Antimicrobial peptides designed by computational analysis of proteomes.

Antimicrobial peptides (AMPs) are promising cationic and amphipathic molecules to fight antibiotic resistance. To search for novel AMPs, we applied a computational strategy to identify peptide sequences within the organisms' proteome, including in-house developed software and artificial intelligence tools. After analyzing 150.450 proteins from eight proteomes of bacteria, plants, a protist, and a nematode, nine peptides were selected and modified to increase their antimicrobial potential. The 18 resulting peptides were validated by bioassays with four pathogenic bacterial species, one yeast species, and two cancer cell-lines. Fourteen of the 18 tested peptides were antimicrobial, with minimum inhibitory concentrations (MICs) values under 10 µM against at least three bacterial species; seven were active against Candida albicans with MICs values under 10 µM; six had a therapeutic index above 20; two peptides were active against A549 cells, and eight were active against MCF-7 cells under 30 µM. This study's most active antimicrobial peptides damage the bacterial cell membrane, including grooves, dents, membrane wrinkling, cell destruction, and leakage of cytoplasmic material. The results confirm that the proposed approach, which uses bioinformatic tools and rational modifications, is highly efficient and allows the discovery, with high accuracy, of potent AMPs encrypted in proteins.

De novo design of short antimicrobial lipopeptides.

The increase in bacterial resistance to antibiotics available leads to the search for new compounds with antimicrobial potential, such as peptides and lipopeptides. In this work, eight short lipopeptides with the structural pattern Cn-X1 X2 X3-NH2 were de novo designed, synthesized by Fmoc solid phase and characterized by instrumental techniques. The results of the in vitro tests indicated that two of them, LIP 4 and LIP 12 display antibacterial activity against 4 pathogenic bacteria with minimum inhibitory concentrations (MIC) between 9.50 and 100 µM and between 8.50 and 10.0 µM, respectively; they did not displayed toxicity to human erythrocytes at concentrations between 3.13 and 50.0 µM. The antibacterial mechanism of action observed by scanning electron microscopy indicate that the cell membrane was the target, causing the formation of blisters and vesicles, with size ranging from 100 to 120 nm. The lipopeptide LIP 12, with higher activity, was stable to proteases of human blood serum.

Hexavalent chromium-reducing bacteria on biosolids from the San Fernando Wastewater Treatment Plant in Medellín (Colombia) / Bacterias aisladas de biosólidos de la PTAR San Fernando en Medellín-Colombia con capacidad para reducir cromo hexavalente

ABSTRACT During the most recent decades, advances have been made to reduce the environmental impact by anthropogenic activities that constantly release toxic components into the environment, generating instability and damage to the health of biological communities. Among the different pollutants, heavy metals are important by virtue of their properties, which hinder their degradation or transformation into other less toxic compounds. Chromium is one of the metals of greatest global interest due to its use in multiple industries. Conventional methods using chromed materials in their processes, not only throw considerable amounts of waste into the environment, but also give little account of the fraction of hexavalent chromium (Cr6+) present in certain ecosystems. Bioremediation has been proposed as an economically viable and environmentally sustainable alternative. This work aimed to evaluate the chromium reduction capacity by bacteria isolated from a biosolids matrix obtained at the San Fernando Wastewater Treatment Plant (WWTP), located in Medellín (Colombia). Biosolids samples were grown in a nutrient agar enriched with different concentrations of Cr6+. The strains presenting the greater tolerance to chromium were isolated to perform reduction tests by triplicate, monitoring the concentration of the metal over time. Seven different bacterial species were obtained, among which Staphylococcus saprophyticus, Ochrobactrum anthropic, and Bacillus cereus showed the greatest ability to reduce Cr6+ (29.0%, 61.1 and 100%, at 96 h) respectively.

