Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom—polyclonal antibodies isolated from the plasma of hyperimmunised animals—which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability

DEVELOPMENT OF A NEW GENERATION OF ANTIVENOMIC APPROACH BY MASS SPECTROMETRY

Introduction Snakebite causes the death of about 150,000 people/year. Envenomations are classically treated by injecting anti-venomous sera. However, these treatments can induce immunological reactions which can present several adverse effects to the patient. Venom compositions strongly differ from species to species, gender and habitat and providing antivenoms targeting a specific venom is then already a challenge. Quantitatively evaluating the efficacy of any antivenom is primordial to improve the production of effective sera and to determine the nature of the toxins indeed bound by the antibodies (Igs). Therefore, mass spectrometry (MS) is important to performed it, ant it is then called ¿antivenomics¿. We plan to exploit the huge potential of magnetic beads and of mass spectrometry to speed up the antivenoms efficacy. Methods Shotgun proteomics was performed with 10¿g Echis ocellatus and Dendroaspis polylepis venoms, which were reduced/alkylated/digested with a mixture of Trypsin/GluC/Chymotrypsin, then analysed using a Q-Exactive¿ Plus Mass Spectrometer and the protein identification performed by PeaksStudio X+ using Uniprot/transcriptomes databases. Next step consists in grafting the antivenom antibodies to magnetic beads and incubating with the crude venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and, more relevantly, those remaining on the beads (recognized by Ig) allows the effectiveness and selectivity of the antivenoms studied to be determined. Preliminary data (results) In Echis ocellatus venom, 82.8% were identified as toxins and 17.2% as non-toxins while for Dendroaspis polylepis venom we obtained 26.2% for toxins, 39.9% for non-toxins and 33.9% for cellular components. The most expressed group of toxins were metalloproteinases for Echis ocellatus venom and three-finger toxins for Dendroaspis polylepis venom. These findings will be important in the efficacy evaluation and targeting the main toxins for the next step of the development the antivenomic approach. Snake venoms of the genus Echis and/or Dendroaspis are responsible for a large proportion of snake envenomations in sub-Saharan Africa.Please explain why your abstract is innovative for mass spectrometry?The optimization of the antivenomics can increase the sensibility of the antivenoms targeting the most important toxins responsible for the symptoms in the envenoming and also improve the treatment response. Keyword :Antivenomic, MALDI, Mass spectrometry, Venom

Venomics Approach Reveals a High Proportion of Lactrodectus-Like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis.

The noble false widow spider Steatoda nobilis originates from the Macaronesian archipelago and has expanded its range globally. Outside of its natural range, it may have a negative impact on native wildlife, and in temperate regions it lives in synanthropic environments where it frequently encounters humans, subsequently leading to envenomations. S. nobilis is the only medically significant spider in Ireland and the UK, and envenomations have resulted in local and systemic neurotoxic symptoms similar to true black widows (genus Latrodectus). S. nobilis is a sister group to Latrodectus which possesses the highly potent neurotoxins called α-latrotoxins that can induce neuromuscular paralysis and is responsible for human fatalities. However, and despite this close relationship, the venom composition of S. nobilis has never been investigated. In this context, a combination of transcriptomic and proteomic cutting-edge approaches has been used to deeply characterise S. nobilis venom. Mining of transcriptome data for the peptides identified by proteomics revealed 240 annotated sequences, of which 118 are related to toxins, 37 as enzymes, 43 as proteins involved in various biological functions, and 42 proteins without any identified function to date. Among the toxins, the most represented in numbers are α-latrotoxins (61), δ-latroinsectotoxins (44) and latrodectins (6), all of which were first characterised from black widow venoms. Transcriptomics alone provided a similar representation to proteomics, thus demonstrating that our approach is highly sensitive and accurate. More precisely, a relative quantification approach revealed that latrodectins are the most concentrated toxin (28%), followed by α-latrotoxins (11%), δ-latroinsectotoxins (11%) and α-latrocrustotoxins (11%). Approximately two-thirds of the venom is composed of Latrodectus-like toxins. Such toxins are highly potent towards the nervous system of vertebrates and likely responsible for the array of symptoms occurring after envenomation by black widows and false widows. Thus, caution should be taken in dismissing S. nobilis as harmless. This work paves the way towards a better understanding of the competitiveness of S. nobilis and its potential medical importance

All size but lacks in venom - Male Steatoda nobilis show less diversity in latrotoxins : A comparative study including full body MALDI Imaging and differential venom proteomics.

