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Antiprotozoal Effect of Snake Venoms and Their Fractions: A Systematic Review

Background: Protozoal infection is a lingering public health issue of great concern, despite efforts to produce drugs and vaccines against it. Recent breakthrough research has discovered alternative antiprotozoal agents encompassing the use of snake venoms and their components to cure these infections. This study collated the existing literature to examine the antiprotozoal effect of snake venoms and their fractions. Methods: We conducted a systematic review following the PRISMA guidelines. The PubMed and Embase databases were searched from their inception until 13 October 2021. Articles were screened at the title, abstract and full-text phases. Some additional studies were obtained through the manual search process. Results: We identified 331 studies via the electronic database and manual searches, of which 55 reporting the antiprotozoal effect of snake venoms and their components were included in the review. Around 38% of studies examined the effect of whole crude venoms, and a similar percentage evaluated the effect of a proportion of enzymatic phospholipase A2 (PLA2). In particular, this review reports around 36 PLA2 activities and 29 snake crude venom activities. We also report the notable phenomenon of synergism with PLA2 isoforms of Bothrops asper. Importantly, limited attention has been given so far to the antiprotozoal efficacies of metalloproteinase, serine protease and three-finger toxins, although these venom components have been identified as significant components of the dominant venom families. Conclusion: This study highlights the impact of snake venoms and their fractions on controlling protozoal infections and suggests the need to examine further the effectiveness of other venom components, such as metalloproteinase, serine protease and three-finger toxins. Future research questions in this field must be redirected toward synergism in snake venom components, based on pharmacological usage and in the context of toxicology. Ascertaining the effects of snake venoms and their components on other protozoal species that have not yet been studied is imperative.


Introduction
The evolutionary origin of snake venom has been traced back to the Cenozoic era [1]. Snake venoms have been identified as one of the most well-characterized animal venoms, with complex compositions of toxic, pharmacologically active proteins and peptides [2]. When compared with the venoms of other animals such as scorpions, spiders and cone snails, snake venoms are considered advanced due to their vast array of larger proteins and peptides that possess medicinal and toxicological effects [3]. About 50-100 components in snake venoms are distributed in dominant and secondary families, presenting multiple alytics, Philadelphia, PA, USA), and duplicates were removed using the built-in "find duplicates" feature. Two authors (Z.U.A. and S.S.M.) compiled the citations separately and later discussed them with the other authors to ensure an accurate literature report. Subsequently, the two authors independently and sequentially assessed the titles, abstracts and full text of the non-duplicated generated citations against the eligibility criteria of the study. Discrepancies in the outcomes of the screening processes between the two authors were resolved by further discussions and consultations with the other authors.

Study Selection and Eligibility Criteria
Articles were included in this review if they assessed the antiprotozoal effect of whole snake venoms or their fractions were published with at least an abstract in English. Restrictions were not placed on the study design; hence, studies of various designs (experimental, quasi-experimental, observational, case-control and case series, among others) were included. We excluded published reviews, review protocols, and conference abstracts.

Data Extraction and Synthesis
To satisfy the set criteria of the review, two authors (Z.U.A. and S.S.M.) independently extracted all the relevant data using a pre-designed Excel sheet. The data extracted included the author details, date of publication, snake species under study, snake venom components or fractions, concentration of snake venom and components used, specificity of venoms and fractions to protozoan species, and snake venom and component-induced activity on protozoa. The extracted data were then compared, and cases of inconsistent outcomes were rectified via further deliberations among the authors. The data analysis followed the synthesis without meta-analysis (SWiM) guidelines [17].

