Microbiología

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12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages
(2020) Moreira, Vanessa; Gutiérrez, José María; Lomonte, Bruno; Ramirez Vinolo, Marco Aurélio; Curi, Rui; Lambeau, Gérard; Teixeira, Catarina de Fátima
The snake venom myotoxin (MT)-III is a group IIA secreted phospholipase A2 (sPLA2) with pro-inflammatory activities. Previous studies have demonstrated that MT-III has the ability to stimulate macrophages to release inflammatory lipid mediators derived from arachidonic acid metabolism. Among them, we highlight prostaglandin (PG)E2 produced by the cyclooxygenase (COX)-2 pathway, through activation of nuclear factor (NF)-κB. However, the mechanisms coordinating this process are not fully understood. This study investigates the regulatory mechanisms exerted by other groups of bioactive eicosanoids derived from 12-lipoxygenase (12-LO), in particular 12-hydroxyeicosatetraenoic (12-HETE), on group IIA sPLA2-induced (i) PGE2 release, (ii) COX-2 expression, and (iii) activation of signaling pathways p38 mitogen-activated protein kinases(p38MAPK), protein C kinase (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB. Stimulation of macrophages with group IIA sPLA2 resulted in release of 12-HETE without modification of 12-LO protein levels. Pre-treatment of these cells with baicalein, a 12-LO inhibitor, decreased the sPLA2-induced PGE2 production, significantly reduced COX-2 expression, and inhibited sPLA2-induced ERK; however, it did not affect p38MAPK or PKC phosphorylation. In turn, sPLA2-induced PGE2 release and COX-2 expression, but not NF-κB activation, was attenuated by pre-treating macrophages with PD98059 an inhibitor of ERK1/2. These results suggest that, in macrophages, group IIA sPLA2-induced PGE2 release and COX-2 protein expression are distinctly mediated through 12-HETE followed by ERK1/2 pathway activation, independently of NF-κB activation. These findings highlight an as yet undescribed mechanism by which 12-HETE regulates one of the distinct signaling pathways for snake venom group IIA sPLA2-induced PGE2 release and COX-2 expression in macrophages.
A biocomputational platform for the automated construction of large-scale mathematical models of miRNA-transcription factor networks for studies on gene dosage compensation
(2016-11-09) Acón, Man Sai; Siles Canales, Francisco; Mora Rodríguez, Rodrigo Antonio
Cancer complexity and resistance is mediated by cell-to-cell heterogeneity, which is the consequence of the enormous instability of its genetic material. It is unknown how cancer cells are able to withstand the effects of these alterations, while normal cells are typically very sensitive. We hypothesize that cancer requires specific type of stability to survive the enormous chromosomal alterations. This stability may be mediated by a group of genes, whose expression is tightly regulated to maintain viability through a process called gene dosage compensation. This mechanism could be mediated by systems-level properties of complex networks of microRNAs (miRNA) and transcription factors (TF), regulating gene expression despite changes in copy number. Therefore, we designed a biocomputational platform to automatically construct large-scale mathematical models regulating the expression of several candidate genes under dosage compensation. This platform has a broader potential application to other scientific questions involving miRNA and TF networks.
A bright future for integrative venomics
(2015-10) Calvete Chornet, Juan José; Lomonte, Bruno
Venomous secretions are produced by a myriad of animal species, from invertebrates to vertebrates. As a general rule, peptides and proteins represent the most abundant and functionally relevant components of these dangerous “cocktails”. It may be argued that the first and indispensable requirement to understand a particular venom is to know its composition, and, to this end, the combination of -omics technologies have emerged as the most powerful tools available to date
A Call for Incorporating Social Research in the Global Struggle against Snakebite
(2015-09-17) Gutiérrez, José María; Burnouf, Thierry; Harrison, Robert A.; Calvete Chornet, Juan José; Brown, Nicholas I.; Jensen, Simon D.; Warrell, David A.; Williams, David J.
