High-Voltage Toxin’Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes
Abstract
:1. Introduction
2. Results
- A. melanocephalus was bound the strongest by all venoms, consistent with this species retaining both the Pythonidae family’s negatively charged amino acids (191D and 195E) and the lack of any positive charges in the orthosteric site.
- M. reticulatus was strongly bound but at a level less than A. melanocephalus, consistent with it having only one negatively charged amino acid in the orthosteric site (195E) due to the secondary loss of 191D.
- P. sebae was bound less strongly than M. reticulatus but higher than P. bivittatus, P. brongersmai, or Python regius, consistent with it retaining only one of the Python genus lysine mutations (191K), with 189K replaced by glutamine (Q).
- P. regius displayed a lower venom binding affinity than P. sebae but bound stronger than P. bivittatus and P. brongersmai, consistent with it having two lysine mutations (189K and 191K), one more than P. sebae but one fewer than P. bivittatus and P. brongersmai.
- P. bivittatus and P. brongersmai were the most resistant to binding by any of the venoms, consistent with both species having three lysine mutations (189K, 191K, and 195K).
- A stepwise replacement of the lysine residues [26] confirmed the relative role of the positively charged amino acids in the evolution of venom resistance in P. bivittatus and P. brongersmai.
3. Discussion
4. Materials and Methods
- DNeasy Blood & Tissue kit (QIAGEN, Carlsbad, CA, USA) was used to isolated the DNA, using the spin column protocol for all species, except for the P. brongersmai, whose data was extracted using the E.Z.N.A. Tissue DNA Kit (Omega Bio-tek, Norcross, GA, USA).
- In total, 25 mg of homogenized tissue samples were mixed with a lysis buffer and Proteinase K solution and 56 °C shake-incubated for 3 h. Several centrifugation steps were undertaken followed by washing with wash buffer solutions.
- Prior to the DNA extraction, the tissues were rinsed with 10% of phosphate-buffered saline (PBS) to remove the 70% ethanol preservative.
- Post-elution, the DNA concentration and purity were determined using the Nanodrop 2000 UV–VIS Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
- The isolated genomic DNA was stored at −20 °C.
- A ~200 base pair range corresponding to chrna1 (muscular nAChR gene) was amplified by locus-specific primer-directed PCR.
- Primers specific for the orthosteric site of the nAChR were designed using the published Python bivittatus CHRNA1 sequence XM_007444717.2.
- ○
- Python-F = 5′ TGAATAACTACATGCCGAGTGG 3′.
- ○
- Python-R = 5′ CGTGGGTAGATAAAATACTAATCC 3′.
- The following were the PCR reaction contents:
- ○
- 25 μL of Taq PCR master mix;
- ○
- 3 μL of each primer (10 μM);
- ○
- 500 ng of DNA;
- ○
- PCR water to adjust to the 50 μL total PCR reaction volume.
- The PCR reaction conditions were as follows:
- ○
- Initial denaturation at 95 °C for 3 min (all subsequent denaturation steps were at 95 °C for 30 s);
- ○
- Annealing was at 55 °C for 30 s;
- ○
- Extension was at 72 °C for 1 min;
- ○
- The PCR steps of denaturation, annealing, and extension were repeated for 35 cycles;
- ○
- Final extension at 72 °C for 10 min.
- Sequencing of the primer-directed locus-specific amplified PCR products was undertaken at the Australian Genome Research Facility, University of Queensland, Australia, and Florida State University’s Core Facilities DNA Sequencing Laboratory, Tallahassee, Florida, using the automated dideoxy sequencing method dual-direction sequencing.
- The sequence reads were aligned and manually curated using the Aliview v.1.1 software (alignment viewer and editor) and Expasy (translate tool) to ascertain the relative absence or presence of the resistance elements in the ligand binding domain of the α1subunit of the nAChR of each of the python species tested.
- The venom samples were sourced from the long-term cryogenic collection of the Adaptive Biotoxicology Lab, University of Queensland, St Lucia, Australia.
- All the venom study protocols of this work were performed with the University of Queensland Biosafety Approval #IBC134BSBS2015 and the University of Queensland Animal Ethics Approval 2021/AE000075.
- The lyophilized crude venom samples were reconstituted with double-deionized water (ddH2O) before use. The centrifugation was performed at 14,000 RCF for 10 min with a temperature of 4 °C.
- Subsequently, the pellet (if any) was discarded, and the supernatant was used to make a working venom stock of 1 mg/mL in 50% of glycerol to preserve the enzymatic action while avoiding freezing upon storage at −20 °C.
- The concentrations of the prepared venom stocks were checked at 280 nm with a NanoDrop 2000 UV–VIS Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
- Uncontrollable postsynthetic thiol oxidation was prevented by the synthetic peptides having a serine doublet in place of the cysteine doublet [44].
- The mimotopes were dissolved in 100% of dimethyl sulfoxide (DMSO) followed by a 1:10 dilution with double-deionized water in order to make 50 µg/mL of working stocks.
- All prepared mimotope stock solutions were stored at −20 °C for future use.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Chandrasekara, U.; Broussard, E.M.; Rokyta, D.R.; Fry, B.G. High-Voltage Toxin’Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes. Toxins 2024, 16, 176. https://doi.org/10.3390/toxins16040176
Chandrasekara U, Broussard EM, Rokyta DR, Fry BG. High-Voltage Toxin’Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes. Toxins. 2024; 16(4):176. https://doi.org/10.3390/toxins16040176
Chicago/Turabian StyleChandrasekara, Uthpala, Emilie M. Broussard, Darin R. Rokyta, and Bryan G. Fry. 2024. "High-Voltage Toxin’Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes" Toxins 16, no. 4: 176. https://doi.org/10.3390/toxins16040176