Envenomation in spiders is an adaptive evolutionary mechanism that is used to protect the territory and to obtain food. Spider bites are a major problem in many Asian, African and other tropical countries. Spiders hide in crevices to predate or hunt insects. They make a web of thread to catch the insects or sting it to paralyze simultaneously. Spiders are arachnids which bear toxins in their mouth and belong to family Arenae. During long evolutionary timescale, spiders have evolved complex venoms that are employed to subdue prey and deter predator [1] . Spider toxins are short basic peptides of 32 - 76 amino acids in length. These are highly toxic in nature. Only exception is Latrotoxin which possess 1401 amino acids with 4 disulphide bridges. Spider toxins show diverse biological effects in animals i.e. scratching, lacrimation, hypertension, salivation, sweating, agitation followed by spastic paralysis of the posterior and anterior extremities. Spider toxins show diverse biological effects in animals such as muscle and respiratory paralysis, integumentary, neuro-inflammatory and neuro-pathological effects in prey. Spider toxins (JZ TX-V) inhibit ion channels present on nerve cell membrane [2] and show platelet aggregation in haemostatic system [3] . These toxins are either acylpolyamine or polypeptides which cause irreversible paralysis in lepidopteran insects by massive transmitter release. Spider toxins also generate insecticidal effect and kill insect larvae at a very low dose 0.05 mg/gm body weight.

Spider venom toxin peptides interact with ligand gated channels and modulate the activity of neuronal ion channels and receptors. These target voltage gated potassium (Kv), calcium (Cav) or sodium (Nav) channels. Spider-venom peptides target vertebrate or invertebrate Na(V) channels. They also target specific Na(V) channel subtypes that are considered to be important analgesic targets. Spider venom peptides bind on purinergic receptors, channels such as acid sensing ion channels, mechanosensitive channels and transient receptors and potassium channels. Spider venom toxins show action agents against more than 65 blockers of vertebrates Cav channels and which of them found are active mainly on Cav 2.2. Venom composition is highly species specific and depends on many climatic and biological factors. Spider venom contains a mixture of biologically active compounds with different biochemical activities. These compounds are enzymes, proteins, polyamines and acids which show multiple biological effects in animals as well as in human. Spider venom toxins generate necrotizing skin lesions, cell necrosis, and show systemic reactions and impose death in animals [4] . Venom toxins show analgesic effect, do inhibition of voltage gated ion channels [5] and generate various channelopathies including epilepsy, arrhythmia, and paralytic myotonic effect with severe pain [2] . Toxin peptides can also become a good source of strong antimicrobials and can replace broad spectrum antibiotics which are highly toxic and show multiple biological effects and responsible for drug resistance in microbes [6] . Spider venom is concerned to be a potential source of modulators for all of drug targets. These are proved much valuable tool for the investigation of structure and function of channels and for drug development [7] . Molecular templates of spider toxins and using recent technologies spider toxins can be used for production of good therapeutic agents mainly antimicrobials, anticancer agents and bio-insecticides. Spider venom includes substances of different chemical nature which possess immense therapeutic potential against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation and erectile dysfunction. Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Present study deals with isolation, purification and characterization of spider venom toxins and generation of polyclonal antibodies and its protective effect in mice model and their antimicrobial susceptibility to certain bacteria and fungi.

Before antimicrobial susceptibility testing, proteins were solubilized in different solubilizing buffers. Triton X- 100 (0.1%) proved to be a good solubilizing agent for toxin/proteins. Higher protein solubilization was observed in the supernatant than in the residue, except TCA (Figure 1). Gel filtration elution pattern displayed two major peaks at 280 nm in fraction no. 43 - 51 and 61 - 90 (Figure 2(a)). Further, estimation of venom proteins in above fractions was determined by Further Lowry’s method (1951). Again two similar peaks were resolved at the same place, a minor one was obtained between fractions 43 - 51 and a major peak between fractions 61 - 90 (Figure 2(b)). Both peaks were eluted with PBS buffer (pH 6.9) (Figure 2(c)). The total yield of protein was 67.3% and specific activity was determined in each fraction (Figure 2(d)). Molecular weights in protein fractions were ranging from 6.2 - 70 kD (Figure 2(e)).

MIC values were 7.5 µg/ml against K. pneumoniae 7.5 µg/ml against E. coli and L. acidophilus, 15.00 µg/ml against B. cereus; 15.00 µg/ml against S. aureus and M. luteus. The results suggest that E. coli and B. cereus were most susceptible to spider toxins while K. pneumoniae was least susceptible. MIC values for tetracycline and ampicillin were 27.5 µg/ml and for ciprofloxin 25 µg/ml against K. pneumoniae. (Table 1). Salmonella typhi 3.75 µg/ml, Vibrio cholerae 3.75 µg/ml, Pseudomonas aeruginosa 15.0 µg/ml respectively, while MIC value obtained against fungal strains i.e. Aspergillus niger 7.5 µg/ml, Candida albicans 7.5 µg/ml, Rhizopus stolonifer 3.75 µg/ml (Table 1).

By agar disc diffusion method the diameter of inhibition zones in mm in presence of Spider toxins at a concentration range of 197.12 - 0.96 µg/ml E. coli 17.86 ± 0.21, Bacillus cereus 19.13 ± 0.21, L. acidophilus 16.83 ± 0.25, Micrococcus luteus 18.46 ± 0.17, S. aeurus 16.23 ± 0.19, Klebsiella pneumoniae 21.83 ± 0.16, Salmonella typhi 16.16 ± 0.21, Vibrio cholerae 18.66 ± 0.21, Pseudomonas aeruginosa 18.66 ± 0.21, Aspergillus niger 22.9 ± 0.24, Candida albicans 24.66 ± 0.28, Rhizopus stolonifer 21.1 ± 0.16 (Table 2).

