Venom Ex Machina? Exploring the Potential of Cell-Free Protein Production for Venom Biodiscovery.

Venoms are a complex cocktail of potent biomolecules and are present in many animal lineages. Owed to their translational potential in biomedicine, agriculture and industrial applications, they have been targeted by several biodiscovery programs in the past. That said, many venomous animals are relatively small and deliver minuscule venom yields. Thus, the most commonly employed activity-guided biodiscovery pipeline cannot be applied effectively. Cell-free protein production may represent an attractive tool to produce selected venom components at high speed and without the creation of genetically modified organisms, promising rapid and highly efficient access to biomolecules for bioactivity studies. However, these methods have only sporadically been used in venom research and their potential remains to be established. Here, we explore the ability of a prokaryote-based cell-free system to produce a range of venom toxins of different types and from various source organisms. We show that only a very limited number of toxins could be expressed in small amounts. Paired with known problems to facilitate correct folding, our preliminary investigation underpins that venom-tailored cell-free systems probably need to be developed before this technology can be employed effectively in venom biodiscovery.

Engineering a wolf spider A-family toxin towards increased antimicrobial activity but low toxicity.

Spider-derived peptides with insecticidal, antimicrobial and/or cytolytic activities, also known as spider venom antimicrobial peptides (AMPs), can be found in the venoms of RTA-clade spiders. They show translational potential as therapeutic leads. A set of 52 AMPs has been described in the Chinese wolf spider (Lycosa shansia), and many have been shown to exhibit antibacterial effects. Here we explored the potential to enhance their antimicrobial activity using bioengineering. We generated a panel of artificial derivatives of an A-family peptide and screened their activity against selected microbial pathogens, vertebrate cells and insects. In several cases, we increased the antimicrobial activity of the derivatives while retaining the low cytotoxicity of the parental molecule. Furthermore, we injected the peptides into adult Drosophila suzukii and found no evidence of insecticidal effects, confirming the low levels of toxicity. Our data therefore suggest that spider venom linear peptides naturally defend the venom gland against microbial colonization and can be modified into more potent antimicrobial agents that could help to battle infectious diseases in the future.

Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

Functional Profiling of the A-Family of Venom Peptides from the Wolf Spider Lycosa shansia.

The venoms of spiders from the RTA (retro-lateral tibia apophysis) clade contain diverse short linear peptides (SLPs) that offer a rich source of therapeutic candidates. Many of these peptides have insecticidal, antimicrobial and/or cytolytic activities, but their biological functions are unclear. Here, we explore the bioactivity of all known members of the A-family of SLPs previously identified in the venom of the Chinese wolf spider (Lycosa shansia). Our broad approach included an in silico analysis of physicochemical properties and bioactivity profiling for cytotoxic, antiviral, insecticidal and antibacterial activities. We found that most members of the A-family can form α-helices and resemble the antibacterial peptides found in frog poison. The peptides we tested showed no cytotoxic, antiviral or insecticidal activities but were able to reduce the growth of bacteria, including clinically relevant strains of Staphylococcus epidermidis and Listeria monocytogenes. The absence of insecticidal activity may suggest that these peptides have no role in prey capture, but their antibacterial activity may help to defend the venom gland against infection.

Bioactivity Profiling of In Silico Predicted Linear Toxins from the Ants Myrmica rubra and Myrmica ruginodis.

The venoms of ants (Formicidae) are a promising source of novel bioactive molecules with potential for clinical and agricultural applications. However, despite the rich diversity of ant species, only a fraction of this vast resource has been thoroughly examined in bioprospecting programs. Previous studies focusing on the venom of Central European ants (subfamily Myrmicinae) identified a number of short linear decapeptides and nonapeptides resembling antimicrobial peptides (AMPs). Here, we describe the in silico approach and bioactivity profiling of 10 novel AMP-like peptides from the fellow Central European myrmicine ants Myrmica rubra and Myrmica ruginodis. Using the sequences of known ant venom peptides as queries, we screened the venom gland transcriptomes of both species. We found transcripts of nine novel decapeptides and one novel nonapeptide. The corresponding peptides were synthesized for bioactivity profiling in a broad panel of assays consisting of tests for cytotoxicity as well as antiviral, insecticidal, and antimicrobial activity. U-MYRTX-Mrug5a showed moderately potent antimicrobial effects against several bacteria, including clinically relevant pathogens such as Listeria monocytogenes and Staphylococcus epidermidis, but high concentrations showed negligible cytotoxicity. U-MYRTX-Mrug5a is, therefore, a probable lead for the development of novel peptide-based antibiotics.