In Africa, Asia, Latin America, and parts of Oceania, envenoming after snakebite is a serious public health problem. Conservative data suggest that between 1.2 and 5.5 million people suffer snakebites every year, resulting in 25,000 to 125,000 deaths and leaving approximately 400,000 victims with permanent sequelae. Despite its significant impact on human health, this disease remains largely neglected by national and international health authorities, funding agencies, pharmaceutical companies, patients’ organizations, and health advocacy groups
A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages
(2013-05-15) Moreira, Vanessa; de Castro Souto, Pollyana Cristina Maggio; Ramirez Vinolo, Marco Aurélio; Lomonte, Bruno; Gutiérrez, José María; Curi, Rui; Teixeira, Catarina de Fátima
The effects of a snakevenom Lys-49phospholipaseA2 (PLA2) homolog named MT-II,devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2(COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages wit hMT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca2+ independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2induced by MT-II. Inhibition of nuclear factor kB (NF-kB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either proteinkinase C (PKC), proteintyrosinekinase (PTK),or extracellular signal-regulated kinase(ERK) pathways abrogated MT-II-induced NF-kB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibit or reduced MT-II-induced COX-2 expression and PGD2 production.Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-kB activation,but affected neither prostaglandins production nor COX-2expression.MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively,our data demonstrate that a catalytically-inactivePLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2expression in macrophages in both in vitro and in vivo models,indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-kB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events.
A cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C
(2005-07-22) Flores Díaz, Marietta; Alape Girón, Alberto; Clark, Graeme; Catimel, Bruno; Hirabayashi, Yoshio; Nice, Ed; Gutiérrez, José María; Titball, Richard; Thelestam, Mónica
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.
A Clostridium difficile Lineage Endemic to Costa Rican Hospitals Is Multidrug Resistant by Acquisition of Chromosomal Mutations and Novel Mobile Genetic Elements
(2017-01) Ramírez Vargas, Gabriel; Quesada Gómez, Carlos; Acuña Amador, Luis Alberto; López Ureña, Diana; Murillo Corrales, Tatiana; Gamboa Coronado, María del Mar; Chaves Olarte, Esteban; Thomson, Nicholas R.; Rodríguez Cavallini, Evelyn; Rodríguez Sánchez, César
The antimicrobial resistance (AMR) rates and levels recorded for Clostridium difficile are on the rise. This study reports the nature, levels, diversity, and genomic context of the antimicrobial resistance of human C. difficile isolates of the NAPCR1/RT012/ST54 genotype, which caused an outbreak in 2009 and is endemic in Costa Rican hospitals. To this end, we determined the susceptibilities of 38 NAPCR1 isolates to 10 antibiotics from seven classes using Etests or macrodilution tests and examined 31NAPCR1 whole-genome sequences to identify single nucleotide polymorphisms (SNPs) and genes that could explain the resistance phenotypes observed. The NAPCR1 isolates were multidrug resistant (MDR) and commonly exhibited very high resistance levels. By sequencing their genomes, we showed that they possessed resistance-associated SNPs in gyrA and rpoB and carried eight to nine acquired antimicrobial resistance (AMR) genes. Most of these genes were located on known or novel mobile genetic elements shared by isolates recovered at different hospitals and at different time points. Metronidazole and vancomycin remain the first-line treatment options for these isolates. Overall, the NAPCR1 lineage showed an enhanced ability to acquire AMR genes through lateral gene transfer. On the basis of this finding, we recommend further vigilance and the adoption of improved control measures to limit the dissemination of this lineage and the emergence of more C. difficile MDR strains.
