Analysis of LruC lipoprotein and identification of peptides candidates for vaccine development and diagnosis of leptospirosis.

Leptospirosis is a public health concern with lethality around 15% of the total cases. The current vaccines against Leptospira infection based on bacterins have several limitations, which require urgent development of new ones. In this context, groundbreaking approaches such as peptide-vaccines could be used to come around with promising results. Our goal was to identify conserved and immunogenic epitopes from the lipoprotein LruC that could interact with Major Histocompatibility Complex (MHC) I and II. LruC is a conserved lipoprotein expressed during leptospirosis that is considered among vaccine candidates and can be used as source for development of peptide-based vaccines. We searched for peptides that would be recognized by antibodies from either serum of hamsters previously immunized with low-LPS bacterin vaccines or from serum of patients diagnosed with leptospirosis. Immuno properties of seven peptides from LruC protein were evaluated in silico and by Dot Blot assay, and validate by ELISA. Preliminary results pointed one promising peptide that was recognized by the sera. In conclusion, the immunoinformatic approach helps the search and screening of peptides, while the Dot Blot assay, a simple and effective tool, helps to test and validate them. Thus, these prospective techniques together were validated to identify and validate potential peptides for further investigation as peptide-based vaccines or diagnostic methods.

Antibodies as Snakebite Antivenoms: Past and Future.

Snakebite envenomation is considered a neglected tropical disease, affecting tens of thousands of people each year. The recommended treatment is the use of antivenom, which is composed of immunoglobulins or immunoglobulin fragments obtained from the plasma of animals hyperimmunized with one (monospecific) or several (polyspecific) venoms. In this review, the efforts made in the improvement of the already available antivenoms and the development of new antivenoms, focusing on snakes of medical importance from sub-Saharan Africa and Latin America, are described. Some antivenoms currently used are composed of whole IgGs, whereas others use F(ab')2 fragments. The classic methods of attaining snake antivenoms are presented, in addition to new strategies to improve their effectiveness. Punctual changes in immunization protocols, in addition to the use of cross-reactivity between venoms from different snakes for the manufacture of more potent and widely used antivenoms, are presented. It is known that venoms are a complex mixture of components; however, advances in the field of antivenoms have shown that there are key toxins that, if effectively blocked, are capable of reversing the condition of in vivo envenomation. These studies provide an opportunity for the use of monoclonal antibodies in the development of new-generation antivenoms. Thus, monoclonal antibodies and their fragments are described as a possible alternative for the production of antivenoms, regardless of the venom. This review also highlights the challenges associated with their development.

COVID-19 induces proatherogenic alterations in moderate to severe non-comorbid patients: A single-center observational study.

Patients with COVID-19 can be asymptomatic or present mild to severe symptoms, leading to respiratory and cardiovascular complications and death. Type 2 diabetes mellitus (T2DM) and obesity are considered risk factors for COVID-19 poor prognosis. In parallel, COVID-19 severe patients exhibit dyslipidemia and alterations in neutrophil to lymphocyte ratio (NLR) associated with disease severity and mortality. To investigate whether such alterations are caused by the infection or results from preexisting comorbidities, this work analyzed dyslipidemia and the hemogram profile of COVID-19 patients according to the severity and compared with patients without T2DM or obesity comorbidities. Dyslipidemia, with a marked decrease in HDL levels, and increased NLR accompanied the disease severity, even in non-T2DM and non-obese patients, indicating that COVID-19 causes the observed alterations. Because decreased hemoglobin is involved in COVID-19 severity, and hemoglobin concentration is associated with metabolic diseases, the erythrogram of patients was also evaluated. We verified a drop in hemoglobin and erythrocyte number in severe patients, independently of T2DM and obesity, which may explain in part the need for artificial ventilation in severe cases. Thus, the control of such parameters (especially HDL levels, NLR, and hemoglobin concentration) could be a good strategy to prevent COVID-19 complications and death.

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors.

BACKGROUND: In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. METHODS: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. RESULTS: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. CONCLUSION: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Design of bioactive peptides derived from CART sequence isolated from the toadfish Thalassophryne nattereri.

The emergence of bacterial resistance due to the indiscriminate use of antibiotics warrants the need for developing new bioactive agents. In this context, antimicrobial peptides are highly useful for managing resistant microbial strains. In this study, we report the isolation and characterization of peptides obtained from the venom of the toadfish Thalassophryne nattereri. These peptides were active against Gram-positive and Gram-negative bacteria and fungi. The primary amino acid sequences showed similarity to Cocaine and Amphetamine Regulated Transcript peptides, and two peptide analogs-Tn CRT2 and Tn CRT3-were designed using the AMPA algorithm based on these sequences. The analogs were subjected to physicochemical analysis and antimicrobial screening and were biologically active at concentrations ranging from 2.1 to 13 µM. Zeta potential analysis showed that the peptide analogs increased the positive charge on the cell surface of Gram-positive and Gram-negative bacteria. The toxicity of Tn CRT2 and Tn CRT3 were analyzed in vitro using a hemolytic assay and tetrazolium salt reduction in fibroblasts and was found to be significant only at high concentrations (up to 40 µM). These results suggest that this methodological approach is appropriate to design novel antimicrobial peptides to fight bacterial infections and represents a new and promising discovery in fish venom.

Leptospira Infection Interferes with the Prothrombinase Complex Assembly during Experimental Leptospirosis.