Literature Search Findings and Study Characteristics
The first search identified 309 articles through the electronic databases (100 in PubMed, and 209 in Embase). Additionally, 22 articles were added via the manual search of the reference lists of the included citations, totaling 331 papers (see Figure 1 for the outcomes of the search processes). After duplicates were removed, 319 studies remained. We screened the titles and abstracts of the 319 records against the stated eligibility criteria of the study. Finally, 55 studies were included for further synthesis and analysis. Figure 1 illustrates the flowchart of the study search and screening processes, and Table 1 presents the characteristics of the included studies.
Furthermore, previous studies [28,69] showed the impact of the trypanocidal activity of LAAOs. However, this may exclude the possibility that the other proteins reported by Chechet et al. [35], which corresponded to those reported by Guidlolin et al. [74], were responsible for the antitrypanosomal activity, either singly or synergistically. According to Peichoto et al. [62], the activity of the crude venom on the protozoal species was due to trimorphin. However, several works [18,40,45,46] suggested the need for further research to ascertain which components possess antiprotozoal efficacy, though crucial information has been reported on them, including their molecular weight and thermal stability [45,46]. Similarly, a need was reported to further investigate the fractions of B. jararaca and C.d. terrificus with antigiardial potential, suggesting that more research will provide details on the mechanisms of action [75].   Borges et al. [29] September 2016 Bothrops pauloensis DTDR IC 50 The toxin showed reasonable cytotoxicity against HeLa cells at a higher concentration; however, the effect was reduced with a reduction in concentration. In addition, the toxin could not elicit effects on the viability of tachyzoites but lessened its adhesion and proliferation when the tachyzoites were treated before infection.  Fernandez et al. [45] August 1994 Cerastes cerastes Naja haje Vipera lebetina The venoms of C. cerastes and N. haje showed a growth inhibition effect on the trypanosomatids.

Antiprotozoal Effect of Snake Venom Components or Fractions
LAAOs are oxidoreductase flavoenzymes that catalyze the stereospecific oxidative deamination of L-amino acids to produce the α-keto acids, NH 3 and H 2 O 2 [25]. They form part of several proteins in ophidians, particularly hemorrhagic venoms. LAAOs have been reported to possess the ability to induce apoptosis in several types of cells [25], including vascular endothelial cells, but the mechanism of action remains unclear. The LAAO activity has been proven to be due to H 2 O 2 production, which, in turn, has been linked with the oxidation of several proteins in the plasma membrane [1]. Our systematic review found different documented antiprotozoal activities of LAAOs. Several researchers reported antileishmanial activity in the respective species [31,32,36,67,70]. Other [25,37,38,42,60] showed their influence on growth inhibition, cytotoxic activity, inhibitory effect, programmed cell death and parasite killing on trypanosomatids. Furthermore, the LAAOs of Bothrops pirajai resulted in maximal inhibition of infection with T. gondii [51].
PLA2s are enzymatic proteins with a low molecular weight. They are responsible for promoting hydrolysis of the 3-sn-phosphoglyceride-dependent calcium 2-acyl ester bond, resulting in lysophospholipids and fatty acid products [1]. The PLA2s of snake venoms may appear to be the same but could have different toxicological efficacies in their myotoxicity, neurotoxicity, anticoagulant activity, hemolysis, hyperalgesia, inflammation, edema, cytotoxicity, hypotension, and parasitic activity [10]. The activity of PLA2s on protozoal species varies across species of snakes and the protozoal organisms involved, as described in Table 1. Previous reports [34,47,71] indicated the inhibitory effects of PLA2s of the respective snake venoms on P. falciparum. According to many other studies [21,58,59,68], various PLA2s inhibited the cellular viability of Leishmania species. In addition, Borges et al. [29] and Borges et al. [30] reported that PLA2s of B. pauloensis inhibited parasite adhesion, intracellular proliferation, parasite viability, intracellular proliferation and proinflammatory cytokine production in T. gondii. Furthermore, the PLA2s of B. pauloensis induced in vitro cell death in L. mexicana [52], and Zieler et al. [72] reported that the PLA2s of C. adamanteus blocked the ookinete adhesion and oocyst formation of both P. gallinaceum and P. falciparum. According to a previous study [63], crotoxin B and its complex from C. durissus cumanensis exerted a cytotoxic effect against the mononuclear cells of P. falciparum, and another [19] reported that the crovirin from C. viridis could inhibit and lyse humaninfective trypanosome species, including the intracellular amastigotes. However, despite the successful antiprotozoal activities of PLA2s on protozoal species, Costa-Torres et al. [38] reported that the PLA2s of B. marajoensis did not promote any inhibition of L. amazonensis or L. chagasi growth. Similarly, Grabner et al. [47] reported that the PLA2s of B. marajoensis did not promote the in vitro inhibition of cellular viability in T. cruzi epimastigote, even at 100 µg/mL. Snake venom metalloproteases (SVMPs) are zinc-dependent proteinases of around 20-110 kDa [76]. They are grouped into P-I, P-II and P-III classes according to their structural domains. These toxins are significant in viper venom compositions and have a substantial role in the toxicity of these venoms. The origin of SVMPs is linked to disintegrin and metalloproteinase (ADAM) proteins, particularly ADAM28 [77], with the P-III class being the most basal structural variant, comprising metalloproteinase, disintegrin-like, and cysteine-rich domains. Subsequently, P-II SVMPs came from P-IIIs and consisted of a metalloprotease and disintegrin domain, with the latter particularly found in venom as a proteolytically processed product [1]. The final class, PI SVMPs, which have only the metalloproteinase domain, evolved on multiple independent occasions in specific lineages due to the loss of the P-II disintegrin coding domain. SVMPs contribute extensively to the hemorrhagic and coagulopathy venom activities following bites by viperid snakes. Their isoform diversity often presents in their venom, likely facilitating synergistic effects such as a simultaneous action on multiple steps of the blood-clotting cascade [1]. Reports [27,52,54] showed the antiprotozoal activities of a metalloproteinase from the Bothrops species on T. gondii, and P. falciparum, which is one of the most threatening and widespread species.

