Systemic toxicity of snake venom metalloproteinases: Multi-omics analyses of kidney and blood plasma disturbances in a mouse model.

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Systemic toxicity of snake venom metalloproteinases: multi-omics analyses of kidney and blood plasma disturbances in a mouse model

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Multiomics Profiling of Toxins in the Venom of the Amazonian Spider Acanthoscurria juruenicola.

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

Multiomics profiling of toxins in the venom of the Amazonian spider Acanthoscurria juruenicola

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

Recombinant Production and Characterization of a New Toxin from Cryptops iheringi Centipede Venom Revealed by Proteome and Transcriptome Analysis.

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Rondonin: antimicrobial properties and mechanism of action.

Infectious diseases are among the major causes of death in the human population. A wide variety of organisms produce antimicrobial peptides (AMPs) as part of their first line of defense. A peptide from Acanthoscurria rondoniae plasma, rondonin-with antifungal activity, a molecular mass of 1236 Da and primary sequence IIIQYEGHKH-was previously studied (UniProt accession number B3EWP8). It showed identity with the C terminus of subunit 'D' of the hemocyanin of the Aphonopelma hentzi spider. This result led us to propose a new pathway of the immune system of arachnids that suggests a new function to hemocyanin: production of antimicrobial peptides. Rondonin does not interact with model membranes and was able to bind to yeast nucleic acids but not bacteria. It was not cytotoxic against mammalian cells. The antifungal activity of rondonin is pH-dependent and peaks at pH ˜ 4-5. The peptide presents synergism with gomesin (spider hemocyte antimicrobial peptide-UniProtKB-P82358) against human yeast pathogens, suggesting a new potential alternative treatment option. Antiviral activity was detected against RNA viruses, measles, H1N1, and encephalomyocarditis. This is the first report of an arthropod hemocyanin fragment with activity against human viruses. Currently, it is vital to invest in the search for natural and synthetic antimicrobial compounds that, above all, present alternative mechanisms of action to first-choice antimicrobials.

Integrative multiomics analysis of Premolis semirufa caterpillar venom in the search for molecules leading to a joint disease.

The joint disease called pararamosis is an occupational disease caused by accidental contact with bristles of the caterpillar Premolis semirufa. The chronic inflammatory process narrows the joint space and causes alterations in bone structure and cartilage degeneration, leading to joint stiffness. Aiming to determine the bristle components that could be responsible for this peculiar envenomation, in this work we have examined the toxin composition of the caterpillar bristles extract and compared it with the differentially expressed genes (DEGs) in synovial biopsies of patients affected with rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteins identified, 129 presented an average of 63% homology with human proteins and shared important conserved domains. Among the human homologous proteins, we identified seven DEGs upregulated in synovial biopsies from RA or OA patients using meta-analysis. This approach allowed us to suggest possible toxins from the pararama bristles that could be responsible for starting the joint disease observed in pararamosis. Moreover, the study of pararamosis, in turn, may lead to the discovery of specific pharmacological targets related to the early stages of articular diseases.

Recombinant production and characterization of a new toxin from cryptops iheringi centipede venom revealed by proteome and transcriptome analysis

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Rondonin: antimicrobial properties and mechanism of action

Infectious diseases are among the major causes of death in the human population. A wide variety of organisms produce antimicrobial peptides (AMPs) as part of their first line of defense. A peptide from Acanthoscurria rondoniae plasma, rondonin – with antifungal activity, a molecular mass of 1,236 Da and primary sequence IIIQYEGHKH – was previously studied (Uniprot accession number B3EWP8). It showed identity with the C-terminus of subunit "D" of the hemocyanin of the Aphonopelma hentzi spider. This result led us to propose a new pathway of the immune system of arachnids that suggests a new function to hemocyanin: production of antimicrobial peptides. Rondonin does not interact with model membranes and was able to bind to yeast nucleic acids but not bacteria. It was not cytotoxic against mammalian cells. The antifungal activity of rondonin is pH dependent and peaks at pH ~4-5. The peptide presents synergism with Gomesin (spider hemocyte antimicrobial peptide - UniProtKB - P82358) against human yeast pathogens, suggesting a new potential alternative treatment option. Antiviral activity was detected against RNA viruses, measles, H1N1 and encephalomyocarditis. This is the first report of an arthropod hemocyanin fragment with activity against human viruses. Currently, it is vital to invest in the search for natural and synthetic antimicrobial compounds that, above all, present alternative mechanisms of action to first-choice antimicrobials.

