Snake venomics and antivenomics of Middle and South America rattlesnakes.Identification of neurotoxin crotoxin as an adaptive trait during Crotalus Durissus invasion of South America

Comunicaciones a congreso

Heterologous hyperimmune polyclonal antibodies against SARS-COV-2: A broad coverage, affordable, and scalable potential immunotherapy for Covid-19

The emergence and dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting COVID-19 pandemic triggered a global public health crisis. Although several SARS-CoV-2 vaccines have been developed, demand far exceeds supply, access to them is inequitable, and thus, populations in low- and middle-income countries are unlikely to be protected soon (1). Furthermore, there are no specific therapies available, which is a challenge for COVID-19 patient care (2). Thus, the appearance of SARS-CoV-2 variants and reports of reinfections associated with immune escape (3, 4) highlight the urgent need for effective and broad coverage COVID-19 therapeutics. Intravenous administration of human or heterologous antibodies is a therapy successfully used in patients with viral respiratory diseases (5). Accordingly, formulations containing SARS-CoV-2 specific antibodies are an attractive therapeutic option for COVID-19 patients (6). SARS-CoV-2 specific antibodies could limit infection by direct virion neutralization and/or by targeting infected cells for elimination via complement or antibody-mediated cytotoxicity (6). Specific SARS-CoV-2 antibody-based therapeutics include convalescent plasma (CP), monoclonal antibodies (mAbs), human polyclonal IgG formulations purified from CP or transgenic animals, and heterologous hyperimmune polyclonal antibodies (pAbs) (6). Although the window for using antibody-based therapeutics varies, clinical data show that they are mainly effective if administered early after symptoms onset (6).Universidad de Costa Rica/[741-C0-198]/UCR/Costa RicaCaja Costarricense del Seguro Social/[]/CCSS/Costa RicaBanco Centroamericano de Integración Económica/[]/BCIE/Costa RicaGerman academic exchange services/[57592642]/DAAD/AlemaniaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de MedicinaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

A cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Membrane-damaging and cytotoxic sphingomyelinases and phospholipases

This chapter presents an overview of the classification, structure, and main physiopathological activities of bacterial sphingomyelinases and phospholipases, providing examples of their roles as virulence factors in several human and animal diseases. Bacterial sphingomyelinases (SMases) and phospholipases (PLases) constitute a heterogeneous group of surface-associated or secreted esterases produced by a variety of intracellular and extracellular pathogens. These enzymes might favor in different ways tissue colonization establishment and progression of the infection, or evasion of the immune response. In several cases, mutant bacterial strains lacking a sphingomyelinase or a phospholipase encoding gene have impaired virulence in experimental animals, demonstrating the role of these enzymes in pathogenicity. However, PLases contribute also to other aspects of bacterial lifestyle, including survival in different environments, and competition with other microorganisms; thus, the multifunctional nature of these enzymes reflects the remarkable adaptability of some bacteria.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Phospholipase C and sphingomyelinase activities of the Clostridium perfringens Alfa-toxin

Alfa-Toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. Alfa-Toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of Alfa-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5–7 range, in agreement with the proposed role of -toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.CIEMIC e Instituto Clodomiro Picado, Universidad de Costa Rica. Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), y Departamento de Bioquímica, Universidad del País Vasco, España.UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Clostridium perfringens Phospholipase C, an Archetypal Bacterial Virulence Factor, induces the Formation of Extracellular Traps by Human Neutrophils

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins, released to the extracellular space to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and is involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen that produces many exotoxins associated with various animal and human diseases, including the necrotizing soft tissue infection called gas gangrene. This work demonstrates that the C. perfringens toxinotype A secretome induces NETs formation (NETosis) in human neutrophils. Antibodies against the C. perfringens phospholipase C (CpPLC) completely abrogate the NETosis-inducing activity of that secretome, and the recombinant CpPLC induces NETs formation in a dose-response manner. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that help other pathogens evade NETs. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/ extracellular signal-regulated kinase (MEK/ERK) pathways, and the production of reactive oxygen species (ROS) by the xanthine oxidase (XO) and the metabolism of arachidonic acid. CpPLC was the first bacterial toxin found to be enzymatically active and is the major virulence factor in the pathogenesis of gas gangrene. This toxin drives the formation of neutrophil/platelet aggregates within the vasculature of the infected tissues, which leads to the circulation's halt and extends the anaerobic environment for C. perfringens growth. It is suggested that this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis, and then escapes and spreads to deeper tissues. Understanding the role of NETs in the thrombotic events occurring in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis

Snake venomics and antivenomics: Proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming

Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact, particularly in Asia, Africa and Latin America. A global initiative, aimed at increasing antivenom production and accessibility, is being promoted by the World Health Organization and others. This work discusses several aspects of antivenom manufacture and control in which the proteomic analysis of snake venoms, for which the term ‘snake venomics’ has been coined, might play a relevant supporting role. Snake venomics has already shown its usefulness for generating knowledge at different levels (ontogenetic, individual, and geographic) on inter- and intraspecies venom variability. This information has applications for the quality control of venom preparations used in antivenom manufacture. Moreover, the design of the best venom mixtures for immunization, aimed at increasing the effectiveness of antivenoms, may also be guided by venom proteome analysis, including molecular studies of the cross-reactivity of antivenoms and heterologous venoms through a recently developed methodological approach termed ‘antivenomics’. Results generated by proteomic protocols should be complemented by preclinical testing of antivenom efficacy using functional neutralization assays. Snake venomics might be also helpful in designing alternative in vitro tests for the assessment of antivenom efficacy that would eventually substitute current in vivo tests.Consejo Superior de Investigaciones Científicas (CSIC), project 2007CR0004CYTED, project 206AC0281Universidad de Costa Rica, project 741-A8-521Consejo Nacional de Rectores (CONARE), Costa RicaMinisterios de Educación y Ciencia and Ciencia e Innovación, Madrid, grants BFU2004-01432 and BFU2007-61563Acciones Integradas 2006CR0010UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de MedicinaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC

PKC activation is required for the cytotoxic effect of CpPLC.

<p>Don Q cells (A) or GM95 cells (B) were treated overnight with GF109203x (GF, 20 µM (A), 10 µM (B)); Safingol (Saf, 10 µM (A), 30 µM (B)); Hispidin (His, 2 µM); P205 (66 µg/ml (A), 100 µg/ml (B) P222 (40 µg/ml); P219 (66 µg/ml); P223 (66 µg/ml) and Rottlerin (Rott, 7,2 µM) or MEM (control) before exposure to CpPLC. Cell viability was determined 18 h later using the neutral red assay. Results are expressed as the percentage of neutral red incorporated by the remaining cells, in comparison with the neutral red incorporated by control cells incubated with each treatment, but not exposed to the toxin. The results represent the average of two-four independent experiments with three replicate samples. (** p<0.001, * p<0.01). (C) Don Q cells were treated without (0 minutes) or with CpPLC for 10, 20, 30 or 60 minutes at 37°C. PMA was used as positive control for PKC activation. Cells were collected and PKC activation was measured using Promega'sPepTag® Non-Radioactive Protein Kinase Assays, according to manufacturer instructions. Results are representative of three independent experiments. (D) Don Q cells were treated without (0 minutes) or with CpPLC (3 ng/ml) for 5, 15, 30 or 60 minutes. Cytosol and membrane fractions were separated as described in the Materials and Methods section, and immunoblotted against PKCβII. After stripping of the blot, actin or clathrin were also detected. Densitometric analysis was performed using Image J software. Results are representative of three independent experiments.</p