RESUMEN

Antimicrobial peptides (AMPs) are recognized for their potential application as new generation antibiotics, however, up to date, they have not been widely commercialized as expected. Although current bioinformatic tools can predict antimicrobial activity based on only amino acid sequences with astounding accuracy, peptide selectivity and potency are not foreseeable. This, in turn, creates a bottleneck not only in the discovery and isolation of promising candidates but, most importantly, in the design and development of novel synthetic peptides. In this paper, we discuss the challenges faced when trying to predict peptide selectivity and potency, based on peptide sequence, structure and relevant biophysical properties such as length, net charge and hydrophobicity. Here, pore-forming alpha-helical antimicrobial peptides family isolated from anurans was used as the case study. Our findings revealed no congruent relationship between the predicted peptide properties and reported microbial assay data, such as minimum inhibitory concentrations against microorganisms and hemolysis. In many instances, the peptides with the best physicochemical properties performed poorly against microbial strains. In some cases, the predicted properties were so similar that differences in activity amongst peptides of the same family could not be projected. Our general conclusion is that antimicrobial peptides of interest must be carefully examined since there is no universal strategy for accurately predicting their behavior.

RESUMEN

Ascaphins are cationic antimicrobial peptides that have been shown to have potential in the treatment of infectious diseases caused by multidrug-resistant pathogens (MDR). However, to date, their principal molecular target and mechanism of action are unknown. Results from peptide prediction software and molecular dynamics simulations confirmed that ascaphin-8 is an alpha-helical peptide. For the first time, the peptide was described as membranotrophic using biophysical approaches including calcein liposome leakage, Laurdan general polarization, and dynamic light scattering. Ascaphin-8's activity and selectivity were modulated by rearranging the spatial distribution of lysine (Var-K5), aspartic acid (Var-D4) residues, or substitution of phenylalanine with tyrosine (Var-Y). The parental peptide and its variants presented high affinity toward the bacterial membrane model (≤2 µM), but lost activity in sterol-enriched membranes (mammal and fungal models, with cholesterol and ergosterol, respectively). The peptide-induced pore size was estimated to be >20 nm in the bacterial model, with no difference among peptides. The same pattern was observed in membrane fluidity (general polarization) assays, where all peptides reduced membrane fluidity of the bacterial model but not in the models containing sterols. The peptides also showed high activity toward MDR bacteria. Moreover, peptide sensitivity of the artificial membrane models compared with pathogenic bacterial isolates were in good agreement.

Asunto(s)

Péptidos Catiónicos Antimicrobianos , Fluidez de la Membrana , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias , Colesterol/química , Mamíferos , Pruebas de Sensibilidad Microbiana , Esteroles/química

RESUMEN

The development of new strategies for achieving stable asymmetric membrane models has turned interleaflet lipid asymmetry into a topic of major interest. Cyclodextrin-mediated lipid exchange constitutes a simple and versatile method for preparing asymmetric membrane models without the need for sophisticated equipment. Here we describe a protocol for preparing asymmetric supported lipid bilayers mimicking membrane rafts by cyclodextrin-mediated lipid exchange and the main guidelines for obtaining structural information and quantitative measures of their mechanical properties using Atomic force microscopy and Force spectroscopy; two powerful techniques that allow membrane characterization at the nanoscale.

Asunto(s)

Membrana Dobles de Lípidos , Ciclodextrinas , Microdominios de Membrana , Microscopía de Fuerza Atómica

RESUMEN

The cytoplasmic membrane is one of the most frequent cell targets of antimicrobial peptides (AMPs) and other biomolecules. Understanding the mechanism of action of AMPs at the molecular level is of utmost importance for designing of new membrane-specific molecules. In particular, the formation of pores, the structure and size of these pores are of great interest and require nanoscale resolution approaches, therefore, biophysical strategies are essential to achieve an understanding of these processes at this scale. In the case of membrane active peptides, pore formation or general membrane disruption is usually the last step before cell death, and so, pore size is generally directly associated to pore structure and stability and loss of cellular homeostasis, implicated in overall peptide activity. Up to date, there has not been a critical review discussing the methods that can be used specifically for estimating the pore dimensions induced by membrane active peptides. In this review we discuss the scope, relevance and popularity of the different biophysical techniques such as liposome leakage experiments, advanced microscopy, neutron or X-ray scattering, electrophysiological techniques and molecular dynamics studies, all of them useful for determining pore structure and dimension.

