Phenotypic dichotomy in Crotalus durissus ruruima venom and potential consequences for clinical management of snakebite envenomations

Abstract

Background: Phenotypic polymorphism in rattlesnake venoms is well-documented, with a dichotomy between hemorrhagic (Type I) and neurotoxic (Type II) venoms. In South America, the Type II phenotype is predominant; however, evidence of Type I venom in Crotalus durissus ruruima raises concerns about the efficacy of the Crotalus antivenom, which is prepared only with Type II venoms. Consequently, the Bothrops-Crotalus antivenom has been proposed as an alternative treatment for envenomation by Type I venoms.

Methodology/principal findings: This study characterizes the dichotomy of C. d. ruruima venom by analyzing the structure of isoforms differentially expressed in Type I and Type II venoms, assessing their biological activities, and evaluating the implications for snakebite clinical management in Roraima State (northern Brazil). Four toxins were differentially expressed between Type I and Type II venoms: two PIII-class SVMPs, predominantly found in Type I venoms, associated with proteolytic and hemorrhagic activity; and two PLA2s, corresponding to Crotoxin A and B chains, prevalent in Type II venoms and related to elevated phospholipase A2 activity, myotoxicity, and increased lethality. The structure of Crotoxin chains was well conserved compared to C. d. terrificus Crotoxin. However, the SVMP sequences exhibited multiple substitutions in functional and immunoreactive regions compared to Bothropasin, resulting in low hemorrhagic activity and limited reactivity/neutralization by the Bothrops antivenom. Conversely, the Crotalus antivenom reacted with high antibody titer and neutralized all activities of both venom subtypes, except for the low hemorrhagic activity induced by Type I venoms.

Conclusions/significance: The efficacy of Bothrops antivenom in snakebites caused by rattlesnakes with Type I venoms remains uncertain. We advocate for a clinical study in Roraima to assess patient outcomes and benefits of Bothrops-Crotalus versus Crotalus antivenoms for these accidents. Meanwhile, administering Bothrops-Crotalus antivenom may be acceptable; however, caution is needed regarding the use of heterologous Bothrops antibodies, which have limited efficacy in treating Crotalus envenomation.

Fig 1. Representative chromatographic profiles of venom samples from C. d. ruruima fractionated by RP-HPLC: Individual venom samples (2 mg) were applied to a Phenomenex C-18 column.

The mobile phases used were 0.1% TFA in water (solution A) or 0.1% TFA in acetonitrile (solution B). Proteins were eluted in a gradient at 2 mL/min (5% B for 5 min, 5–15% B over 10 min, 15–45% B over 60 min, 45–70% B over 10 min, 70–100% over 5 min, and 100% B over 10 min). Separation was monitored at 214 nm. A – chromatogram representative of venoms CDR 4, CDR 9, CDR 13, and CDR 15; B – chromatogram representative of venoms CDR 1, CDR 2, CDR 3, CDR 5, CDR 6, CDR 7, CDR 10, CDR 14, SB 831, SB 833, and SB 1130; C – chromatogram representative of venoms CDR 8, CDR 11, CDR 12, and SB 834. The figure highlights the peaks that characteristically elute the basic subunit of Crotoxin (CTX), acidic forms of phospholipases A2 (acidic PLA2), and snake venom metalloproteinases (SVMP).

Fig 2. Distribution of toxin families in the proteomes of Type I, Type II, and intermediate type venoms of C. d. ruruima: Individual samples of C. d. ruruima venom were subjected to shotgun proteomic analysis, and the spectra were analyzed according to the master set annotated from the transcriptome data of C. d. ruruima.

Pie charts represent the average composition of the venom from each phenotype, classified by toxin family. Relative expression was estimated by normalized total spectra counts (NTSC) by Scaffold 5.0.

Fig 3. Average number of spectra of toxin isoforms in the venoms of C. d. ruruima from different phenotypes: Individual venom samples underwent shotgun proteomics analysis using the toxins master set annotated from the transcriptome data of C. d. ruruima.

Bars represent the mean ± SD of normalized total spectra counts (NTSC) obtained via Scaffold 5.0 for each isoform in Type I (yellow bars, n = 4), Type II (blue bars, n = 11), or intermediate-type venoms (grey bars, n = 4). An asterisk (*) denotes the isoforms for which differences were statistically significant between groups of venoms (p < 0.01), indicating their predominance in Type I (*) or Type II (*) venom samples.

Fig 4. Geographical distribution of Type I, Type II, and intermediate venoms.

An elevation map of the collection area illustrates the collection points of young individuals (circles) and adults (triangles) with venoms characterized as Type I (red), Type II (blue), or intermediate (orange) venoms. A: map of South America with Roraima State highlighted in green; B: map of Roraima State with the collection area highlighted as a pink square; C: collection area. Maps of geographical records were created using QGIS 3.34.4-Prizren, and the base layer of the map is available at https://ibge.gov.br/geociencias/downloads-geociencias.html.

Fig 5. Comparison of violin plots for differentially expressed isoforms based on snake characteristics.

The violins illustrate the distribution of normalized total spectra counts (NTSC) from the isoforms Cdr_SVMPIII_01 and 03, Cdr_PLA_01 and 03, Cdr_LAO_01, Cdr_SVSP_12, and Cdr_CTL_09 in samples categorized by venom color (A), snake geographical location (B), ontogenetic stage (C), or sex (D). An asterisk (*) indicates the isoforms for which differences were statistically significant between venom groups (p < 0.01), highlighting their predominance in Type I (*) or Type II (*) venom samples.

