Comparative Biochemical, Structural, and Functional Analysis of Recombinant Phospholipases D from Three Loxosceles Spider Venoms.

Spiders of Loxosceles genus are widely distributed and their venoms contain phospholipases D (PLDs), which degrade phospholipids and trigger inflammatory responses, dermonecrosis, hematological changes, and renal injuries. Biochemical, functional, and structural properties of three recombinant PLDs from L. intermedia, L. laeta, and L. gaucho, the principal species clinically relevant in South America, were analyzed. Sera against L. gaucho and L. laeta PLDs strongly cross-reacted with other PLDs, but sera against L. intermedia PLD mostly reacted with homologous molecules, suggesting underlying structural and functional differences. PLDs presented a similar secondary structure profile but distinct melting temperatures. Different methods demonstrated that all PLDs cleave sphingomyelin and lysophosphatidylcholine, but L. gaucho and L. laeta PLDs excelled. L. gaucho PLD showed greater "in vitro" hemolytic activity. L. gaucho and L. laeta PLDs were more lethal in assays with mice and crickets. Molecular dynamics simulations correlated their biochemical activities with differences in sequences and conformations of specific surface loops, which play roles in protein stability and in modulating interactions with the membrane. Despite the high similarity, PLDs from L. gaucho and L. laeta venoms are more active than L. intermedia PLD, requiring special attention from physicians when these two species prevail in endemic regions.

Immunoprophylactic properties of the Corynebacterium pseudotuberculosis-derived MBP:PLD:CP40 fusion protein.

Caseous lymphadenitis (CLA) is a disease that affects small ruminants, and the best way to prevent its spread on a herd is through immunoprophylaxis. Thus, we aimed to evaluate the MBP:PLD:CP40 fusion protein as a new CLA immunogen. The fusion protein was constructed by combining Corynebacterium pseudotuberculosis PLD and CP40 proteins with maltose-binding protein (MBP) as an intrinsic adjuvant. The antigenicity, allergenic potential, prediction of B epitopes, binding to MHC receptors, and docking on the Toll-Like 2 receptor were evaluated in silico. MBP:PLD:CP40 was expressed and purified. 40 BALB/c were divided into four groups (G1 - control, G2 - Saponin, G3 - MBP:PLD:CP40, and G4 - rPLD + rCP40). Total IgG, IgG1, and IgG2a were quantified, and the expressions of cytokines after splenocyte in vitro stimulation were assessed. Mice were challenged 42 days after the first immunization. The in silico analysis showed that MBP:PLD:CP40 has immunogenic potential, does not have allergic properties, and can dock on the TRL2 receptor. MBP:PLD:CP40 stimulated the production of IgG1 antibodies in a fivefold proportion to IgG2a, and TNF and IL-17 were significantly expressed in response to the antigenic stimuli. When rPLD and rCP40 were used together for immunization, they could induce IFN-γ and IL-12, but with no detectable antibody production. The G3 and G4 groups presented a survival of 57.14% and 42.86%, respectively, while the G1 and G2 mice were all dead 15 days after the challenge. MBP:PLD:CP40 partially protected the mice against C. pseudotuberculosis infection and can be considered a potential new CLA immunogen. KEY POINTS: • The fusion protein induced more IgG1 than IgG2a antibodies; • The fusion protein also induced the expression of the TNF and IL-17 cytokines; • Mice inoculated with MBP:PLD:CP40 presented a 57.14% survival.

Staphylococcus aureus Exfoliative Toxin E, Oligomeric State and Flip of P186: Implications for Its Action Mechanism.

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.

Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis.

The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium.

Crystal structure of mature 2S albumin from Moringa oleifera seeds.

2S albumins, the seed storage proteins, are the primary sources of carbon and nitrogen and are involved in plant defense. The mature form of Moringa oleifera (M. oleifera), a chitin binding protein isoform 3-1 (mMo-CBP3-1) a thermostable antifungal, antibacterial, flocculating 2S albumin is widely used for the treatment of water and is potentially interesting for the development of both antifungal drugs and transgenic crops. The crystal structure of mMo-CBP3-1 determined at 1.7 Å resolution demonstrated that it is comprised of two proteolytically processed α-helical chains, stabilized by four disulfide bridges that is stable, resistant to pH changes and has a melting temperature (TM) of approximately 98 °C. The surface arginines and the polyglutamine motif are the key structural factors for the observed flocculating, antibacterial and antifungal activities. This represents the first crystal structure of a 2S albumin and the model of the pro-protein indicates the structural changes that occur upon formation of mMo-CBP3-1 and determines the structural motif and charge distribution patterns for the diverse observed activities.

