrophila, an important pathogen of reptiles and amphibians. Two antibacterial components from the venom of an Australian elapid, Pseudechis australis (Australian king brown or mulga snake) were purified to homogeneity. The proteins, designated LAO1 and LAO2, had potent antibacterial properties associated with L-amino acid oxidase activity. Both had native and subunit mol. wts of 142,000 and 56,000, respectively. Antibacterial activity correlated with enzymatic activity and was eliminated with catalase. LAO1 and LAO2 had 244 and 113 units of L-amino acid oxidase activity/mg protein, respectively. Compared to tetracycline, a drug of choice for Aeromonas infections in humans, reptiles and amphibians, the in vitro antibacterial effects of LAO1 and LAO2 were respectively 70 and 17.5 times more effective (on a molar basis).

A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus.

Therapeutic antisera against Micrurus nigrocinctus venom were tested for protection against lethality, as well as for inhibition of the nicotinic acetylcholine receptor (AchR)-binding and neutralization of phospholipase A2 (PLA2) activities of the homologous venom. Protection against venom lethality did not correlate with inhibition of AchR-binding activity, whereas there was a significant correlation between antisera potency and inhibition of PLA2 activity (r = 0.82, n = 10, P < 0.02). Inhibition of PLA2 activity could be useful in assessing the protective efficacy of M. nigrocinctus antisera during antivenom production. Micrurus nigrocinctus nigrocinctus venom proteins were fractionated by cation-exchange chromatography on Mono S FPLC and fractions assayed for lethality, AchR-binding and PLA2 activities. Antisera were titrated by enzyme-linked immunoassay (ELISA) against a crude M. n. nigrocinctus venom, two FPLC lethal fractions containing AchR-binding activity, and two toxins purified from M. n. nigrocinctus venom. No correlation was found between protective efficacy and the ELISA titer against any of these antigens. Compared to other elapid venoms that contain few toxins as major components, M. n. nigrocinctus venom appears to be more complex and its lethal effect is likely to be due to the combined effect of several neurotoxins.

Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin.

Twelve monoclonal antibodies against cobrotoxin from Naja naja atra venom were tested for cross-reactivity with eight different snake toxins, binding to linear epitopes, prevention of cobrotoxin binding to acetylcholine receptor (AchR) in vitro, and protection in mice concomitantly given a lethal dose of cobrotoxin. The antibodies were highly specific, as evidenced by little reactivity with other snake toxins. None of the monoclonal antibodies bound to reduced cobrotoxin or synthesized 8-mer regions spanning the whole molecule, thus suggesting the recognition of conformational epitopes. The in vitro binding of toxin to AchR was competitively inhibited (23-79%) with a 1.66:1 mole ratio of antibody:AchR. Preincubation of monoclonal antibody with toxin before adding AchR (3:1 mole ratio of AchR:antibody) inhibited the in vitro binding of toxin to AchR by 20-80%. Monoclonal antibodies added after the preincubation of toxin with AchR did not dissociate the toxin-AchR complex. An antibody:toxin mole ratio of 2.5:1, with 6 micrograms of cobrotoxin, delayed the time to death of mice 3.7-23.8-fold over control mice. The monoclonal antibodies that most effectively prevented in vitro binding of toxin to AchR also provided the longest delay in time to death in mice.

Telemetric evaluation of body temperature and physical activity as predictors of mortality in a murine model of staphylococcal enterotoxic shock.