Asunto(s)

Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Proteínas Citotóxicas Formadoras de Poros/química , Liposomas/química

RESUMEN

Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate important cellular functions. Notwithstanding, the asymmetric transbilayer distribution of phospholipids that exists in the membrane confers the two leaflets different potentials to form lateral domains as next to no sphingolipids are present in the inner leaflet. How the physical properties of one leaflet can influence the properties of the other and its importance on signal transduction across the membrane are questions still unresolved. In this work, we combined AFM imaging and Force spectroscopy measurements to assess domain formation and to study the nanomechanical properties of asymmetric supported lipid bilayers (SLBs) mimicking membrane rafts. Asymmetric SLBs were formed by incorporating N-palmitoyl-sphingomyelin (16:0SM) into the outer leaflet of preformed 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Cholesterol SLBs through methyl-ß-cyclodextrin-mediated lipid exchange. Lipid domains were detected after incorporation of 16:0SM though their phase state varied from gel to liquid ordered (Lo) phase if the procedure was performed at 24 or 37 °C, respectively. When comparing symmetric and asymmetric Lo domains, differences in size and morphology were observed, with asymmetric domains being smaller and more interconnected. Both types of Lo domains showed similar mechanical stability in terms of rupture forces and Young's moduli. Notably, force curves in asymmetric domains presented two rupture events that could be attributed to the sequential rupture of a liquid disordered (Ld) and a Lo phase. Interleaflet coupling in asymmetric Lo domains could also be inferred from those measurements. The experimental approach outlined here would significantly enhance the applicability of membrane models.

Asunto(s)

Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Esfingolípidos/química

RESUMEN

In recent years, lipopeptides (LPs) have attracted a lot of attention in the pharmaceutical industry due to their broad-spectrum of antimicrobial activity against a variety of pathogens and their unique mode of action. This class of compounds has enormous potential for application as an alternative to conventional antibiotics and for pest control. Understanding how LPs work from a structural and biophysical standpoint through investigating their interaction with cell membranes is crucial for the rational design of these biomolecules. Various analytical techniques have been developed for studying intramolecular interactions with high resolution. However, these tools have been barely exploited in lipopeptide-lipid interactions studies. These biophysical approaches would give precise insight on these interactions. Here, we reviewed these state-of-the-art analytical techniques. Knowledge at this level is indispensable for understanding LPs activity and particularly their potential specificity, which is relevant information for safe application. Additionally, the principle of each analytical technique is presented and the information acquired is discussed. The key challenges, such as the selection of the membrane model are also been briefly reviewed.

Asunto(s)

Antibacterianos/metabolismo , Membrana Celular/metabolismo , Lípidos/química , Lipopéptidos/metabolismo , Animales , Biofisica , Humanos

RESUMEN

The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Liposomas Unilamelares/química , Animales , Péptidos Catiónicos Antimicrobianos/química , Bacterias , Membrana Celular/química , Membrana Eritrocítica/efectos de los fármacos , Hongos , Fluidez de la Membrana , Potenciales de la Membrana , Esteroles/química , Viscosidad

RESUMEN

Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains - which proteolyse cytoskeleton proteins - and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.

Asunto(s)

Micropartículas Derivadas de Células/metabolismo , Eritrocitos/efectos de los fármacos , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/toxicidad , Proteínas Hemolisinas/toxicidad , Micropartículas Derivadas de Células/efectos de los fármacos , Eritrocitos/citología , Eritrocitos/metabolismo , Escherichia coli/metabolismo , Infecciones por Escherichia coli/fisiopatología , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólisis/efectos de los fármacos , Humanos , Fosfatidilserinas/metabolismo

RESUMEN

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K+ ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K+ ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

Asunto(s)

Adenilil Ciclasas/genética , Proteínas Bacterianas/toxicidad , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Proteínas de Insectos/genética , Sistema de Señalización de MAP Quinasas , Mariposas Nocturnas/efectos de los fármacos , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Insectos/metabolismo , Larva/efectos de los fármacos , Larva/genética , Larva/microbiología , Mariposas Nocturnas/genética , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/microbiología

Asunto(s)

Calcio/metabolismo , Eritrocitos/química , Proteínas de Escherichia coli/farmacología , Proteínas Hemolisinas/farmacología , Acilación , Adenosina Trifosfato/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/ultraestructura , Proteínas de Escherichia coli/química , Proteínas Hemolisinas/química , Líquido Intracelular/química , Transporte Iónico , Procesamiento Proteico-Postraduccional , Conejos , Receptores Purinérgicos/fisiología , Imagen de Lapso de Tiempo

RESUMEN

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.