The Noble false widow spider Steatoda nobilis (Sn) is a member of the Therididae family, akin the ¿true¿ Black widows of the genus Latrodectus. Sn is rapidly expanding its geographic range throughout Europe and parts of the Americas, particularly in and around human dwellings [Dugon, 2017]. This species has also been shown to be of medical significance in the UK and in Ireland, where a growing number of severe cases of envenomations has occurred over the past five years [Dunbar, 2021]. A recent study showed clear similarities between the venom composition of female Sn and the venom of Latrodectus [Dunbar 2020]. In particular, both venoms contain latrotoxins and latrodectins, which could explain the latrodectism-like symptomatology observed after envenomation by Sn. In this present study, we investigated the venom composition of male Sn and compared the results with the venom of females, using a proteo-transcriptomic approach. We also present the first whole-body imaging of a spider using MALDI mass spectrometry. This proof-of-concept allows to compare the anatomy of females and males based on molecular markers and could allow to identify regionalization on toxin production in venom glands when applied to other venomous animal groups

Venomics approach reveals a high proportion of Lactrodectus-like toxins in Steatoda nobilis venom - First link to post-bite symptomology

The Noble false widow spider Steatoda nobilis has expanded its range globally and may represent a potential threat to native ecosystems and public health. Envenomations can result in local and systemic neurotoxic symptoms, similar to true black widows (genus Latrodectus). We used transcriptomic and proteomic cutting-edge approaches to deeply characterise S. nobilis venom. Among the toxins, the most represented in numbers are α-latrotoxins, -latroinsectotoxins and latrodectins, which were first characterised from black widow venoms. Approximately two-thirds of the venom is composed of Latrodectus-like toxins. We present symptomology from 23 cases (15 unpublished) of S.nobilis envenomations confirming necrosis and Latrodectus-like symptoms such as debilitating pain, tremors, fatigue, nausea and hypotension. The continued rising numbers of S. nobilis will undoubtedly result in further bites and this study will help provide the medical community with a better understanding of the potential medical outcomes from bites by this species and alert them to the possibility of medically important outcomes

Antivenomics by mass spectrometry: use of magnetic beads.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, and up to 4 times more people suffer long-term morbidity, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenoms containing antitoxin antibodies (Igs), produced by hyperimmunized animals. As antitoxin Igs represent only 10-15% of the total antivenom Ig content, such treatments have poor efficacy and the administration of animal Igs may induce immunological adverse reactions, possibly severe for the patient. Moreover, venom compositions strongly differ from species, gender, and habitat, explaining why providing broadly effective antivenoms is a real challenge. An effort has been made to reduce snakebite deaths and disability by 50% by 2030 [1]. In this context, quantitatively evaluating the toxin-binding (/neutralizing) capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called ‘antivenomics’ methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant [2], but their preparation and their lifetime represent strong constraints to the increase of antivenom evaluation throughput. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper Echis ocellatus

Steatoda nobilis : A comparative study including full body MALDI Imaging and application to Venomics field.

The Noble false widow spider Steatoda nobilis is a member of the Therididae family, akin the ¿true¿ Black widows of the genus Latrodectus. Sn is rapidly expanding its geographic range throughout Europe and parts of the Americas, particularly in and around human dwellings. Sn has also been shown to be of medical significance in the UK and in Ireland, where a growing number of severe cases of envenomation has occurred over the past five years [Dunbar, 2021]. To illustrate the comparison of both male and female Sn profiles, we additionally present the first whole-body imaging of a spider using MALDI mass spectrometry. This proof-of concept allows to compare the anatomy of females and males based on molecular markers (specific m/z distribution on spider slices)

Development of an innovative methodology of antivenomics approach, combining the use of magnetic beads and mass spectrometry.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenom, which is antitoxin antibodies (Igs) collected from immunized animals. However, these treatments may induce immunological reactions including severe adverse reaction to the patient. Moreover, venom compositions strongly differ from species, gender and habitat, explaining why providing broadly effective antivenoms is a real challenge. In this context, quantitatively evaluating the toxin-binding capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called “antivenomics” methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant, but their preparation and their lifetime represent constraints to the throughput of antivenom evaluation. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper, Echis ocellatus

Antivenomics by mass spectrometry : use of magnetic beads.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenom, which is antitoxin antibodies (Igs) collected from immunized animals. However, these treatments may induce immunological reactions including severe adverse reaction to the patient. Moreover, venom compositions strongly differ from species, gender and habitat, explaining why providing broadly effective antivenoms is a real challenge. In this context, quantitatively evaluating the toxin-binding capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called ¿antivenomics¿ methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant, but their preparation and their lifetime represent constraints to the throughput of antivenom evaluation. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper, Echis ocellatus