The search for an antiviral lead molecule to combat the neglected emerging Oropouche virus.

Oropouche virus (OROV) is a member of the Peribunyaviridae family and the causative agent of a dengue-like febrile illness transmitted by mosquitoes. Although mild symptoms generally occur, complications such as encephalitis and meningitis may develop. A lack of proper diagnosis, makes it a potential candidate for new epidemics and outbreaks like other known arboviruses such as Dengue, Yellow Fever and Zika virus. The study of natural molecules as potential antiviral compounds is a promising alternative for antiviral therapies. Wedelolactone (WDL) has been demonstrated to inhibit some viral proteins and virus replication, making it useful to target a wide range of viruses. In this study, we report the in silico effects of WDL on the OROV N-terminal polymerase and its potential inhibitory effects on several steps of viral infection in mammalian cells in vitro, which revealed that WDL indeed acts as a potential inhibitor molecule against OROV infection.

Necrotic activity of ExhC from Mammaliicoccus sciuri is mediated by specific amino acid residues.

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, ETs are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintain tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs. Previous experiments have implicated the ExhC79-128 fragment in causing necrosis. Site-directed mutagenesis of specific residues within this fragment were studied and led to the design of an ExhC variant containing four-point mutations (ExhCmut4) lacking necrotic potential but retaining nearly wild-type (wt) levels of enzymatic activity. Moreover, the determination of the ExhCwt and ExhCmut4 crystal structures identified the conformation in the necrosis-linked region. These results constitute an important step toward the understanding of the mechanisms underlying the necrotic and epidermolytic activity of ExhC.

Brown Spider Venom Phospholipase-D Activity upon Different Lipid Substrates.

Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme's substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.

Purification, crystallization and preliminary X-ray diffraction analysis of a class P-III metalloproteinase (BmMP-III) from the venom of Bothrops moojeni.

Snake-venom metalloproteinases (SVMPs) comprise a family of haemostatically active toxins which can cause haemorrhage, coagulopathy, inhibition of platelet aggregation and inflammatory response. These effects are attributed to the proteolytic action of SVMPs on extracellular matrix components, plasma proteins and cell-surface proteins. SVMPs are classified into four classes (P-I to P-IV) based on their domain structures. In order to understand the multiple roles played by the domains of P-III SVMPs, a P-III SVMP (BmMP-III) from the venom of Bothrops moojeni was purified, characterized and crystallized. The crystals belonged to space group I4(1)22, with unit-cell parameters a = b = 108.16, c = 196.09 Å. Initially, flash-cooled crystals diffracted poorly to a resolution of about 10 Å. However, a significant improvement in the diffraction resolution was observed upon annealing and a complete data set was collected to 3.3 Šresolution. The asymmetric unit contained one molecule and the structure was determined and partially refined to an R factor of 34%. Structural comparisons indicated that the cysteine-rich domain can adopt different conformations in relation to the catalytic domain, which may modulate the enzyme activity.

Transcriptomics Applied to the Study of Chagas Disease Vectors.

Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi, and is transmitted mainly by the feces of contaminated triatomines. Knowledge of the biological, ecological, behavioral, genetic, taxonomic, and systematic aspects of these vectors can contribute to the planning of vector control programs, because all species are considered to be potential vectors of Chagas disease. Transcriptomic studies, in general, provided a new view of the physiology of triatomines (aiding in the knowledge of reproductive aspects of the hematophagy process and even the immune system and the sensory apparatus) and even contributed, as a new tool, to the taxonomy and systematics of these insects. Thus, we conducted a review of the transcriptomic studies on Chagas disease vectors.

Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge.

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom.

Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12(1)1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 Å, ß = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively.

Omics Tools Applied to the Study of Chagas Disease Vectors: Cytogenomics and Genomics.

