Roderich D. Süssmuth

The medical threat of mamba envenoming in sub-Saharan Africa revealed by genus-wide analysis of venom composition, toxicity and antivenomics profiling of available antivenoms

Mambas (genus Dendroaspis) are among the most feared of the medically important elapid snakes found in sub-Saharan Africa, but many facets of their biology, including the diversity of venom composition, remain relatively understudied. Here, we present a reconstruction of mamba phylogeny, alongside genus-wide venom gland transcriptomic and high-resolution top-down venomic analyses. Whereas the green mambas, D. viridis, D. angusticeps, D. j. jamesoni and D. j. kaimosae, express 3FTx-predominant venoms, black mamba (D. polylepis) venom is dominated by dendrotoxins I and K. The divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas makes it plausible that this major difference in venom composition is due to dietary variation. The pattern of intrageneric venom variability across Dendroaspis represented a valuable opportunity to investigate, in a genus-wide context, the variant toxicity of the venom, and the degree of paraspecific cross-reactivity between antivenoms and mamba venoms. To this end, the immunological profiles of the five mamba venoms were assessed against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview of which available antivenoms may be more efficacious in neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the notably different potency and pharmacological profiles of Dendroaspis venoms at locus resolution. This understanding will allow selection and design of toxin immunogens with a view to generating a safer and more efficacious pan-specific antivenom against any mamba envenomation.nnnBIOLOGICAL SIGNIFICANCEnThe mambas (genus Dendroaspis) comprise five especially notorious medically important venomous snakes endemic to sub-Saharan Africa. Their highly potent venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Previous studies on mamba venoms have focused on the biochemical and pharmacological characterisation of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species, but there has been little work on overall venom composition or comparisons between them. Only very recently an overview of the composition of the venom of two Dendroaspis species, D. angusticeps and D. polylepis, has been unveiled through venomics approaches. Here we present the first genus-wide transcriptomic-proteomic analysis of mamba venom composition. The transcriptomic analyses described in this paper have contributed 29 (D. polylepis), 23 (D. angusticeps), 40 (D. viridis), 25 (D. j. jamesoni) and 21 (D. j. kaimosae), novel full-length toxin sequences to the non-redundant Dendroaspis sequence database. The mamba genus-wide venomic analysis demonstrated that major D. polylepis venom components are Kunitz-fold family toxins. This feature is unique in relation to the relatively conserved three-finger toxin (3FTx)-dominated venom compositions of the green mambas. Venom variation was interpreted in the context of dietary variation due to the divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas. Additionally, the degree of cross-reactivity conservation of mamba venoms was assessed by antivenomics against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview to infer which available antivenoms may be capable of neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the pharmacological profiles of mamba venoms at locus resolution. This understanding will contribute to the generation of a safer and more efficacious pan-Dendroaspis therapeutic antivenom against any mamba envenomation.

Quantitative Expression of Hepatobiliary Transporters and Functional Uptake of Substrates in Hepatic 2D Sandwich-Cultures: A Comparative Evaluation of Upcyte and Primary Human Hepatocytes