Discussion
A total of 55 articles on the antiprotozoal effect of snake venoms and their components were identified through our systematic search of the existing literature. The majority were on the antiprotozoal efficacy of PLA2s. Over 70% of the snake species reported were vipers, with very few reports on the Colubridae species [78]. A significant proportion (around 20%) constituted species of the Elapidae family. PLA2s form a considerable component in the venoms of vipers and elapids [78], due to their biomedical importance over others [79]. PLA2s have catalytically active and inactive components. Asp49-PLA2s are the catalytically active component, and Lys49-PLA2s are the catalytically inactive component, which can facilitate pharmacological effects regardless of catalytic activity [80,81]. Findings on both the catalytically active and catalytically inactive PLA2s were reported in our study. The mediation of antiprotozoal effects by PLA2s could occur through the interaction of either PLA2 phospholipids or PLA2 proteins. Interestingly, the commonly described receptors in the cell membranes are the vascular endothelial growth factor receptor-2 (VEGFR-2), Mtype receptors, and nucleolin [82,83]. Bregge-Silva et al. [31] reported synergism involving the PLA2 isoforms of B. asper, which resulted in around a 10-fold increase in antiplasmodial activity during the association of AS49-PLA2 and LYS49-PLA2.
Synergism is an important phenomenon that occurs in snake venoms, leading to evolving strategies to potentiate toxicities. Synergism exists between toxins or toxin complexes in various snake venoms, with PLA2s (toxins or subunits) the primary enablers [84]. Snake venoms can induce considerable toxicity, which may be due to many toxins' cumulative or synergistic roles. Their compositions function together, directly or indirectly, and result in improved toxicity and pharmacological efficacy. Most synergisms of toxins have been noticed where SVSPs, PLA2s, 3FTxs and SVMPs were co-administered [84]. Synergism involving two PLA2s in B. asper has also been reported [85]. The ASP49-PLA2 and LYS49-PLA2 homologs were reported to have acted synergistically, leading to an increase in Ca 2+ ions in the plasma membrane, in turn resulting in the rapid death of myotubes. Another study reported a synergistic phenomenon between the myotoxins of ASP49-PLA2 and LYS49-PLA2, which resulted in irreversible membrane and overall cell damage [86].
Concerning the antiprotozoal activity of whole crude venoms, variations in their activity and composition are not uncommon, leading to their unique potentials in biomedical research [79]. The past literature has noted that variations in snake venoms' biochemical makeup occur even among closely related species and within species [87][88][89]. For instance, in pit vipers and adders, intra-genus or intra-specific variation in venoms has been documented [87,90]. These diversities are attributed to diet [87,[91][92][93] or topography [94,95]. Other attributable factors include repetitions in toxin-encoding genes, production processes [96][97][98][99][100], and functional and structural diversifications [75,88,101,102]. For example, venom from Laticauda semifasciata (a sea snake) does not have a complex composition, and it has just two prominent families of proteins, 3FTxs and PLA2s. However, the venoms of rattlesnakes and mambas can have 50-100 peptides or proteins, representing around 10-20 protein families [84]. Generally, the predominant protein families in snake venoms significantly comprise phylogenetic trends. The venoms of cobras, kraits, mambas and hydrophids in particular have more negligible toxins, such as 3FTxs and PLA2s. In contrast, viperid venoms are made up of more significant fractions with enzymatic activities such as snake venom metalloproteinase and snake venom serine protease [84]. For instance, the venom of C. durissus terrificus is composed of amino acids, small peptides, carbohydrates, lipids, biogenic amines, and enzymes, whereas that of B. jararaca has peptides, serine, and metalloproteases as its constituents [75]. Hence, the activity of venoms varies with the difference in concentrations and compositions.
Aside from the role of snake species in the antiprotozoal effect, parasites also present contributing factors. Promastigotes and amastigotes are physiologically different in their sensitivity to drugs, with amastigotes having the greater capability to accumulate drugs [75]. Furthermore, Podešvová et al. [52] reported that variations in the compositions of parasite membranes could also be responsible for the differences in the activities of snake venoms and their fractions. Additionally, mechanisms including post-translational modifications, protein stability, and folding may likely influence toxin activity on parasites [52].

