Papers by Marietta Flores-Díaz
Frontiers in Cellular and Infection Microbiology, Oct 26, 2023
Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins relea... more Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP 3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene 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 gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.
Journal of Biological Chemistry, Sep 1, 1998
Toxicon, Dec 1, 2003
Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfring... more Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfringens and characterized by severe myonecrosis, intravascular leukocyte accumulation, and significant thrombosis. Several lines of evidence indicate that C. perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in this disease. This toxin is a Zn2+ metalloenzyme with lecithinase and sphingomyelinase activities. Its three dimensional structure shows two domains, an N-terminal domain which contains the active site, and a C-terminal domain required for the Ca2+dependent interaction with membranes. Cp-PLC displays several biological activities: it increases capillary permeability, induces platelet aggregation, hemolysis, myonecrosis, decreases cardiac contractility, and is lethal. Experiments with genetically engineered Cp-PLC variants have revealed that the sphingomyelinase activity and the C-terminal domain are required for toxicity. The myotoxicity of Cp-PLC is largely dependent on its membrane damaging effect. In addition, it has been suggested that the alterations in the blood flow induced by this toxin also contribute to muscle damage. In gas gangrene, Cp-PLC dysregulates transduction pathways in endothelial cells, platelets and neutrophils leading to the uncontrolled production of several intercellular mediators and adhesion molecules. Thus, Cp-PLC alters the traffic of neutrophils to the infected tissue and promotes thrombotic events, enhancing the conditions for anaerobic growth.
Journal of Proteome Research, Jun 17, 2008
We report the comparative proteomic characterization of the venoms of adult and newborn specimens... more We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA 2, serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA 2 molecules than in the neonates. In addition, the ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecological and taxonomical implications, the geographical venom variability reported here may have an impact in the treatment of bite victims and in the selection of specimens for antivenom production. The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different geographical location and age has long been appreciated by herpetologist and toxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the first detailed characterization of individual and ontogenetic venom protein profile variations in two geographical isolated B. asper populations, and highlights the necessity of using pooled venoms as a statistically representative venom for antivenom production.
Cellular Microbiology, Dec 13, 2013
Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pat... more Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane.
Springer eBooks, 2016
Poultry products represent over 30% of animal protein consumption worldwide, and its demand is gr... more Poultry products represent over 30% of animal protein consumption worldwide, and its demand is growing considerably. The current global annual production of poultry meat and eggs is more than 115 million tons and 70 million tons, respectively. Necrotic enteritis (NE) in poultry is a reemerging infectious disease caused by certain strains of Clostridium perfringens. This bacterium is found in limited quantities as a normal inhabitant of the birds' gut; however, under certain circumstances, a pathogenic strain proliferates and secretes a variety of bacteriocins, mucins, and adhesins that favors bacteria colonization and establishment. Once bacterial population reaches a certain density, toxin production is triggered which induces mucosal damage. NE generates a dramatic reduction of production levels and a significant increase in mortality in flocks of broilers and laying hens, leading to annual economic losses for the poultry industry estimated to be over $6 billion. NE may manifest as an acute or chronic enterotoxemia. Acute infection is associated with higher rates of mortality and chronic infection, with loss of weight and productivity in sick animals. There is evidence that immunization with formalin-inactivated crude supernatants, native or modified toxins, or other proteins induces partial protection against NE. This review summarizes the findings concerning virulence factors associated with NE pathogenesis and the efforts oriented to develop rational strategies to prevent and control this disease.
Journal of Proteome Research, Nov 24, 2009
Intraspecific snake venom variations have implications in the preparation of venom pools for the ... more Intraspecific snake venom variations have implications in the preparation of venom pools for the generation of antivenoms. The impact of such variation in the cross-reactivity of antivenoms against Bothrops asper venom was assessed by comparing two commercial and four experimental antivenoms. All antivenoms showed similar immunorecognition pattern toward the venoms from adult and neonate specimens. They completely immunodepleted most P-III snake venom metalloproteinases (SVMPs), l-amino acid oxidases, serine proteinases, DC fragments, cysteine-rich secretory proteins (CRISPs), and C-type lectin-like proteins, and partially immunodepleted medium-sized disintegrins, phospholipases A(2) (PLA(2)s), some serine proteinases, and P-I SVMPs. Although all antivenoms abrogated the lethal, hemorrhagic, coagulant, proteinase, and PLA(2) venoms activities, monospecific experimental antivenoms were more effective than the polyspecific experimental antivenom. In addition, the commercial antivenoms, produced in horses subjected to repeated immunization cycles, showed higher neutralization than experimental polyspecific antivenom, produced by a single round of immunization. Overall, a conspicuous pattern of cross-neutralization was evident for all effects by all antivenoms, and monospecific antivenoms raised against venom from the Caribbean population were effective against venom from the Pacific population, indicating that geographic variations in venom proteomes of B. asper from Costa Rica do not result in overt variations in immunological cross-reactivity between antivenoms.
Toxicon, 2017
Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blo... more Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikreinekinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surfacelocated epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms.
The Comprehensive Sourcebook of Bacterial Protein Toxins, 2015
This chapter presents an overview of the classification, structure, and main physiopathological a... more 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.