BACKGROUND AND PURPOSE: Hypothermia and death are used as experimental markers in murine models of staphylococcal enterotoxic shock. This study determined whether body temperature and physical activity, monitored telemetrically, could predict impending death and provide an earlier, more humane experimental endpoint. METHODS: The study consisted of two iterations (experiments 1 and 2) to determine reproducibility of the model. Each experiment consisted of 24 BALB/c mice surgically implanted with intra-abdominal telemetry transmitters and then injected intraperitoneally with sublethal or lethal doses of staphylococcal enterotoxin B (SEB) and/or lipopolysaccharide (LPS). Core body temperature and physical activity were continuously monitored in all mice for 10 days before, and 5 days after, injections. Additionally, in experiment 2, subcutaneous temperatures were compared with core body temperatures obtained by telemetry. RESULTS: Body temperature and physical activity were reduced in mice after administration of SEB and LPS, or LPS alone, but not SEB only. There was a significant (P < 0.05) correlation between mortality and body temperature (P = 0.0077), but not physical activity (P = 0.97). CONCLUSION: Body temperature proved to be an early indicator of mortality in this murine model of staphylococcal enterotoxic shock.

Epitope mapping studies of snake venom phospholipase A2 using monoclonal antibodies.

Fifteen different monoclonal antibodies developed against pseudexin, a snake venom phospholipase A2 with presynaptic neurotoxicity, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin were synthesized by using alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies bound to toxin peptides and were detected with an enzyme-linked immunosorbent assay. Three of the bound antibodies recognized a site important in calcium binding and the interlocking of dimeric forms of snake venom phospholipase A2. Analogous regions from other phospholipases were synthesized and probed with the four reactive antibodies. A good correlation was found between the reactivity of whole molecule phospholipases and peptide regions with the antibodies. Monoclonal antibodies neutralizing the lethal or enzymatic effects of pseudexin did not recognize any linear epitopes.

Snake venom phospholipases A(2): a novel tool against bacterial diseases.

The majority of snake venom phospholipases A(2) (svPLA(2)s) are toxic and induce a wide spectrum of biological effects. They are cysteine-rich proteins that contain 119-134 amino acids and share similar structures and functions. About 50% of the residues are incorporated into α-helices, whereas only 10% are in ß-sheets. Fourteen conserved cysteines form a network of seven disulfide bridges that stabilize the tertiary structure. They show a high degree of sequence and structural similarity, and are believed to have a common calcium- dependent catalytic mechanism. Additionally, svPLA(2)s display an array of biological actions that are either dependent or independent of catalysis. The PLA(2)s of mammalian origin also exert potent bactericidal activity by binding to anionic surfaces and enzymatic degradation of phospholipids in the target membranes, preferentially of Gram-positive species. The bactericidal activity against Gram-negatives by svPLA(2) requires a synergistic action with bactericidal/permeability-increasing protein (BPI), but is equally dependent on enzymatic- based membrane degradation. Several hypotheses account for the bactericidal properties of svPLA(2)s, which include "fatal depolarization" of the bacterial membrane, creation of physical holes in the membrane, scrambling of normal distribution of lipids between the bilayer leaflets, and damage of critical intracellular targets after internalization of the peptide. The present review discusses several svPLA(2)s and derived peptides that exhibit strong bactericidal activity. The reports demonstrate that svPLA(2)-derived peptides have the potential to counteract microbial infections. In fact, the C-terminal cationic/hydrophobic segment (residues 115-129) of svPLA(2)s is bactericidal. Thus identification of the bactericidal sites in svPLA(2)s has potential for developing novel antimicrobials.

Antimicrobial proteins from snake venoms: direct bacterial damage and activation of innate immunity against Staphylococcus aureus skin infection.