Strengths
This systematic review was conducted following an extensive literature search of the pertinent PubMed and Embase databases. Relevant citations were extracted using the reference lists of the included studies to ensure robust coverage of the existing literature. The systematic review covered studies on the antiprotozoal effect of crude venoms and their components from clinical studies and scientific reports. No restrictions were placed on the year of publication to ensure the thorough collation of relevant information. Equally, the study inclusion criteria were not restricted to snake species or components, to provide detailed information to the research community on the research question and the gaps in the literature.

Limitations
Despite the strengths of our systematic review, it has some limitations. First, we restricted inclusion to studies published in English, thereby limiting the ability to incorporate relevant data from studies in languages other than English. Additionally, incorporating a meta-analysis on the antiprotozoal efficacy of venoms and their fractions would have improved the quality of our work, which could be considered in future studies.

Conclusions
This systematic review provides a general overview of the antiprotozoal effect of snake venoms and their components. We found varying antiprotozoal activities, presenting outstanding breakthroughs in the quest for alternative therapies for lingering protozoal infections. However, several variations were documented, including the concentrations of the crude venoms and fractions used, IC50 dosages, protozoan species, and antiprotozoal activities. These findings present challenges as to how the reviewed snake venoms and their fractions could serve as alternative antiprotozoal agents for many protozoal species, if not all. An excellent approach to this dilemma could be gearing research efforts toward understanding the relationships between venom components in the context of synergism, rather than toward studies on individual units, mainly because venomous snake species are numerous. Future studies also need to focus on other snake venom components that have received little attention. We recommend that other protozoan species should be subjected to trials with crude snake venoms and their fractions.
Author Contributions: Conceptualization, data curation, formal analysis, formal analysis, investigation, methodology, project administration, resources, validation, visualization, writing-original draft, writing-review and editing: Z.U.A.; conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing-original draft, writing-review and editing: S.S.M.; Conceptualization, data curation, formal analysis, formal analysis, investigation, methodology, project administration, resources, validation, visualization, writing-original draft, writing-review and editing: D.H.; conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing-original draft, writing-review and editing: U.M.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
All data used in this study can be obtained from the public domain.