Presence of a neprilysin on Avicularia juruensis (Mygalomorphae: Theraphosidae) venom

Spiders are the most successful and diversified group of venomous animals. Currently, there are more than 49,000 species distributed almost all over the world. This broad distribution suggests that they have efficient strategies to improve their survival; one of them is the production of highly elaborate venoms, which are a heterogeneous mixture of molecules like inorganic salts, peptides, proteins, and enzymes. Considering this, this study aimed to analyze the venom of the spider Avicularia juruensis (Mygalomorphae: Theraphosidae) searching for proteolytic enzymes. Using zymography, electrophoresis, transcriptomics and proteomics approaches we identified one neprilysin able to degrade casein, that we named “Ajur_Neprilysin”. Neprilysins are metalloendopeptidases whose presence has already been described in animal venoms, however, its function has not yet been elucidated. Our results showed for the first time one non-bacterial neprilysin which can cleave casein and suggest that its role in envenomation is to degrade the extracellular matrix, facilitating the access of other toxins to their targets, as well as digestive fluids. Moreover, this discovery contributes to increasing the knowledge about little-studied species, since the Ajur_Neprilysin is the second neprilysin found in the venom from a mygalomorph spider.

Integrative multiomics analysis of Premolis semirufa caterpillar venom in the search for molecules leading to a joint disease

The joint disease called pararamosis is an occupational disease caused by accidental contact with bristles of the caterpillar Premolis semirufa. The chronic inflammatory process narrows the joint space and causes alterations in bone structure and cartilage degeneration, leading to joint stiffness. Aiming to determine the bristle components that could be responsible for this peculiar envenomation, in this work we have examined the toxin composition of the caterpillar bristles extract and compared it with the differentially expressed genes (DEGs) in synovial biopsies of patients affected with rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteins identified, 129 presented an average of 63% homology with human proteins and shared important conserved domains. Among the human homologous proteins, we identified seven DEGs upregulated in synovial biopsies from RA or OA patients using meta-analysis. This approach allowed us to suggest possible toxins from the pararama bristles that could be responsible for starting the joint disease observed in pararamosis. Moreover, the study of pararamosis, in turn, may lead to the discovery of specific pharmacological targets related to the early stages of articular diseases.

A Multiomics Approach Unravels New Toxins With Possible In Silico Antimicrobial, Antiviral, and Antitumoral Activities in the Venom of Acanthoscurria rondoniae.

The Araneae order is considered one of the most successful groups among venomous animals in the world. An important factor for this success is the production of venoms, a refined biological fluid rich in proteins, short peptides and cysteine-rich peptides (CRPs). These toxins may present pharmacologically relevant biological actions, as antimicrobial, antiviral and anticancer activities, for instance. Therefore, there is an increasing interest in the exploration of venom toxins for therapeutic reasons, such as drug development. However, the process of peptide sequencing and mainly the evaluation of potential biological activities of these peptides are laborious, considering the low yield of venom extraction and the high variability of toxins present in spider venoms. Here we show a robust methodology for identification, sequencing, and initial screening of potential bioactive peptides found in the venom of Acanthoscurria rondoniae. This methodology consists in a multiomics approach involving proteomics, peptidomics and transcriptomics analyses allied to in silico predictions of antibacterial, antifungal, antiviral, and anticancer activities. Through the application of this strategy, a total of 92,889 venom gland transcripts were assembled and 84 novel toxins were identified at the protein level, including seven short peptides and 10 fully sequenced CRPs (belonging to seven toxin families). In silico analysis suggests that seven CRPs families may have potential antimicrobial or antiviral activities, while two CRPs and four short peptides are potentially anticancer. Taken together, our results demonstrate an effective multiomics strategy for the discovery of new toxins and in silico screening of potential bioactivities. This strategy may be useful in toxin discovery, as well as in the screening of possible activities for the vast diversity of molecules produced by venomous animals.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model.