A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus
(1997-04) Alape Girón, Alberto; Miranda Arrieta, Keyna; Cortés Bratti, Ximena; Stiles, Bradley G.; Gutiérrez, José María
Therapeutic antisera against Micrurus nigrocinctus venom were tested for protection against lethality, as well as for inhibition of the nicotinic acetylcholine receptor (AchR)-binding and neutralization of phospholipase A2 (PLA2) activities of the homologous venom. Protection against venom lethality did not correlate with inhibition of AchR-binding activity, whereas there was a significant correlation between antisera potency and inhibition of PLA2 activity (r = 0.82, n = 10, P < 0.02). Inhibition of PLA2 activity could be useful in assessing the protective efficacy of M. nigrocinctus antisera during antivenom production. Micrurus nigrocinctus nigrocinctus venom proteins were fractionated by cation-exchange chromatography on Mono S FPLC and fractions assayed for lethality, AchR-binding and PLA2 activities. Antisera were titrated by enzyme-linked immunoassay (ELISA) against a crude M. n. nigrocinctus venom, two FPLC lethal fractions containing AchR-binding activity, and two toxins purified from M. n. nigrocinctus venom. No correlation was found between protective efficacy and the ELISA titer against any of these antigens. Compared to other elapid venoms that contain few toxins as major components, M. n. nigrocinctus venom appears to be more complex and its lethal effect is likely to be due to the combined effect of several neurotoxins.
A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipases A2
(2015-10) Yunes Quartino, Pablo J.; Portela, Madelón; Lima, Analía; Durán, Rosario; Lomonte, Bruno; Fidelio, Gerardo Daniel
We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of theΠopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venomenzymes is a newisoenzyme, partially sequenced by amass spectrometry approach.We also included the basicmyotoxin sPLA2-III fromBothrops asper. Results obtained with the isochoricmethod and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m−1 and 10 mN·m−1, termed here as LR20/10 index. This index differentiates quite well “high surface pressure” from “low surface pressure” sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.
A cryptic palm-pitviper species (Squamata: Viperidae: Bothriechis) from the Costa Rican highlands, with notes on the variation within B. nigroviridis
(2016-07-15) Doan, Tiffany M.; Mason, Andrew J.; Castoe, Todd A.; Sasa Marín, Mahmood; Parkinson, Christopher L.
Middle America is one of the most biodiverse regions in the world, harboring an exceptional number of rare and endemic species. This is especially true of Middle American cloud forests, where montane specialists occupy restricted, high-elevation ranges making them attractive candidates for investigating historical biogeography and speciation. One such highland- restricted species, the black speckled palm-pitviper (Bothriechis nigroviridis), occupies the Central, Tilarán, and Talamanca Cordilleras in Costa Rica and Panama. In this study, we investigate the genetic and morphological variation among populations of B. nigroviridis by inferring a multilocus phylogeny (21 individuals) and analyzing meristic scale characters with a principal component analysis (64 individuals). We find B. nigroviridis sensu stricto to be composed of two deeply divergent lineages, one with a restricted range in the northern and central Cordillera Talamanca and the other ranging throughout the Central, Tilarán, and Talamanca Cordilleras. Furthermore, these two lineages are morphologically distinct, with previously unrecognized differences in several characters allowing us to name and diagnose a new species B. nubestris sp. nov. We also examine the genetic and morphological variation within B. nigroviridis and discuss biogeographic hypotheses that may have led to the diversification of Bothriechis lineages.