Toxicon, 2009
Bothrops asper is responsible for the vast majority of snakebite accidents in Central America and... more Bothrops asper is responsible for the vast majority of snakebite accidents in Central America and several studies have demonstrated that specific toxic and enzymatic activities of its venom vary with the geographic origin and age of the specimens. Variability in venom proteins and enzymes between specimens from the Caribbean and the Pacific versants of Costa Rica has been reported since 1964. Recently, we performed a comparative proteomic characterization of the venoms from one population of each versant. Proteins belonging to several families, including disintegrin, phospholipases A 2 , serine proteinases, C-type lectins, CRISP, L-amino acid oxidase, and Zn 2þ-dependent metalloproteinases show a variable degree of relative occurrence in the venoms of both populations. The occurrence of prominent differences in the protein profile between venoms from adults and newborns, and among venom samples from individual specimens of the same region or developmental stage, further demonstrated the existence of geographic, ontogenetic and individual variability in the venom proteome of this species. These findings provide new insights towards understanding the biology of B. asper, contribute to a deeper understanding of the pathology induced by its venom and underscore the importance of the use of venoms pooled from specimens from both regions for producing antivenom exhibiting the broadest cross-reactivity. Furthermore, knowledge of the protein composition of B. asper venom paves the way for detailed future structure-function studies of individual toxins as well as for the development of new protocols to study the reactivity of therapeutic antivenoms.
Toxicon, 2003
Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfring... more Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfringens and characterized by severe myonecrosis, intravascular leukocyte accumulation, and significant thrombosis. Several lines of evidence indicate that C. perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in this disease. This toxin is a Zn2+ metalloenzyme with lecithinase and sphingomyelinase activities. Its three dimensional structure shows two domains, an N-terminal domain which contains the active site, and a C-terminal domain required for the Ca2+dependent interaction with membranes. Cp-PLC displays several biological activities: it increases capillary permeability, induces platelet aggregation, hemolysis, myonecrosis, decreases cardiac contractility, and is lethal. Experiments with genetically engineered Cp-PLC variants have revealed that the sphingomyelinase activity and the C-terminal domain are required for toxicity. The myotoxicity of Cp-PLC is largely dependent on its membrane damaging effect. In addition, it has been suggested that the alterations in the blood flow induced by this toxin also contribute to muscle damage. In gas gangrene, Cp-PLC dysregulates transduction pathways in endothelial cells, platelets and neutrophils leading to the uncontrolled production of several intercellular mediators and adhesion molecules. Thus, Cp-PLC alters the traffic of neutrophils to the infected tissue and promotes thrombotic events, enhancing the conditions for anaerobic growth.
Chemistry and Physics of Lipids, 2009
Alpha-toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of ... more Alpha-toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. Alpha-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 alpha-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 alpha-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.
Archives of Biochemistry and Biophysics, 2000
A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perf... more A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens ␣-toxin was constructed. Three mutants were identified which encoded ␣-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate NPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the aminoand carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.
Acta Crystallographica Section D Biological Crystallography, 2010
Cellular Microbiology, 2013
Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pat... more Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane.
SARS-CoV-2 variants of concern (VoC) show reduced neutralization by vaccine-induced and therapeut... more SARS-CoV-2 variants of concern (VoC) show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies. We tested therapeutic equine polyclonal antibodies (pAbs) against four VoC (alpha, beta, epsilon and gamma). We show that equine pAbs efficiently neutralize VoC, suggesting they are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.
Frontiers in Medicine, 2021
Journal of Neuroscience Research, 2019
Considering the etiology of multiple sclerosis (MS) is still unknown, experimental models resembl... more Considering the etiology of multiple sclerosis (MS) is still unknown, experimental models resembling specific aspects of this immune-mediated demyelinating human disease have been developed to increase the understanding of processes related to pathogenesis, disease evolution, evaluation of therapeutic interventions, and demyelination and remyelination mechanisms. Based on the nature of the investigation, biological models may include in vitro, in vivo, and ex vivo assessments. Even though these approaches have disclosed valuable information, every disease animal model has limitations and can only replicate specific features of MS. In vitro and ex vivo models generally do not reflect what occurs in the organism, and in vivo animal models are more likely used; nevertheless, they are able to reproduce only certain stages of the disease. In vivo MS disease animal models in mammals include: experimental autoimmune encephalomyelitis, viral encephalomyelitis, and induced demyelination. This review examines and describes the most common biological disease animal models for the study of MS, their specific characteristics and limitations.
Infection and Immunity, 2019
Gas gangrene, or clostridial myonecrosis, is usually caused by Clostridium perfringens and may oc... more Gas gangrene, or clostridial myonecrosis, is usually caused by Clostridium perfringens and may occur spontaneously in association with diabetes mellitus, peripheral vascular disease, or some malignancies but more often after contamination of a deep surgical or traumatic lesion. If not controlled, clostridial myonecrosis results in multiorgan failure, shock, and death, but very little is known about the muscle regeneration process that follows myonecrosis when the infection is controlled.
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Papers by Marietta Flores-Díaz