Chagas disease is an illness caused by the protozoan Trypanosoma cruzi that is distributed in 21 countries of Latin America. The main way of transmission of T. cruzi is through the feces of triatomines infected with the parasite. With technological advances came new technologies called omics. In the pre-genomic era, the omics science was based on cytogenomic studies of triatomines. With the Rhodnius prolixus genome sequencing project, new omics tools were applied to understand the organism at a systemic level and not just from a genomic point of view. Thus, the present review aims to put together the cytogenomic and genomic information available in the literature for Chagas disease vectors. Here, we review all studies related to cytogenomics and genomics of Chagas disease vectors, contributing to the direction of further research with these insect vectors, because it was evident that most studies focus on cytogenomic knowledge of the species. Given the importance of genomic studies, which contributed to the knowledge of taxonomy, systematics, as well as the vector's biology, the need to apply these techniques in other genera and species of Triatominae subfamily is emphasized.

Brown Spiders' Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms.

Phospholipases-D (PLDs) found in Loxosceles spiders' venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents-L. gaucho and L. laeta-were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

A protective vaccine against the toxic activities following Brown spider accidents based on recombinant mutated phospholipases D as antigens.

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.

Berberine associated photodynamic therapy promotes autophagy and apoptosis via ROS generation in renal carcinoma cells.

Renal cell carcinoma (RCC) consists of the most lethal common urological cancer and the clinical practice has shown that resistant RCC to commons therapies is extremely high. Berberine is an isoquinoline alkaloid, presents in different kinds of plants and it has long been used in Chinese medicine. It has several properties, such as antioxidant, anti-inflammatory, anti-diabetic, anti-microbial and anti-cancer. Moreover, berberine has photosensitive characteristics and its association with photodynamic therapy (PDT) is effective against tumor cells. This study aimed to evaluate the effects of berberine associated with PDT in renal carcinoma cell lines. The cellular viability assay showed increased cytotoxicity in concentration and time-dependent manner. Berberine presented efficient internalization in all cell lines analyzed. In addition, after treatment with berberine associated with PDT, it was observed a high phototoxicity effect with less than 20 % of viable cells. In this study we observed that the increase of reactive oxygen species (ROS) levels was accompanied by an increase of autophagy levels and apoptosis by caspase 3 activity, suggesting cell death by both mechanisms. Additionally, three target genes of anti-cancer drugs were differentially expressed in 786-O cells, being that Vascular Endothelial Growth Factor-D (FIGF) and Human Telomerase Reverse Transcriptase (TERT) gene presented low expression and Polo Like Kinase 3 (PLK3) presented overexpression after treatment with berberine associated with PDT. In this study, the proposed treatment triggered metabolites changes related to cell proliferation, tumorigenesis and angiogenesis. Thus, it was possible to suggest that berberine has promising potential as a photosensitizing agent in a photodynamic therapy, because it induced significant anticancer effects on renal carcinoma cells.

Forty Years of the Description of Brown Spider Venom Phospholipases-D.

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

rSodC is a potential antigen to diagnose Corynebacterium pseudotuberculosis by enzyme-linked immunoassay.

Caseous lymphadenitis (CL) is a chronic infectious disease that affects sheep and goats. Many serological tests have been developed to detect the disease; one of the most widely used is the enzyme-linked immunosorbent assay (ELISA), due to its advantages, which include acceptable cost-effectiveness, applicability, sensitivity and specificity. ELISA formulations using recombinant proteins can exhibit significant sensitivity and specificity when using a single purified antigen. DTxR, Trx, TrxR, LexA, SodC, SpaC, NanH, and PknG recombinant proteins can be considered target proteins for ELISA development due to its extracellular or on the cell surface location, which allows a better recognition by the immune system. Therefore, the objectives of this study were to evaluate the antigenic reactivity of Corynebacterium pseudotuberculosis recombinant proteins in goat and sheep serum. Of eight proteins evaluated, rSodC was selected for validation assays with small ruminant serum samples from the semiarid region of the state of Bahia, Brazil. Validation assays with goat serum samples showed that ELISA-rSodC presented sensitivity and specificity of 96% and 94%, respectively. Validation assays with sheep serum showed that ELISA-rSodC exhibited sensitivity and specificity of 95% and 98%, respectively. Analysis of 756 field serum samples showed that rSodC identified 95 positive samples (23%) in goats and 75 positive samples (21%) in sheep. The ELISA with recombinant SodC protein developed in this study discriminated positive and negative serum samples with high levels of sensitivity and specificity. This formulation is promising for epidemiological surveys and CL control programs.Trial registration AEC No 4958051018. 12/18/2018, retrospectively registered.

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major.

Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host-parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 angstrom resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 angstrom. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3(2)21.

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep.

Caseous lymphadenitis (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false-negative results. This study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.