In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens.

Antimicrobial peptides (AMPs) are being explored as alternatives to traditional antibiotics to combat the rising antimicrobial resistance. Insects have proven to be a valuable source of new, potent AMPs with large structural diversity. For example, the black soldier fly has one of the largest AMP repertoires ever recorded in insects. Currently, however, this AMP collection has not yet undergone antimicrobial evaluation or in-depth in vitro characterization. This study evaluated the activity of a library of 36 black soldier fly AMPs against a panel of human pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumigatus) and a human cell line (MRC5-SV2). The activity profile of two cecropins (Hill-Cec1 and Hill-Cec10) with potent Gram-negative activity, was further explored by characterizing their hemolysis, time-to-kill kinetics, membrane-permeabilization properties, and anti-biofilm activity. Hill-Cec1 and Hill-Cec10 also showed high activity against other bacterial species, including Klebsiella pneumoniae and multi-drug resistant P. aeruginosa. Both AMPs are bactericidal and have a rapid onset of action with membrane-permeabilizing effects. Hill-Cec1 and Hill-Cec10 were also able to prevent P. aeruginosa biofilm formation, but no relevant effect was seen on biofilm eradication. Overall, Hill-Cec1 and Hill-Cec10 are promising leads for new antimicrobial development to treat critical infections caused by Gram-negative pathogens such as P. aeruginosa. IMPORTANCE With the ever growing antimicrobial resistance, finding new candidates for antimicrobial drug development is indispensable. Antimicrobial peptides have steadily gained attention as alternatives for conventional antibiotics, due to some highly desirable characteristics, such as their low propensity for resistance development. With this article, we aim to upgrade the knowledge on the activity of black soldier fly antimicrobial peptides and their potential as future therapeutics. To achieve this, we have evaluated for the first time a library of 36 synthetically produced peptides from the black soldier fly against a range of human pathogens and a human cell line. Two selected peptides have undergone additional testing to characterize their antimicrobial profile against P. aeruginosa, a clinically important Gram-negative pathogen with a high established resistance. Overall, this research has contributed to the search for new peptide drug leads to combat the rising antimicrobial resistance.

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

Tribolium castaneum defensin 1 kills Moraxella catarrhalisin an in vitro infection model but does not harm commensal bacteria.

Moraxella catarrhalis is a bacterial pathogen that causes respiratory tract infections in humans. The increasing prevalence of antibiotic-resistant M. catarrhalis strains has created a demand for alternative treatment options. We therefore tested 23 insect antimicrobial peptides (AMPs) for their activity against M. catarrhalis in a human in vitro infection model with primary macrophages, and against commensal bacteria. Effects on bacterial growth were determined by colony counting and growth curve analysis. The inflammatory macrophage response was characterized by qPCR and multiplex ELISA. Eleven of the AMPs were active against M. catarrhalis. Defensin 1 from the red flour beetle Tribolium castaneum significantly inhibited bacterial growth and reduced the number of colony forming units. This AMP also showed antibacterial activity in the in vitro infection model, reducing cytokine expression and release by macrophages. Defensin 1 had no effect on the commensal bacteria Escherichia coli and Enterococcus faecalis. However, sarcotoxin 1 C from the green bottle fly Lucilia sericata was active against M. catarrhalis and E. coli, but not against E. faecalis. The ability of T. castaneum defensin 1 to inhibit M. catarrhalis but not selected commensal bacteria, and the absence of cytotoxic or inflammatory effects against human blood-derived macrophages, suggests this AMP may be suitable for development as a new therapeutic lead against antibiotic-resistant M. catarrhalis.