The innate immune system is the first line of defense against microbial diseases. Antimicrobial proteins produced by snake venoms have recently attracted significant attention due to their relevance to bacterial infection and potential development into new therapeutic agents. Staphylococcus aureus is one of the major human pathogens causing a variety of infections involving pneumonia, toxic shock syndrome, and skin lesions. With the recent emergence of methicillin (MRSA) and vancomycin (VRSA) resistance, S. aureus infection is a serious clinical problem that will have a grave socio-economic impact in the near future. Although S. aureus susceptibility to innate antimicrobial peptides has been reported recently, the protective effect of snake venom phospholipase A2 (svPLA2) proteins on the skin from S. aureus infection has been understudied. This review details the protective function of svPLA2s derived from venoms against skin infections caused by S. aureus. We have demonstrated in vivo that local application of svPLA2 provides complete clearance of S. aureus within 2 weeks after treatment compared to fusidic acid ointment (FAO). In vitro experiments also demonstrate that svPLA2 proteins have inhibitory (bacteriostatic) and killing (bactericidal) effects on S. aureus in a dose-dependant manner. The mechanism of bacterial membrane damage and perturbation was clearly evidenced by electron microscopic studies. In summary, svPLA2s from Viperidae and Elapidae snakes are novel molecules that can activate important mechanisms of innate immunity in animals to endow them with protection against skin infection caused by S. aureus.

Staphylococcus aureus: the toxic presence of a pathogen extraordinaire.

Staphylococcus aureus is a facultative, Gram-positive coccus well known for its disease-causing capabilities. In particular, methicillin and vancomycin resistant strains of S. aureus (MRSA and VRSA, respectively) isolated globally represent daunting medical challenges for the 21(st) Century. This bacterium causes numerous illnesses in humans such as food poisoning, skin infections, osteomyelitis, endocarditis, pneumonia, enterocolitis, toxic shock, and autoimmune disorders. A few of the many virulence factors attributed to S. aureus include antibiotic resistance, capsule, coagulase, lipase, hyaluronidase, protein A, fibronectin-binding protein, and multiple toxins with diverse activities. One family of protein toxins is the staphylococcal enterotoxins (SEs) and related toxic shock syndrome toxin-1 (TSST-1) that act as superantigens. There are more than twenty different SEs described to date with varying amino acid sequences, common conformations, and similar biological effects. By definition, very low (picomolar) concentrations of these superantigenic toxins activate specific T-cell subsets after binding to major histocompatibility complex class II. Activated T-cells vigorously proliferate and release proinflammatory cytokines plus chemokines that can elicit fever, hypotension, and other ailments which include a potentially lethal shock. In vitro and in vivo models are available for studying the SEs and TSST-1, thus providing important tools for understanding modes of action and subsequently countering these toxins via experimental vaccines or therapeutics. This review succinctly presents the pathogenic ways of S. aureus, with a toxic twist. There will be a particular focus upon the biological and biochemical properties of, plus current neutralization strategies targeting, staphylcoccocal superantigens like the SEs and TSST-1.

Characterization of alpha-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies.

The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies.

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies.

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A2 (PLA2S) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA2. Antibodies neutralizing lethal or enzymatic effects of PLA2 did not recognize linear epitopes.

Pentoxifylline inhibits superantigen-induced toxic shock and cytokine release.

Tumor necrosis factor alpha (TNF-alpha) is a critical cytokine that mediates the toxic effects of bacterial superantigens like staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1). Pentoxifylline, an anti-inflammatory agent that inhibits endotoxemia and lipopolysaccharide (LPS)-induced release of TNF-alpha, was tested for its ability to inhibit SEB- and TSST-1-induced activation of human peripheral blood mononuclear cells (PBMCs) in vitro and toxin-mediated shock in mice. Stimulation of PBMCs by SEB or TSST-1 was effectively blocked by pentoxifylline (10 mM), as evidenced by the inhibition of TNF-alpha, interleukin 1beta (IL-1beta), gamma interferon (IFN-gamma), and T-cell proliferation. The levels of TNF-alpha, IL-1alpha, and IFN-gamma in serum after an SEB or TSST-1 injection were significantly lower in mice given pentoxifylline (5.5 mg/animal) versus control mice. Additionally, pentoxifylline diminished the lethal effects and temperature fluctuations elicited by SEB and TSST-1. Thus, in addition to treating endotoxemias, the cumulative in vitro and in vivo data suggest that pentoxifylline may also be useful in abrogating the ill effects of staphylococcal enterotoxins and TSST-1.