Deficient functional expression of drug transporters incapacitates most hepatic cell lines as a reliable tool for evaluating transporter-mediated drug-drug interactions. Recently, genetically modified cells (referred to as upcyte hepatocytes) have emerged as an expandable, noncancerous source of human hepatic cells. Herein, we quantified mRNA and protein levels of key hepatobiliary transporters and we assessed associated uptake activity in short- and long-term cultures of upcyte human hepatocytes (UHH) in comparison to cryopreserved primary human hepatocytes (cPHH). Expression of canalicular efflux pumps, such as MRD1/ABCB1, MATE1/SLC47A1, and MRP2/ABCC2, was relatively well preserved in UHH. By contrast, long-term cultivation of UHH in a two-dimensional sandwich configuration [sandwich-cultured upcyte human hepatocytes (SCUHH)] was required to upregulate organic anion-transporting polypeptide OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, NTCP/SLC10A1, and OCT1/SLC22A1 mRNA expression, which correlated well with respective protein abundances. However, mRNA and protein levels of sinusoidal solute carrier transporters, except for NTCP and OATP2B1, remained low in SCUHH compared to sandwich-cultured cPHH. OCT1- and NTCP-mediated uptake of N-methyl-4-phenylpyridinium acetate and taurocholate was demonstrated in both hepatic models, whereas active uptake of OATP1B1/1B3-selective marker substrates, paralleled by markedly reduced SLCO1B1/1B3 expression, were not detectable in SCUHH. Uptake studies under Na+-depletion and excess of taurocholate confirmed the presence of functional NTCP protein and indicated that NTCP, apart from OATP2B1, contributed substantially to the overall hepatic uptake of rosuvastatin in SCUHH. In conclusion, our data suggest that SCUHH, despite their limitation for evaluating OATP1B1/1B3-mediated transport processes, retain NTCP, OATP2B1, and OCT1 transport activities and thus may be considered as a tool for elucidating compensatory uptake pathways for OATP1B1/1B3 substrates.

The anti-staphylococcal lipolanthines are ribosomally synthesized lipopeptides

AbstractThe potent antibacterial lanthipeptide microvionin, isolated from a culture of Microbacterium arborescens, exhibits a new triamino-dicarboxylic acid moiety, termed avionin, and an unprecedented N-terminal guanidino fatty acid. We identified the corresponding biosynthetic gene cluster and reconstituted central steps of avionin biosynthesis in vitro. Genome mining and isolation of nocavionin from Nocardia terpenica revealed a widespread distribution of this lanthipeptide class, termed lipolanthines, which may be useful as future antimicrobial drugs.The antibacterial microvionin contains two new lanthipeptide modifications, a triamino-dicarboxylic acid (avionin) and an N-terminal guanidino fatty acid, that lead to the establishment of the lipolanthine natural product class.n

Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides

BackgroundFungal cyclodepsipeptides (CDPs) are non-ribosomally synthesized peptides produced by a variety of filamentous fungi and are of interest to the pharmaceutical industry due to their anticancer, antimicrobial and anthelmintic bioactivities. However, both chemical synthesis and isolation of CDPs from their natural producers are limited due to high costs and comparatively low yields. These challenges might be overcome by heterologous expression of the respective CDP-synthesizing genes in a suitable fungal host. The well-established industrial fungus Aspergillus niger was recently genetically reprogrammed to overproduce the cyclodepsipeptide enniatin B in g/L scale, suggesting that it can generally serve as a high production strain for natural products such as CDPs. In this study, we thus aimed to determine whether other CDPs such as beauvericin and bassianolide can be produced with high titres in A. niger, and whether the generated expression strains can be used to synthesize new-to-nature CDP derivatives.ResultsThe beauvericin and bassianolide synthetases were expressed under control of the tuneable Tet-on promoter, and titres of about 350–600xa0mg/L for bassianolide and beauvericin were achieved when using optimized feeding conditions, respectively. These are the highest concentrations ever reported for both compounds, whether isolated from natural or heterologous expression systems. We also show that the newly established Tet-on based expression strains can be used to produce new-to-nature beauvericin derivatives by precursor directed biosynthesis, including the compounds 12-hydroxyvalerate-beauvericin and bromo-beauvericin. By feeding deuterated variants of one of the necessary precursors (d-hydroxyisovalerate), we were able to purify deuterated analogues of beauvericin and bassianolide from the respective A. niger expression strains. These deuterated compounds could potentially be used as internal standards in stable isotope dilution analyses to evaluate and quantify fungal spoilage of food and feed products.ConclusionIn this study, we show that the product portfolio of A. niger can be expanded from enniatin to other CDPs such as beauvericin and bassianolide, as well as derivatives thereof. This illustrates the capability of A. niger to produce a range of different peptide natural products in titres high enough to become industrially relevant.