A eficácia do antiveneno botrópico-crotálico na neutralização das principais atividades do veneno de Bothrops jararacussu
(1992) Dos Santos, María Cristina; Gonçalves, Luís Roberto de Camargo; Fortes Dias, Consuelo L.; Cury, Yara; Gutiérrez, José María; Furtado, María de Fátima
Myonecrosis is one of the effects of Bothrops jararacussu venom, from which a myotoxin was isolated showing structural homology to phospholipase A2 (PLA2), but without enzimatic activity. Such myotoxic activity is also present in the Crotalus durissus terrificus venom, and is atributed to crotoxin and to PLA2 (crotoxin B), the basic component of the crotoxin complex. The Bothrops jararacussu venom showed three proteins with immunologic identity to PLA2 from crotoxin. The bothropic (AB) and the bothropic/crotalic (AB/C) antivenoms, two commercial polyvalent antivenoms produced at Instituto Butantan, were compared in order to assess their capacity for neutralization of the lethal, hemorrhagic, coagulant and myotoxic activities of Bothrops jararacussu venom. Both antivenoms showed the same level of hemorrhagic activity neutralization. However, AB/C was about three times more efficient than AB in neutralizing the myotoxic activity, and two times more potent for neutralization of lethality and coagulant activity of Bothrops jararacussu venom. These data suggest that the use of AB/C could be of value in the treatment of patients bitten by snakes of this species
A first perturbome of Pseudomonas aeruginosa: Identification of core genes related to multiple perturbations by a machine learning approach
(2021) Molina Mora, José Arturo; Montero Manso, Pablo; García Batan, Raquel; Campos Sánchez, Rebeca; Vilar Fernández, José; García Santamaría, Fernando
Tolerance to stress conditions is vital for organismal survival, including bacteria under specific environmental conditions, antibiotics, and other perturbations. Some studies have described common modulation and shared genes during stress response to different types of disturbances (termed as perturbome), leading to the idea of central control at the molecular level. We implemented a robust machine learning approach to identify and describe genes associated with multiple perturbations or perturbome in a Pseudomonas aeruginosa PAO1 model. Using microarray datasets from the Gene Expression Omnibus (GEO), we evaluated six approaches to rank and select genes: using two methodologies, data single partition (SP method) or multiple partitions (MP method) for training and testing datasets, we evaluated three classification algorithms (SVM Support Vector Machine, KNN KNearest neighbor and RF Random Forest). Gene expression patterns and topological features at the systems level were included to describe the perturbome elements. We were able to select and describe 46 core response genes associated with multiple perturbations in P. aeruginosa PAO1 and it can be considered a first report of the P. aeruginosa perturbome. Molecular annotations, patterns in expression levels, and topological features in molecular networks revealed biological functions of biosynthesis, binding, and metabolism, many of them related to DNA damage repair and aerobic respiration in the context of tolerance to stress. We also discuss different issues related to implemented and assessed algorithms, including data partitioning, classification approaches, and metrics. Altogether, this work offers a different and robust framework to select genes using a machine learning approach.
A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques
(2020-01-09) Arias Arias, Jorge Luis; MacPherson, Derek J.; Hill, Maureen E.; Hardy, Jeanne A.; Mora Rodríguez, Rodrigo Antonio
The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for thera- peutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus–host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivi- rus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B–NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluo- rescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus inter- nal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbec- co’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus–host interactions and facilitate future applications in antiviral drug research to manage flavivi- rus infections.
A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging
(2021-06-22) Arias Arias, Jorge Luis; Corrales Aguilar, Eugenia; Mora Rodríguez, Rodrigo Antonio
Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immuno- labeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA ﬂuorescent dyes with differen- tial cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identiﬁcation using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This ﬂuorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern ﬂuorescence microscopy and image analysis approaches for future applications in virology
A geographical sampling method for surveys of mosquito larvae in an urban area using high-resolution satellite imagery
(2008-06-01) Troyo Rodríguez, Adriana; Fuller, Douglas O.; Calderón Arguedas, Ólger; Beier, John C.
Entomological surveys in urban areas are often biased by selecting houses or locations with known high vector densities. A sampling strategy was developed for Puntarenas, Costa Rica, using high-resolution satellite imagery. Grids from the Advanced Spaceborne Thermal Emission and Reflection Radiometer and a QuickBird classified land cover map were used to determine the optimal final grid area for surveys. A random sample (10% of cells) was selected, and sample suitability was assessed by comparing the mean percentage of tree cover between sample and total cells. Sample cells were used to obtain entomological data from 581 locations: 26.3% of all locations positive for mosquito larvae were not households, they contained 29.5% of mosquito-positive habitats and 16% of Aedes aegypti pupae collected. Entomological indices for Ae. aegypti (pupae per person, Breteau index, container index, location index) were slightly lower when only household data were analyzed. High-resolution satellite imagery and geographical information systems appear useful for evaluating urban sites and randomly selecting locations for accurate entomological surveys.