Fig 6. Alignment of and crotoxin subunits from C. d. ruruima venom: Crotoxin sequences Cdr_PLA_01 and Cdr_PLA_03 were aligned with the sequences of highest identity from venoms of Crotalus spp corresponding to crotoxin A subunit from Crotalus durissus terrificus (P08878.1); crotoxin Bc subunit from Crotalus tzabcan (A0A193CHJ5.1); crotoxin Bd subunit from Crotalus durissus terrificus (COHM14.1); crotoxin Bb subunit from Crotalus durissus terrificus (P0CG56.1); and crotoxin Ba subunit from Crotalus durissus terrificus (P24027.1) using the ClustalW program.

The residues in red denote the substitutions compared to the C. d. ruruima sequence.

Fig 7. Alignment of SVMP sequences differentially expressed in C. d. ruruima venom: Cdr_SVMPIII_03 (A) and Cdr_SVMPIII_01 (B) sequences were aligned with a PIII-class SVMP from Crotalus durissus durissus (Q2QA02.1) and Bothropasin, a PIII-class SVMP from Bothrops jararaca (O93523.2) venoms using the ClustalW program.

The residues in red denote the substitutions compared to the C. d. ruruima sequence and residues in blue correspond to the Zn-binding motif.

Fig 8. Enzymatic activities of the C. d. ruruima venom pools: venom samples from Cdr-SVMP, Cdr-CTX, C. d. terrificus (Cdt), and B. jararaca (Bjar) were subjected to the enzymatic assay of SVMPs through the hydrolysis of the FRET substrate (Abz-AGLA-EDDnp) (A), PLA2 activity through the hydrolysis of the chromogenic substrate (NOBA) (B), and SVSP by hydrolysis of the chromogenic synthetic substrate (L-BAPNA) (C). Readings obtained from wells containing only PBS were used as a reaction blank.

The experiments were repeated three times in duplicate, and the data are represented as mean ± SD of individual tests (n = 6). Symbols denote significant difference (p < 0.05) related to B. jararaca venom (δ), C. d. terrificus venom (#), and between Cdr-CTX and Cdr-SVMP (&).

Fig 9. Functional activities of the C. d. ruruima venom pools: Venom samples from Cdr-SVMP, Cdr-CTX, C. d. terrificus, and B. jararaca were subjected to the hemorrhagic activity test by measuring the halo induced in the dorsal skin of mice three hours after the intradermal injection of 50 μg of the venoms (A); myotoxic activity by measuring creatine kinase levels in the serum of mice three hours after the injection of 20 μg of the venoms into the gastrocnemius muscle (B); and procoagulant activity, expressed as the increase in absorbance at 650 nm due to clot formation over five minutes, as indicated in the Methods section (C).

A and B: the data represent the mean ± SD of two independent experiments involving five mice each (n = 10). Symbols denote significant difference (p < 0.05) related to PBS (*), B. jararaca venom (δ), C. d. terrificus venom (#), and between Cdr-CTX and Cdr-SVMP (&). C: The plot represents a representative experiment out of three repetitions.

Fig 10. Survival time of mice injected with C. d. ruruima venom pools: Groups of five mice were injected ip with 50 μg/mouse of Cdr-SVMP, Cdr-CTX, C. d. terrificus (Cdt), or B. jararaca (Bjar) venom pools, and the number of survived mice was observed at 30, 90, 120, and 150 min and then at 4, 6, 24, and 48 h.

The result is representative of two independent experiments.

Fig 11. Molecular modelling and epitope prediction of Cdr_SVMPIII_03 and Cdr_SVMPIII_01 sequences: Three-dimensional structures of Cdr_SVMPIIIs were modelled using the I-TASSER package (https://www.rcsb.org/3d-view), and the predicted epitopes were obtained from the Epitope Prediction package provided by DTU (https://services.healthtech.dtu.dk/).

Illustrations depict the Cdr_SVMPIII_03 (A, C, D, E) and Cdr_SVMPIII_01 (B, F, G, H) structures overlapping with the Bothropasin structure (3dslA.pdb) (A, B). Regions with amino acid substitutions compared to Bothropasin are highlighted in red (C, F), epitopes predicted for reactivity with antibodies are highlighted in blue (D, G), and merged substitutions and epitopes are shown (E, H).

Fig 12. Reactivity of antigens from C. d. ruruima venom pools with antivenoms produced at the Butantan Institute.

Venom samples of Cdr-CTX (1), Cdr-SVMP (2), C. d. terrificus (3), B. jararaca (4), crotoxin, isolated from C. d. terrificus venom (5), and bathroxrhagin, an SVMP isolated from B. atrox venom (6), were fractionated by SDS-PAGE on a 12.5% acrylamide gel under non-reducing conditions and stained with Coomassie blue (A) or transferred to nitrocellulose membranes, which were then incubated with Bothrops, Crotalus, or Bothrops-Crotalus antivenoms as primary antibodies, followed by incubation with peroxidase-labelled anti-horse IgG. Reactive bands were detected by incubation with 4-chloro-a-naphthol and H₂O₂ (B). The numbers on the left indicate the mobility of the molecular mass markers in kDa.

Fig 13. Neutralization of the toxic activities of C. d. ruruima venom pools by antivenoms.

Venom samples of Cdr-SVMP (A, C, E) and Cdr-CTX (B, D, F) were incubated with Bothrops (SAB), Crotalus (SAC), Bothrops-Crotalus (SABC) antivenoms or with PBS (control) for 30 minutes at 37 °C, then centrifuged. The supernatants were tested to determine the survival time (A, B) as well as the residual procoagulant (C, D), hemorrhagic (E), and myotoxic (F) activities as described in the Methods. A, B, C, and D: the graphs are representative of two independent experiments; E and F: the data represent the mean ± SD of two independent experiments involving five mice each (n = 10). Symbols denote significant difference (p < 0.05) related to positive control PBS/venom group (*), or negative control antivenom only group (#).