Biology subjects

Hardes, K.

Publications and source records attributed to Hardes, K..

7 recordsLinked to original sources

bioRxiv2026

Ontogenetic variation in composition and bioactivity of common adder (Vipera berus) venom revealed by genome-guided proteomics and in vitro functional assays

1Ontogenetic shifts in diet are well documented in snakes and are increasingly linked to age-related venom variation. The common adder, Vipera berus, exhibits a dietary transition from predominantly ectothermic prey in its early life to increasingly incorporating endothermic prey as an adult. Here, we investigate whether this dietary shift is reflected in age-related changes in the venom composition and bioactivity of V. berus. Venoms from captive-bred V. berus from Germany were obtained and pooled across five age groups, from neonates to adults. Venom profiles were assessed by SDS-PAGE and genome-guided shotgun proteomics, with quantification based on normalized spectral abundance factors (NSAF) using a toxin-gene catalogue generated from a novel V. berus genome assembly. In parallel, we assayed general protease and PLA2 activities, as well as FXa-, thrombin-, and plasmin-like activities, and cytotoxicity toward mammalian cell lines. We identified two distinct age-related venom phenotypes (ontotypes): an svMP/CTL-rich ontotype A ([≤]1 year) and an svSP/PLA2-rich ontotype B ([≥]2 years). Functionally, protease activity decreased with age, whereas thrombin-like, plasmin-like and PLA2 activities, and cytotoxicity, increased. Our findings indicate an ontogenetic shift in composition and activities of V. berus venom that parallels dietary transitions and potentially reflect adaptation to differing prey physiologies.

biochemistry↗

bioRxiv2025

A genome-guided atlas to the composition, activity, and β-bungarotoxin dimerization in many-banded krait venom inferred by functional venomics

1Snakebite is a neglected tropical disease claiming [~]140,000 lives every year. One of the most medically relevant snakes of Asia is the many-banded krait (Bungarus multicinctus). Approximately 8% of the global human population is at risk of being envenomated by this species, able to cause fatal neurotoxicity. Here, we present a proteogenomic and functional assessment of the B. multicinctus venom via genome-guided bottom-up and top-down proteomics, combined with traditional protein profiling and bioassays. We report its venom profile alongside the primary structures of its toxins, revealing a relatively simple venom containing 55 components from 16 protein families. It is largely composed by three-finger toxins and phospholipase A2, besides acetylcholinesterase and snake venom metalloprotease. Top-down data unveiled the diversity of the highly lethal {beta}-bungarotoxins and allowed us to infer the complex dimerization patterning of these multi-domain neurotoxins. Our functional analysis revealed that B. multicinctus venom exerts potent phospholipase A2 and acetylcholinesterase activities, but protease activity as well as effects on cell viability and release of second messengers were virtually absent. This suggests, that B. multicinctus venom causes its devastating neurotoxic symptoms due to a heavy reliance on phospholipase A2 and acetylcholinesterase, but without impairing viability of neurons nor via interference of second messenger release. Antibacterial and antiviral screens further revealed activity against some pathogenic microbes that warrant further translational investigations. A comparison to previously published venom proteomes of B. multicinctus and its congeners suggests, that intraspecific venom variation occurs more widely in kraits than previously acknowledged and deserves higher attention. Overall, our investigation provides pivotal new insights into the biochemistry and pathophysiology of one of earths most lethal snakes and represents an important resource to inform future proteogenomic and functional studies in krait venom and beyond.

molecular biology↗

bioRxiv2025

Low extent of sex-based venom variation in Earth's most widespread viper, the common adder (Vipera berus)

Snake venom is an ecologically critical functional trait, primarily applied for foraging and accordingly shaped by selective pressures. Recent insights underpinned the high variability of snake venoms down to the intraspecific level, with regional, ontogenetic, and seasonal variation being mostly investigated. In contrast, sex-based venom variation has received considerably less attention so far, and its influence on venom compositions in vipers is virtually unknown. The common adder (Vipera berus) is a promising model species to explore this subject because of a described sexual dimorphism and its wide distribution, which promises a noteworthy degree of adaptability and venom plasticity. Here, we tested for sex-based venom variation in Central European V. berus by comparing venom profiles and bioactivity. Proteomics, paired with SDS-PAGE and RP-HPLC, revealed highly similar venom profiles. Likewise, phospholipases A2 and proteases bioactivity profiling, and bioassays targeting the coagulation cascade and mammalian cell lines revealed similar activity spectra. Hence, our analysis does not show a noteworthy extent of sex-based intraspecific venom variation in V. berus. We further discuss our data in light of the species venom profile at larger geographic scales, its clinical relevance, and the need for more standardized approaches when investigating venom variability. Our work provides novel insights into the venom biology of Earths most widespread viper, and serves as a foundation upon which future works can build.

biochemistry↗

bioRxiv2024

Comparative venom analysis between melanistic and normally-colored phenotypes of the common adder (Vipera berus)

1Snake venom is an ecologically-relevant functional trait directly linked with a snakes fitness and survival, facilitating predation and defense. Snake venom variation occurs at all taxonomic levels, but the study of variation at intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in color phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such "hell adders" are considered more toxic than their normally-colored conspecifics. However, these claims have never been formally tested. Here, we provide the first comparative analysis of venoms from melanistic and normally-colored common adders. Specifically, we compared the venom profiles by SDS-PAGE and RP-HPLC, and tested the protease, phospholipase A2 and cytotoxic activities of the venoms of both phenotypes. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 {micro}g/ml). These minor differences between the venoms of melanistic and normally-colored adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally-colored conspecifics appears rooted entirely in folklore.

molecular biology↗

bioRxiv2024

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

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 can be modified into more potent antimicrobial agents that could help to battle infectious diseases in the future.

bioengineering↗

bioRxiv2020

TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

In December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines. In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2 site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.

microbiology↗

bioRxiv2018

A novel cell-based sensor detecting the activity of individual basic proprotein convertases

The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides and do not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. We report here PCific, a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. PCific consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in cis. Screening of selected candidate peptidomimetic inhibitors revealed that PCific can readily distinguish between general and selective PC inhibitors in a high-throughput screening format.

biochemistry↗