A geographical sampling strategy for field surveys in an urban area using high-resolution satellite imagery
(2006-11) Troyo Rodríguez, Adriana; Fuller, Douglas O.; Calderón Arguedas, Ólger; Beier, John C.
Field evaluations for studying the epidemiology of vector-borne diseases like dengue in urban areas are often restricted to selection of households and buildings for field surveys. Therefore, the resulting sampling frame may exclude specific locations within the urban environment that contain vector habitats and thus may bias the results. A sampling strategy was developed for field surveys in an urban area using high-resolution satellite imagery. The site selected was Puntarenas, a city affected by dengue on the Pacific coast of Costa Rica, for which high-resolution satellite imagery was available from the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER, 15 m spatial resolution) and QuickBird (0.6 m and 2.4 m spatial resolution for panchromatic and multispectral bands, respectively). Grids obtained from the ASTER imagery and a cover map generated from the QuickBird multispectral bands were used to determine the optimal grid area of 10 000 m2 , which contain 136 houses. A final grid 42 by 42 pixels (100.8 x 100.8 m) was created using the multispectral Quickbird imagery, and cells that had an area less than 90% within one specific locality of Puntarenas were excluded. The remaining cells were grouped according to locality and a random sample (10%) was selected from each. This sample of cells would be used for field data collection on specific mosquito larval habitats by evaluating the entire area within the geographical limits of each cell. To assess the suitability of the selected grid cells, the proportion of tree area (“tree” class Kappa = 0.91) was extracted for the individual cells from the QuickBird cover map. The mean percentage of tree cover in each locality and total area was compared between the selected sample cells and the total cells of the Puntarenas image. Overall, the sample adequately represented the total area and most of the individual localities in terms of tree cover. In 8 of 10 localities the difference between the estimate (sample) and the real percentage of tree cover was less than 3%. These results show that high-resolution satellite imagery and geographical information systems are useful in evaluating urban areas and randomly selecting sections for field data collection on mosquito larval habitats that are practical, representative, and will reduce bias.
A group IIA-secreted phospholipase A2 from snake venom induces lipid body formation in macrophages: the roles of intracellular phospholipases A2 and distinct signaling pathways
(2011-07) Leiguez, Elbio; Pavan Zuliani, Juliana; Marques Cianciarullo, Aurora; Fernandes, Cristina Maria; Gutiérrez, José María; Teixeira, Catarina de Fátima
We investigated the ability of the sPLA(2), known as MT-III, isolated from the viperid snake Bothrops asper, to induce LB formation in macrophages and the major cellular signaling pathways involved in this process. The effects of MT-III on ADRP localization and expression and macrophage ultrastructure were assessed. Our results showed that this sPLA(2) induced a marked increase in LB numbers in macrophages, induced the recruitment of ADRP in macrophages, and up-regulated ADRP expression. Ultrastructural analysis showed the presence of weakly and strongly osmiophilic LBs in sPLA(2)-stimulated cells. Enlargement of the ER and Golgi cisterns was also observed. Pretreatment of cells with H7 or staurosporine (PKC inhibitors), LY294002 or wortmannin (PI3K inhibitors), SB202190 or PD98059 (p38(MAPK) and ERK1/2 inhibitors, respectively), or Pyr-2 or Bel (cPLA(2) and iPLA(2) inhibitors, respectively) significantly reduced sPLA(2)-induced LB formation. Herbimycin (a PTK inhibitor) and indomethacin or etoricoxib (COX inhibitors) failed to alter sPLA(2)-induced effects. In conclusion, our results show for the first time the ability of a venom sPLA(2) to induce the formation of LBs and the expression of ADRP in macrophages. Venom PLA(2)-induced LB formation is dependent on PKC, PI3K, p38(MAPK), ERK1/2, cPLA(2), and iPLA(2) signaling pathways but not on PTK, COX-1, or COX-2 pathways. Activation of the ER and Golgi complex may play an important role in the formation of LBs induced by this sPLA(2) in macrophages.