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

HIV-1 genetic diversity and divergence and its correlation with disease progression among antiretroviral naïve recently infected individuals.

HIV-1 genetic diversity evolution was deeply characterized during the first year of infection among recently-infected patients using deep sequencing technology and correlated with disease progression surrogate markers. RNA and DNA samples from twenty-five individuals (13 female) encoding the protease and reverse transcriptase regions of the pol gene, and the V3 region of the env gene were evaluated at recent infection and during established infection. Infection by a unique HIV-1 strain was inferred in 70.1% of the individuals, with no differences between genders. Infections by multiple strains were associated with higher viral loads and faster CD4+ T cell declines. Either low or high levels of viral loads accompanied low levels of genetic diversity and lower selective pressure. With massive sequence data from 3 distinct genomic HIV-1 regions from plasma and PBMCs over time, we propose a model for HIV-1 genetic diversity, which correlates to basal viral loads of patients.

A multiomics approach unravels new toxins with possible in silico antimicrobial, antiviral, and antitumoral activities in the venom of Acanthoscurria rondoniae

The Araneae order is considered one of the most successful groups among venomous animals in the world. An important factor for this success is the production of venoms, a refined biological fluid rich in proteins, short peptides and cysteine-rich peptides (CRPs). These toxins may present pharmacologically relevant biological actions, as antimicrobial, antiviral and anticancer activities, for instance. Therefore, there is an increasing interest in the exploration of venom toxins for therapeutic reasons, such as drug development. However, the process of peptide sequencing and mainly the evaluation of potential biological activities of these peptides are laborious, considering the low yield of venom extraction and the high variability of toxins present in spider venoms. Here we show a robust methodology for identification, sequencing, and initial screening of potential bioactive peptides found in the venom of Acanthoscurria rondoniae. This methodology consists in a multiomics approach involving proteomics, peptidomics and transcriptomics analyses allied to in silico predictions of antibacterial, antifungal, antiviral, and anticancer activities. Through the application of this strategy, a total of 92,889 venom gland transcripts were assembled and 84 novel toxins were identified at the protein level, including seven short peptides and 10 fully sequenced CRPs (belonging to seven toxin families). In silico analysis suggests that seven CRPs families may have potential antimicrobial or antiviral activities, while two CRPs and four short peptides are potentially anticancer. Taken together, our results demonstrate an effective multiomics strategy for the discovery of new toxins and in silico screening of potential bioactivities. This strategy may be useful in toxin discovery, as well as in the screening of possible activities for the vast diversity of molecules produced by venomous animals.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

HIV-1 genetic diversity and divergence and its correlation with disease progression among antiretroviral naïve recently infected individuals

HIV-1 genetic diversity evolution was deeply characterized during the first year of infection among recently-infected patients using deep sequencing technology and correlated with disease progression surrogate markers. RNA and DNA samples from twenty-five individuals (13 female) encoding the protease and reverse transcriptase regions of the pol gene, and the V3 region of the env gene were evaluated at recent infection and during established infection. Infection by a unique HIV-1 strain was inferred in 70.1% of the individuals, with no differences between genders. Infections by multiple strains were associated with higher viral loads and faster CD4+ T cell declines. Either low or high levels of viral loads accompanied low levels of genetic diversity and lower selective pressure. With massive sequence data from 3 distinct genomic HIV-1 regions from plasma and PBMCs over time, we propose a model for HIV-1 genetic diversity, which correlates to basal viral loads of patients.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.