Leptospirosis is a worldwide zoonotic and neglected infectious disease of human and veterinary concern, caused by pathogenic Leptospira species. Although bleeding is a common symptom of severe leptospirosis, the cause of hemorrhage is not completely understood. In severe infections, modulation of hemostasis by pathogens is an important virulence mechanism, and hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. Here, we analyze the coagulation status of experimentally infected hamsters in an attempt to determine coagulation interferences and the origin of leptospirosis hemorrhagic symptomatology. Hamsters were experimentally infected with L. interrogans. The lungs, kidneys, and livers were collected for culture, histopathology, and coagulation assays. L. interrogans infection disturbs normal coagulation in the organs of animals. Our results suggest the presence of a thrombin-like factor or FX activator, which is able to activate FII in the leptospirosis organ extracts. The activity of those factors is accelerated in the prothrombinase complex. Additionally, we show for the first time that live leptospires act as a surface for the prothrombinase complex assembly. Our results contribute to the understanding of leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments in the severe manifestations of the disease.

Amblyomin-X having a Kunitz-type homologous domain, is a noncompetitive inhibitor of FXa and induces anticoagulation in vitro and in vivo.

BACKGROUND: Cancer has long been associated with thrombosis and many of the standard chemotherapeutics used to treat cancer are pro-thrombotic. Thus, the identification of novel selective anticancer drugs that also have antithrombotic properties is of enormous significance. Amblyomin-X is an anticancer protein derived from the salivary glands of the Amblyomma cajennense tick. METHODS: In this work, we determined the inhibition profile of Amblyomin-X and its effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), using various approaches such as, kinetic analyses, amidolytic assays, SDS-PAGE, and mass spectrometry. RESULTS: Amblyomin-X inhibited factor Xa, prothrombinase and tenase activities. It was hydrolyzed by trypsin and plasmin. MS/MS data of tryptic hydrolysate of Amblyomin-X suggested the presence of Cys(8)-Cys(59) and Cys(19)-Cys(42) but not Cys(34)-Cys(55) disulfide bond. Instead of Cys(34)-Cys(55), two noncanonical Cys(34)-Cys(74) and Cys(55)-Cys(74) disulfide bonds were identified. Furthermore, when Amblyomin-X (1mg/kg) injected in rabbits, it prolonged aPTT and PT. CONCLUSION: Amblyomin-X is a noncompetitive inhibitor (Ki=3.9µM) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The disulfide Cys(34)-Cys(55) bond probably scrambles with interchain seventh free cysteine residues (Cys(74)) of Amblyomin-X. The prolongation of PT and aPTT is reversible. GENERAL SIGNIFICANCE: In term of anticoagulant property, this is structural and functional characterization of Amblyomin-X. All together, these results and previous findings suggest that Amblyomin-X has a potential to become an anticancer drug with antithrombotic property.

The Kallikrein Inhibitor from Bauhinia bauhinioides (BbKI) shows antithrombotic properties in venous and arterial thrombosis models.

The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (Kiapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis.

Effects of compounds from Passiflora edulis Sims f. flavicarpa juice on blood coagulation and on proteolytic enzymes.

Passion fruit (Passiflora edulis Sims f. flavicarpa) is popularly known for its sedative and calming properties and is consumed as a fresh fruit or as a juice. The clinical observation of blood incoagulability associated with excessive consumption of passion fruit juice, in a patient treated with warfarin, prompted the current study to investigate in vitro the presence of blood clotting inhibitors in Passiflora edulis Sims f. flavicarpa extract. After purification process, two compounds of distinct molecular weight and inhibitory action were better characterized. One is a trypsin inhibitor similar to inhibitors from Bowman-Birk family, named PeTI-I12, and other is a compound active in coagulation that prolongs aPTT and PT, but does not change TT. The aim of this study is to provide evidence that passion fruit extract's components play a role on hemostasis and therefore may be relevant in the handling of patients treated with anticoagulants or suffering hemorrhagic diseases.

Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases.

Spider venom sphingomyelinases D catalyze the hydrolysis of sphingomyelin via an Mg(2+) ion-dependent acid-base catalytic mechanism which involves two histidines. In the crystal structure of the sulfate free enzyme determined at 1.85A resolution, the metal ion is tetrahedrally coordinated instead of the trigonal-bipyramidal coordination observed in the sulfate bound form. The observed hyperpolarized state of His47 requires a revision of the previously suggested catalytic mechanism. Molecular modeling indicates that the fundamental structural features important for catalysis are fully conserved in both classes of SMases D and that the Class II SMases D contain an additional intra-chain disulphide bridge (Cys53-Cys201). Structural analysis suggests that the highly homologous enzyme from Loxosceles bonetti is unable to hydrolyze sphingomyelin due to the 95Gly-->Asn and 134Pro-->Glu mutations that modify the local charge and hydrophobicity of the interfacial face. Structural and sequence comparisons confirm the evolutionary relationship between sphingomyelinases D and the glicerophosphodiester phosphoesterases which utilize a similar catalytic mechanism.

Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom.

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide-phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intermedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (alpha/beta)8 barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro-Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding.

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: purification and biological characterization.

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED50 of 0.270 microg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.

Conformational changes of Loxosceles venom sphingomyelinases monitored by circular dichroism.

Envenomation by arachnids of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and hemolysis. We have previously identified in L. intermedia venom two highly homologous proteins with sphingomyelinase activity, termed P1 and P2, responsible for all these pathological events, and also an inactive isoform P3. The toxins P1 and P2 displayed 85% identity with each other at the amino acid level and showed a 57% identity with SMase I, an active toxin from L. laeta venom. Circular dichroism was used to determine and compare the solution structure of the active and inactive isoforms. Effects of pH and temperature change on the CD spectra of the toxins were investigated and correlated with the biological activities. This study sheds new light on the structure-function relationship of homologous proteins with distinct biological properties and represents the first report on the structure-function relationship of Loxosceles sphingomyelinases D.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.