A hybrid mathematical modeling approach of the metabolic fate of a fluorescent sphingolipid analogue to predict cancer chemosensitivity
(2018-06) Molina Mora, José Arturo; Kop Montero, Mariana; Quirós Fernández, Isaac; Quirós Barrantes, Steve; Crespo Mariño, Juan Luis; Mora Rodríguez, Rodrigo Antonio
Sphingolipid (SL) metabolism is a complex biological system that produces and transforms ceramides and other molecules able to modulate other cellular processes, including survival or death pathways key to cell fate decisions. This signaling pathway integrates several types of stress signals, including chemotherapy, into changes in the activity of its metabolic enzymes, altering thereby the cellular composition of bioactive SLs. Therefore, the SL pathway is a promising sensor of chemosensitivity in cancer and a target hub to overcome resistance. However, there is still a gap in our understanding of how chemotherapeutic drugs can disturb the SL pathway in order to control cellular fate. We propose to bridge this gap by a systems biology approach to integrate i) a dynamic model of SL analogue (BODIPY-FL fluorescent-sphingomyelin analogue, SM-BOD) metabolism, ii) a Gaussian mixture model (GMM) of the fluorescence features to identify how the SL pathway senses the effect of chemotherapy and iii) a fuzzy logic model (FLM) to associate SL composition with cell viability by semi-quantitative rules. Altogether, this hybrid model approach was able to predict the cell viability of double experimental perturbations with chemotherapy, indicating that the SL pathway is a promising sensor to design strategies to overcome drug resistance in cancer.
A lipidomic perspective of the action of group IIA secreted phospholipase A2 on human monocytes: lipid droplet biogenesis and activation of cytosolic phospholipase A2α
(2020) Rodríguez, Juan Pablo; Leiguez, Elbio; Guijas, Carlos; Lomonte, Bruno; Gutiérrez, José María; Teixeira, Catarina de Fátima; Balboa, María A.; Balsinde Rodríguez, Jesús
Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2α. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.
A Lys49 phospholipase A2 homologue from Bothrops asper snake venom induces proliferation, apoptosis and necrosis in a lymphoblastoid cell line
(2005-04) Mora Rodríguez, Rodrigo Antonio; Valverde Rojas, Berta E.; Díaz Oreiro, Cecilia; Lomonte, Bruno; Gutiérrez, José María
Lys49 phospholipase A2 homologues are abundant in viperid snake venoms. These proteins have substitutions at the calcium-binding loop and catalytic center which render them enzymatically inactive; however, they display a series of toxic activities, particularly cytotoxicity upon various cell lines in vitro. In this study we explored whether myotoxin II (MT-II), a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops asper, is capable of inducing various effects in a single cell type, using the lymphoblastoid B cell line CRL-8062 as a model. Cells were incubated with varying concentrations of MT-II for 24 and 48 h, time intervals that are more prolonged than the usual incubation times previously used in the characterization of this toxin. Results indicate that MT-II induces proliferation at low concentrations (0.5–5.0 μg/mL). Apoptosis was predominant at higher toxin levels (5–25 μg/mL), whereas necrosis, associated with overt plasma membrane disruption, occurred at concentrations ≥25 μg/mL, and was the predominant effect at higher MT-II concentrations (50 μg/mL). It is concluded that a single phospholipase A2 homologue can induce markedly different effects on a single cell line, depending on the concentration used, an observation that may have implications for the action of this type of venom component in vivo.

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