In the present study, antimicrobial activity of purified spider toxins was determined in vitro and compared with broad-spectrum antibiotics. From antimicrobial susceptibility tests, it was found that antibiotics were found less active than the spider venom toxins against the test bacteria and fungi. From the antimicrobial bioassays significantly lower MIC values were observed in spider toxins than the broad spectrum antibiotics. MIC values determined were 7.5 µg/ml against K. pneumonia, and E. coli and 15 µg/ml against L. acidophilus, 15 µg/ml against B. cereus; 3.75 µg/ml against S. aureus and 7.5 against µg/ml M. luteus. Both E. coli, S. aeurus, and L. acidophilus were found most susceptible to spider toxins while Aspergillus niger was least susceptible. MIC values for tetracycline were 12.5 µg/ml and ampicillin were 13.75 µg/ml and for ciprofloxin 25 µg/ml against K. pneumoniae (Table 1). Salmonella typhi and Vibrio cholerae 24.69 µg/ml, Pseudomonas aeruginosa 15 µg/ml

Positive controls are 1. tetracycline, 2. ampicillin & 3. ciprofloxacin and Tests represents spider toxins, “-” negative control is DMSO, Gsf Griseofulvin, ND = not done.

Values are expressed as mean ± SD (N = 3) and values followed by same letter are not significantly different at the p < 0.05 determined by Duncan’s Multiple Range Test. Negative control is DMSO, Positive control are 1. tetracycline, 2. ampicillin & 3. ciprofloxacin and T represents spider toxins. IZD = inhibition zone diameters.

respectively, while MIC value obtained against fungal strains i.e. Aspergillus niger and Candida albicans 7.5 µg/ml, Rhizopus stolonifer 3.75 µg/ml (Table 1). Similar activity was reported in the spider venom peptide Lycosin-II against clinically isolated bacterial pathogens [12] and Latarcins, the versatile spider venom peptides [13] . Spider venom possesses proteinaceous toxins which show remarkable insecticidal and antibacterial activities [14] . Lycosin-I as a shows much higher antipathogenic action in vitro and is used as an alternative antimicrobial drug for treatment of multidrug-resistant Acinetobacter baumannii infections [12] [15] , notably, expression of a spider toxin in tobacco inhibits the growth of microbes and insects [16] . Lycosin-I, as is a cationic and amphiphilic peptide isolated from the venom of the spider Lycosa singorensis [17] strongly act upon lipid membrane, lyses it and obstructs its integrity. Spider toxins also showed gating modifier action that works on mechanosensitive channels according to membrane thickness [18] . Antimicrobial peptides from arachnid venoms showed better microbicidal activity in the presence of commercial antibiotic due to additive effect on membrane lysis [19] . A single glycine to alanine substitution in antimicrobial peptide latarcin 2a increases interactions with lipid membrane [20] . Similarly, L- to D-amino acid substitution affects deamidation activity and membrane interactions [21] . From the inhibition zone diameter assays the IZDs obtained were larger in case of spider venom than antibiotics Similar action of spider toxins was observed in agar disc diffusion bioassay in which diameter of inhibition zones was E. coli 17.86 ± 0.21, Bacillus cereus 19.13 ± 0.21, L. acidophilus 16.83 ± 0.25, Micrococcus luteus 18.46 ± 0.17, S. aeurus 16.23 ± 0.19, Klebsiella pneuminiae 21.83 ± 0.16, Salmonella typhi 16.16 ± 0.21, Vibrio cholerae 18.66 ± 0.21, Pseudomonas aeruginosa 18.66 ± 0.21, Aspergillus niger 22.9 ± 0.24, Candida albicans 24.66 ± 0.28, Rhizopus stolonifer 21.1 ± 0.16 (Table 2). Inhibition zone diameters in mm for tetracycline, ampicillin and ciprofloxacin against S. aureus, K. pneumoniae, E. coli, L. acidophilus and M. luteus, Streptococcus pneumoniae and Bacillus cereus were 13.73 ± 0.22, 10.63 ± 0.25, 11.16 ± 0.21; 12.3 ± 0.19, 14.23 ± 0.21, 9.80 ± 0.18; 13.8 ± 0.12, 16.43 ± 0.28, 9.86 ± 0.17; respectively (Table 2). Triton X-100 (0.1%) proved to be a good solubilizing agent for toxin/proteins. Higher protein solubilization was observed in the supernatant than in the residue, except TCA (Figure 1). Spider toxins show haemolytic and and insecticidal in nature [22] . Because of their strong cytolytic and cytotoxic activity [23] , Juruin isolated from the venom of the Amazonian Pink Toe spider, Avicularia juruensis, shows antifungal activity [24] , while cupiennin 1a isolated from neotropical wandering spider Cupiennius salei exhibits remarkably broad, spectrum activity against bacteria [25] , protozoan parasites, insects, and shows cytolytic action against human cancer cells [26] . Similar antimicrobial potential was seen in LyeTx I, [27] latarcin [28] and synthetic analogues of peptides from the venom of the Central Asian spider Lachesana tarabaevi [22] . Here it can be concluded that spider toxins are potential antimicrobial agents and display broad spectrum activity due to their complex structure. These can be used as antimicrobial therapeutic agent and can easily replace highly toxic drugs which put adverse effect on patients after their administration [26] [29] .

Ravi Kumar Gupta,Ravi Kant Upadhyay, (2016) Antimicrobial Activity of Purified Toxins from Crossopriza lyoni (Spider) against Certain Bacteria and Fungi. Journal of Biosciences and Medicines,04,1-9. doi: 10.4236/jbm.2016.48001