RESUMEN En las últimas décadas se ha trabajado activamente para reducir el impacto ambiental generado por las actividades antrópicas que constantemente liberan componentes tóxicos al ambiente generando inestabilidad y daños en la salud de las comunidades biológicas. Entre los diferentes contaminantes, los metales pesados revisten importancia en virtud de sus propiedades, que dificultan su degradación o transformación en otros compuestos menos tóxicos. El cromo es uno de los metales de mayor interés a nivel global por su uso en múltiples industrias. Los métodos convencionales que utilizan materiales cromados en sus procesos, no sólo arrojan cantidades considerables de residuos al ambiente, sino que dan poca cuenta de la fracción de Cr6+ presente en determinados ecosistemas. La biorremediación se ha propuesto como una alternativa económicamente viable y ambientalmente sostenible. El propósito del presente trabajo fue evaluar la capacidad de reducción de cromo por bacterias, aisladas de una matriz de biosólidos de la Planta de tratamiento de aguas residuales (PTAR) San Fernando en la ciudad de Medellín-Colombia. Muestras de biosólidos se cultivaron en Agar Nutritivo enriquecido con diferentes concentraciones de Cr6+. Las cepas que presentaron mayor tolerancia al cromo fueron aisladas para realizar ensayos de reducción por triplicado, monitoreando la concentración del metal en el tiempo. Se obtuvieron siete especies bacterianas diferentes dentro de las cuales se destacaron Staphylococcus saprophyticus, Ochrobactrum anthropi y Bacillus cereus por la capacidad de reducir Cr6+ a 96 h con eficiencias de 29.0%, 61.1% y 100%, respectivamente.

Cytolytic activity of peptides derived from the Cry11Bb insecticidal toxin of B. thuringiensis subsp. medellin.

A few Bacillus thuringiensis Cry proteins, known as parasporins, have demonstrated cell proliferation inhibition of human cancer cells in vitro after protease activation. In this work, eight peptides derived from the Cry11Bb protoxin produced by B. thuringiensis subsp. medellin were selected and evaluated to investigate their membrane permeabilization and cytolytic activities, using red blood cells and cancer cell lines A549, MCF-7 and Caco-2, respectively. The most active peptides permeabilized red blood cells in a membrane potential-dependent manner. Half maximal inhibitory concentration in cancer cells was in the range 0.78-7.63 µM. At the same time, at peptides concentration of 25 µM, the hemolysis percentage varied in the range of 4.6-32.4%. The peptides BTM-P1 and BTM-P4 in D form had the lowest IC50 values on the MCF-7 cell line and they are considered as the most promising peptides among the evaluated. Fluorescence microscopy using AnnexinV-FLUOS staining indicates that the possible cause of MCF-7 cell death by peptide BTM-P1, is apoptosis. Real time PCR analysis showed an increased transcription of p53 in MCF-7 cells, thus confirming the probable pro-apoptotic effect of the peptide BTM-P1. In general, this study suggests that the cytolytic activity of the polycationic peptides derived from the Cry11Bb protoxin could be mediated by a pro-apoptotic mechanism that might include potential-dependent membrane permeabilization. Further studies might be accomplished to establish whether the peptides are cytolytic to other cancer cell lines and to solid tumors.

Design of antimicrobial and cytolytic peptides by computational analysis of bacterial, algal, and invertebrate proteomes.

The increase of antibiotic resistance in bacterial species has raised the need to search for novel antimicrobial molecules. Antimicrobial peptides are molecules that commonly display an amphipathic character. In this work, we developed a computational strategy to search for new peptide sequences within the proteome of any organism that includes in-house developed software and the use of artificial intelligence tools available online. Eleven peptides were selected after analyzing 63,343 proteins from the proteomes of bacteria, algae and invertebrates. Then, we validated the results by means of several assays which were carried out against five (5) pathogenic bacterial species and two (2) cancer cell lines. As a result, we found that ten of the peptides were antimicrobial, with minimum inhibitory concentration values between 4 and [Formula: see text]. Furthermore, two of the more active peptides were also cytotoxic to human red blood cells and cancer cells. In general, the antimicrobial peptides we discovered produced damage on the bacterial cell membrane that included membrane wrinkling, cell blebbing, and leakage of cytoplasmic material. Based on these results, we concluded that the computational approach proposed for finding sequences encrypted in proteins is appropriate for the discovery of selective and non-selective antimicrobial and anticancer peptides.

