Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/c mice
Introduction
The mortality caused by Hemiscorpius Lepturus (HL) scorpion sting is a very critical problem in medicine that is especially common in the south and southwest of Iran, including Khuzestan Province (Dehghani and valaie, 2005). Almost all species of scorpions in Iran, with the exception of H. lepturus, belong to the large Buthidae family. Although HL is only responsible for 15% of scorpion stings, it causes the highest fatality and is to blame for 95% of the deaths caused by scorpion sting (Pipelzadeh et al., 2007). The symptoms of HL sting include cutaneous necrosis, the nephrotic syndrome, renal failure and early death (Vazirianzadeh et al., 2013, Swanson and Vetter, 2006). HL venom contains the peptides hemiclacin and hemitoxin and phospholipase D proteins such as heminecrolysin with hemolytic effects on red blood cells (Shahbazzadeh et al., 2007; Srairi-Abid et al., 2008; Borchani et al., 2011a,b). The toxicity and death caused by this scorpion venom are attributed to phospholipase D activities, including recombinant phospholipase D1 (rPLD1) activities (Dehghani et al., 2009; Rafizadeh et al., 2013). Recombinant PLD1 is a homologue of 32-kDa phospholipases D, which has no hemolytic effects on red blood cells and is often detected in the venom of Loxosceles (Chaim et al., 2006). The dermonecrotic toxin from rPLD1 in HL scorpions and the RecDT5 in Loxosceles intermedia species are the two main toxin molecules responsible for scorpion-related clinical symptoms and deaths in humans (Torabi et al., 2017a; Futrell, 1992). Dermonecrotic toxins are identified and characterized as phospholipase D and are capable of Sphingomyelin hydrolysis, and since they compose the main outer layer of the plasma membrane, they can also convert it into choline and ceramide 1-phosphate (da Silveira et al., 2006; 2007). Due to the painless nature of HL sting and the gradual emergence of symptoms in the stung, the patients often fail to go to the hospital or seek care very late (Radmanesh, 1998). These conditions create sufficient time for the toxin to affect the body, leading to critical consequences and ultimately death. According to epidemiological findings, despite the recent advances in the production of polyvalent antivenoms, the mortality rate among the victims of HL sting has not yet been dramatically modified (Vazirianzadeh et al., 2012). In addition, there is still no consensus on the effectiveness of an antivenom in the treatment of the symptoms of HL infection, which may be attributed to several factors, including late arrival to the hospital due to the painless nature of the sting and its low immunogenic potential (Jalali et al., 2012).
The present study was designed to investigate the antigenic toxoid properties of recombinant phospholipase D1 derived from H. lepturus scorpion venom glands cDNA library as an immunogen in BALB/c mice for neutralizing the lethal effect of rPLD1 toxin and HL whole venom.
Section snippets
Expression and purification of recombinant phospholipase D1
The H. lepturus phospholipase D1 gene was expressed and purified according to the previous method (Torabi et al., 2017a). In brief, the expression vector pET22b that contains the DNA fragment of rPLD1 was transformed into the bacteria E. coli BL21. Bacterial cultures were grown overnight in LB broth medium containing Ampicillin at 37 °C. When the cells reached the density of Ab 600 = 0.6, IPTG was added (Thermo Fisher Scientific Co. Waltham, MA, USA). The cells were grown for an additional
Development of recombinant phospholipase D1 (rPLD1) toxoid
The rPLD1 toxoid was developed using formalin 0.25%. When the formalin concentration was increased, no protein bands were observed in the supernatant of the formalin-treated toxin. The toxoid obtained by formalin 0.25% was subjected to Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE) for protein analysis. The rPLD1 toxoid had a single-band identical with rPLD1 toxin with the expected molecular weight of 32 KDa. The correct-size protein-band shows the suitability of the formalin
Discussion
Compared to other scorpions' venom, the venom of H. lepturus consists of a mixture of peptides, proteins and enzymes such as phospholipases, metalloproteases, hyaluronidases, potassium channel toxins, calcium channel toxins, antimicrobial peptides (AMPs), venom proteins, venom toxins, allergens, La1-like peptides, proteases and scorpion-like peptides (Torabi et al., 2017b). The venom of H. lepturus is very potent and possesses hemotoxins and cytotoxins. Phospholipases hydrolyze the ester bond
Conclusion
An interesting finding of this study is that rPLD1 toxoid can stimulate circulating antibodies significantly and effectively to neutralize the lethal effects of whole venom in mice. Generally, rPLD1 toxoid may be a useful tool for the production of active immunization (through vaccine) or passive immunization (by sero-therapeutic and Fab-therapeutic means in horses) against H. lepturus stings. The former is a therapeutic measure, while the latter is rather a preventative strategy. Furthermore,
Conflicts of interest
The authors declare that they have no conflicts of interest with respect to this report.
Ethical statement
Male BALB/c mice (18–20 g) were purchased from Pasteur Institute of Iran. The animals were allowed to adapt for a week in standard conditions with a dark/light cycle of 12 h. The room temperature was 22 ± 1 °C and the relative humidity adjusted at 50 ± 5%. The animals were received a standard pellet diet and fresh tap water. All experiments were approved by Ethical Committee of the Pasteur Institute of Iran (code number IR.PII.REC.1394.38).
Acknowledgements
The present article is part of a PhD thesis by Narges Safari-Foroushani approved by Pasteur Institute of Iran. This research has received no grants from funding agencies in the public, commercial or not-for-profit sectors.
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