Comparative Venomics of the Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni from Turkey Provides Insights into Kinship

The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors. While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the subspecies, an accurate classification of Vipera ammodytes transcaucasiana is still part of the ongoing research. The comparative analysis of the viper venoms on the proteome level reveals a close relationship between the Vipera ammodytes subspecies, which could be considered to clarify the classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of some venom components could be indicating interspecific variations of the two studied subspecies or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity screening with the crude venoms against several human cancerous and non-cancerous cell lines, which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA MB 231 breast cancer cells.

Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells

Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.

Comprehensive snake venomics of the Okinawa Habu pit viper, Protobothrops flavoviridis, by complementary mass spectrometry-guided approaches

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5′-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Propiconazole is an activator of AHR and causes concentration additive effects with an established AHR ligand

Consumers are exposed to pesticide residues and other food contaminants via the diet. Both can exert adverse effects on different target organs via the activation of nuclear receptor pathways. Hepatotoxic effects of the widely used triazole fungicide propiconazole (Pi) are generally attributed to the activation of the constitutive androstane receptor (CAR) or the pregnane X receptor (PXR). We now investigated the effects of Pi on the aryl hydrocarbon receptor (AHR) and possible mixture toxicity when Pi is present in combination with BbF, an AHR ligand. In silico docking simulations indicate that Pi can bind to human AHR. Subsequent dual luciferase reporter gene assays in human HepG2 cells showed that Pi activates the AHR in vitro. This concentration-dependent activation was confirmed by real-time RT-PCR analyses of the model AHR target genes CYP1A1 and CYP1A2 in human HepaRG and HepG2 cells. In addition, induction of CYP1A1 protein levels and enzyme activity were recorded. Similarly, increased mRNA expression and enzyme activity of Cyp1a1 and Cyp1a2 was observed in livers of rats treated with Pi for 28 days via the diet. Gene expression analysis in AHR-knockout HepaRG cells showed no induction of CYP1A1 and CYP1A2, whereas gene expression in CAR-, and PXR-knockout cells was induced. Finally, mixture effects of Pi and BbF were analyzed in human cell lines: modeling of concentration–response curves revealed concentration additivity. In conclusion, our results demonstrate that the triazole Pi is an activator of AHR in silico, in vitro and in vivo and causes additive effects with an established AHR ligand.

Streptomyces AcH 505 triggers production of a salicylic acid analogue in the fungal pathogen Heterobasidion abietinum that enhances infection of Norway spruce seedlings

The necrotrophic fungus Heterobasidion spp. is the causal agent of ‘annosum root rot’ of Norway spruce. In the presence of the rhizosphere bacterium Streptomyces AcH 505, enhanced colonization of Norway spruce roots with Heterobasidion abietinum 331 has previously been observed. By analyzing dual cultures of H. abietinum 331 and Streptomyces AcH 505 with HPLC, a fungal metabolite was identified that was increased in the presence of Streptomyces AcH 505. Likewise, challenge of H. abietum 331 with common antifungals produced by soil streptomycetes rendered the same effect. The structure of the compound, 5-formylsalicylic acid (5-FSA), was elucidated by HPLC-HR-ESI-Orbitrap-mass spectrometry and NMR spectroscopy. Based on in vivo measurements of maximum photosystem II efficiency of Norway spruce seedlings, 5-FSA did not influence plant vitality. However, when challenged with H. abietinum 331, ergosterol amounts in infected roots increased significantly for 5-FSA pre-treated seedlings. The severity of the infection was comparable to that observed in the presence of Streptomyces AcH 505. 5-FSA is a structural analogue of salicylic acid, an important signalling molecule active in plant defence. Thus, the expression of two defence-response related marker genes (PR1, Hel) was analysed in 5-FSA treated Arabidopsis thaliana seedlings by Northern blot analysis. The transcription of both marker genes was altered, indicating that 5-FSA is perceived by Arabidopsis in a similar manner to salicylic acid and is able to interfere with Arabidopsis defence signalling. The role of 5-FSA as a potential virulence factor of H. abietinum 331 in the presence of Streptomyces AcH 505 is discussed.