Aislamiento de una cepa genotipo 3 del virus de la hepatitis E obtenida de heces humanas / Cell culture isolation of hepatitis e virus genotype 3 strain obtained from human feces

ABSTRACT Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/µL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/µL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

RESUMEN El virus de la hepatitis E (HEV) se considera como una de las principales causas de hepatitis viral aguda en el mundo; cada año ocurren aproximadamente 20 millones de infecciones y 57 000 muertes. Debido a la ausencia de un modelo de cultivo celular consenso, se sabe poco sobre el ciclo replicativo del virus. La línea celular A549 se considera susceptible al genotipo 3 de HEV, pero tanto la cepa viral como las condiciones del cultivo celular podrían afectar el aislamiento viral in vitro. Por tanto nos propusimos aislar in vitro una cepa genotipo 3 del HEV. Para ello, se inocularon células A549 con una cepa HEV-3 identificada previamente por caracterización genética, y se incubó durante dos horas a 37 °C. Cinco días después de la infección, las células se pasaron (subcultivaron) por primera vez, y se realizaron pases seriados cada cuatro días en promedio, durante 41 días. En cada pase se evalúo la replicación del HEV mediante RT-qPCR. La reinfección de la línea celular con progenie viral derivada de monocapas de A549 infectadas se evaluó mediante inmunofluorescencia y RT-qPCR. Se detectó ARN viral en cada pase a partir de monocapas, y el pico máximo se alcanzó a los 26 días post infección (2 x 106 copias/µL). En el ensayo de reinfección, se detectó antígeno de cápside perinuclearmente y formando focos, y se detectaron 1 x 104 copias/µL de RNA viral a las 96 horas post infección. El HEV recuperado de lisado de monocapas fue infeccioso. Este aislado viral ofrece una herramienta importante para estudiar aspectos desconocidos de la infección por HEV.

Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling.

The Cry11 family belongs to a large group of δ-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an α-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes.

A case of incidental infection of Hepatitis E virus (HEV) genotype 1 in a domestic pig.

Hepatitis E virus (HEV) infection involving zoonotic genotypes is a public health problem in high-income and non-endemic developing countries. Herein we report the detection of a human genotype 1 (HEV-1) strain infecting a domestic pig, which is not considered a natural reservoir of this genotype. Viral load was quantified in stool by Real-Time qPCR and sequence analyses were performed. Infectivity of the HEV-1 strain was assesed by in vitro isolation in A549 cell line. Results suggest that certain epidemiological settings might favour accidental spillover infection and thus influence the host range restriction of HEV.

In silico design of polycationic antimicrobial peptides active against Pseudomonas aeruginosa and Staphylococcus aureus.

Antimicrobial peptides (AMPs) have the potential to become valuable antimicrobial drugs in the coming years, since they offer wide spectrum of action, rapid bactericidal activity, and low probability for resistance development in comparison with traditional antibiotics. The search and improvement of methodologies for discovering new AMPs to treat resistant bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are needed for further development of antimicrobial products. In this work, the software Peptide ID 1.0® was used to find new antimicrobial peptide candidates encrypted in proteins, considering the physicochemical parameters characteristics of AMPs such as positive net charge, hydrophobicity, and sequence length, among others. From the selected protein fragments, new AMPs were designed after conservative and semi-conservative modifications and amidation of the C-terminal region. In vitro studies of the antimicrobial activity of the newly designed peptides showed that two peptides, P3-B and P3-C, were active against P. aeruginosa Escherichia coli and A. baumannii with low minimum inhibitory concentrations. Peptide P3-C was also active against K. pneumoniae and S. aureus. Furthermore, bactericidal activity and information on the possible mechanisms of action are described according to the scanning electron microscopy studies.

DNA secondary structure formation by DNA shuffling of the conserved domains of the Cry protein of Bacillus thuringiensis.

BACKGROUND: The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy [Formula: see text] were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G. RESULTS: The results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA. CONCLUSION: These results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.