Asunto(s)

Canales de Calcio/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/fisiología , Factores de Transcripción NFATC/fisiología , Proteínas de Neoplasias/fisiología , Calcio/metabolismo , Silenciador del Gen , Histamina/química , Humanos , Inflamación , Interleucina-8/metabolismo , Interleucinas/metabolismo , Proteína ORAI1 , Proteína ORAI2 , Interferencia de ARN , Transducción de Señal , Molécula de Interacción Estromal 1

RESUMEN

Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.

Asunto(s)

Proteínas Bacterianas/metabolismo , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Cadherinas/química , Membrana Celular , Endotoxinas/química , Ensayo de Inmunoadsorción Enzimática , Proteínas Hemolisinas/química , Manduca/metabolismo , Microvellosidades , Unión Proteica , Receptores de Superficie Celular , Tripsina/metabolismo

RESUMEN

Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1.

Asunto(s)

Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Insectos/efectos de los fármacos , Insecticidas/farmacología , Animales , Toxinas de Bacillus thuringiensis , Western Blotting , Línea Celular , Larva/efectos de los fármacos , Manduca/efectos de los fármacos

RESUMEN

BACKGROUND: Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity. METHODS: Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured. RESULTS: A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26-Α29), and K4N Δ(K12-Q18; Ν26-Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25µM and 5 to 25µM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900µM for their EC50, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells. CONCLUSIONS: These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections. GENERAL SIGNIFICANCE: Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.

Asunto(s)

Antiinfecciosos , Antimaláricos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Péptidos , Plasmodium berghei/crecimiento & desarrollo , Venenos de Escorpión , Staphylococcus aureus/crecimiento & desarrollo , Animales , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/farmacología , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología

RESUMEN

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

Asunto(s)

Aedes/crecimiento & desarrollo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Secuencia de Bases , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Unión Proteica , Interferencia de ARN , Resonancia por Plasmón de Superficie

RESUMEN

BACKGROUND: Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins. METHODOLOGY/PRINCIPAL FINDINGS: The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors. CONCLUSIONS/SIGNIFICANCE: We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.

Asunto(s)

Bacillus thuringiensis/patogenicidad , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Multimerización de Proteína , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Endotoxinas/toxicidad , Genes Dominantes , Proteínas Hemolisinas/toxicidad , Manduca/microbiología , Mutación

RESUMEN

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a ß-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the ß-sheet, allowing insertion of ß-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of ß-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.

Asunto(s)

Proteínas Bacterianas/química , Endotoxinas/química , Proteínas Hemolisinas/química , Insecticidas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aedes/efectos de los fármacos , Animales , Toxinas de Bacillus thuringiensis , Larva/efectos de los fármacos , Liposomas/química , Membranas/química , Modelos Químicos , Conformación Proteica , Multimerización de Proteína

RESUMEN

Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

Asunto(s)

Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Endotoxinas/metabolismo , Células Epiteliales/metabolismo , Proteínas Hemolisinas/metabolismo , Insectos/metabolismo , Multimerización de Proteína , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Membrana Celular/química , Endotoxinas/química , Células Epiteliales/química , Proteínas Hemolisinas/química , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Insectos/química , Insecticidas/química , Insecticidas/metabolismo , Precursores de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo

RESUMEN

Cry11Aa and Cyt1Aa of Bacillus thuringiensis are active against mosquitoes and show synergism. Cyt1Aa functions as a membrane receptor inducing Cry11Aa oligomerization. Here we characterized Cry11Aa helix alpha-3 mutants impaired in oligomerization and toxicity against Aedes aegypti, indicating that oligomerization of Cry11Aa is important for toxin action. Cyt1Aa did not recover the insecticidal activity of Cry11Aa mutants.

Asunto(s)

Aedes/efectos de los fármacos , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Endotoxinas/metabolismo , Endotoxinas/toxicidad , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidad , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Multimerización de Proteína , Eliminación de Secuencia

RESUMEN

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix alpha-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Farmacorresistencia Microbiana/efectos de los fármacos , Endotoxinas/genética , Endotoxinas/metabolismo , Ingeniería Genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Insectos/efectos de los fármacos , Larva/efectos de los fármacos , Aedes/efectos de los fármacos , Animales , Anopheles/efectos de los fármacos , Toxinas de Bacillus thuringiensis , Bioensayo , Western Blotting , Antígenos CD13/metabolismo , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Insectos/metabolismo , Insecticidas/farmacología , Larva/metabolismo , Larva/microbiología , Membrana Dobles de Lípidos , Manduca/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Mutación/genética , Control Biológico de Vectores , Multimerización de Proteína , Triptófano