In-silico design and molecular docking evaluation of peptides derivatives from bacteriocins and porcine beta defensin-2 as inhibitors of Hepatitis E virus capsid protein.

Hepatitis E virus (HEV) is considered the main etiological agent that causes acute hepatitis. It is estimated that 20 million cases occur annually worldwide, reaching mortality rates of 28% in pregnant women. To date, available treatments and vaccines have not been entirely effective. In this study, six antiviral peptides derived from the sequences of porcine Beta-Defensin-2 and bacteriocins Nisin and Subtilosin were generate using in silico tools in order to propose new antiviral agents. Through the use of molecular docking, interactions between the HEV capsid protein and the six new antiviral peptide candidates were evaluated. A peptide of 15 residues derived from Subtilosin showed the best docking energy (-7.0 kcal/mol) with the capsid protein. This is the first report to our knowledge involving a non-well study viral protein interacting with peptides susceptibles to being synthesized, and that could be subsequently evaluated in vitro; moreover, this study provide novel information on the nature of the dimerization pocket of the HEV capsid protein, and could help to understand the first steps in the viral replication cycle, needed for the virus entry to the host cell.

InverPep: A database of invertebrate antimicrobial peptides.

OBJECTIVES: The aim of this work was to construct InverPep, a database specialised in experimentally validated antimicrobial peptides (AMPs) from invertebrates. METHODS: AMP data contained in InverPep were manually curated from other databases and the scientific literature. MySQL was integrated with the development platform Laravel; this framework allows to integrate programming in PHP with HTML and was used to design the InverPep web page's interface. InverPep contains 18 separated fields, including InverPep code, phylum and species source, peptide name, sequence, peptide length, secondary structure, molar mass, charge, isoelectric point, hydrophobicity, Boman index, aliphatic index and percentage of hydrophobic amino acids. CALCAMPI, an algorithm to calculate the physicochemical properties of multiple peptides simultaneously, was programmed in PERL language. RESULTS: To date, InverPep contains 702 experimentally validated AMPs from invertebrate species. All of the peptides contain information associated with their source, physicochemical properties, secondary structure, biological activity and links to external literature. Most AMPs in InverPep have a length between 10 and 50 amino acids, a positive charge, a Boman index between 0 and 2 kcal/mol, and 30-50% hydrophobic amino acids. InverPep includes 33 AMPs not reported in other databases. Besides, CALCAMPI and statistical analysis of InverPep data is presented. The InverPep database is available in English and Spanish. CONCLUSIONS: InverPep is a useful database to study invertebrate AMPs and its information could be used for the design of new peptides. The user-friendly interface of InverPep and its information can be freely accessed via a web-based browser at http://ciencias.medellin.unal.edu.co/gruposdeinvestigacion/prospeccionydisenobiomoleculas/InverPep/public/home_en.

Identificación, clonación y actividad lactonasa de la proteína recombinante de N-ácil homoserina lactonasa (AiiA) de Bacillus thuringiensis cepa 147-115-16 / Identification, cloning and lactonase activity of recombinant protein of N-acyl homoserine lactonase (AiiA) from Bacillus thuringiensis 147-115-16 strain

The quorum-quenching N-acyl homoserine lactonases are a family of bacterial metalloenzymes that participate in degradation of N-acyl homoserine lactones (AHLs), disrupting the quorum sensing system of gram negative bacterial species. From a collection of Bacillus thuringiensis strains isolated in Colombia from plants and exhibiting toxic activity against lepidopteran insects, 310 bacterial isolates were tested to determine lactonase activity by using biosensor systems in presence of synthetic N-hexanoyl-L-homoserine lactone (C6-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). From them, 251 strains showed degrading activity to both C6-HSL and C8-HSL, 57% exhibited degrading activity to C6-HSL and 43% to C8-HSL. One B. thuringiensis strain, denoted as 147-115-16, that exhibit high degrading activity to C6-HSL and C8-HSL, was able to attenuate soft rot symptoms in infected potato slices with Pectobacterium carotovorum. This strain contains an homologous of the aiiA gene that was cloned, sequenced and expressed in Esherichia coli DE3. The recombinant protein AiiA147-11516 display activity to C6-HSL, C8-HSL, N-(β-ketocaproyl) (3-O-C6-HSL) and N-3-oxo-dodecanoyl (3-O-C12-HSL). The recombinant strain in the presence of P. caratovorum cultures was able to attenuate the infection, suggesting that it interferes either on the accumulation or response to the AHLs signals. Acording to this data and based on previous report from recombinant AiiA147-11516, this enzyme exhibit activity to wide range of catalytic substrates suggesting its industrial application in the disease control programs by plants transformation.

Las N-acíl homoserina lactonasas son una familia de metaloenzimas bacterianas que participan en la degradación de N-acil homoserina lactonas (AHLs) interrumpiendo el sistema de detección de quórum sensing de bacterias Gram negativas. A partir de una colección de cepas de Bacillus thuringiensis aisladas del filoplano de plantas colombianas que presentaron actividad tóxina contra insectos lepidópteros, 310 fueron probadas para determinar actividad lactonasa mediante el uso de sistemas de biosensores en presencia de N-hexanoilo-L-homoserina lactona (C6-HSL) y la N-octanoilo-L-homoserina lactona (C8-HSL) sintéticas. De estas cepas, el 251 mostraron actividad para ambas lactonas y de estas, el 57% mostró actividad a C6-HSL y el 43% a C8-HSL. Una cepa de B. thuringiensis- denominada 147-115-16- que mostró alta actividad para C6-HSL y C8-HSL, fue capaz de atenuar los síntomas de la pudrición blanda en rodajas de papa infectadas con Pectobacterium carotovorum. Esta cepa contiene un gen homólogo a aiiA, el cual este fue clonado, secuenciado y expresado en Escherichia coli DE3. La proteína recombinante AiiA147-11516 exhibe actividad para C6-HSL y C8-HSL, así como para N-(β-cetocaproil) (3-O-C6-HSL) y N-3-oxo-dodecanoil (3-O-C12-HSL). La cepa recombinante en presencia de P. caratovorum fue capaz de atenuar la infección, sugiriendo que interfiere con la acumulación o respuesta de las señales AHLs. Según estos datos y basados en el reporte previo sobre la actividad hidrolítica de la proteína recombinante AiiA147-11516, esta enzima posee un actividad contra un amplio número de sustratos lo cual sugiere su aplicación en la industria en el control de enfermedades, mediante la transformación de plantas.

Enzymatic hydrolysis of molecules associated with bacterial quorum sensing using an acyl homoserine lactonase from a novel Bacillus thuringiensis strain.

N-acyl homoserine lactones are key components of quorum sensing, the bacterial communication system. This communication mechanism regulates the expression of genes, including those involved in virulence and biofilm formation. This system can be interrupted by the action of enzymes that hydrolyze the signaling molecules. In this work, we studied the enzymatic properties of a recombinant AHL-lactonase from Bacillus thuringiensis strain 147-11516, using substrates with acyl chains of different length (C4-HSL, C6-HSL, C7-HSL, C8-HSL and C10-HSL), we also investigated the effect of pH (5.0­9.0), temperature (20­70 °C), concentration of monovalent, divalent and trivalent metals ions (0.2 and 2.0 mM) and EDTA. The results showed that the recombinant AHL-lactonase had biological activity in alkaline pH conditions (8.0) and high temperature (47 % of hydrolyzed substrate at 60 °C). The recombinant AHL-lactonase has activity on substrates with different acyl chain length. However, the activity of the recombinant enzyme was decreased in the two concentrations of all metal ions evaluated but was not inhibited by EDTA. The affinity of the enzyme for all substrates tested and its performance, in the evaluated conditions, suggest that the AHL-lactonase from B. thuringiensis strain 147-11516 could be used as a strategy for disruption of the Gram-negative bacteria communication system under normal and challenging conditions.

Electrical hypothesis of toxicity of the Cry toxins for mosquito larvae.

Many electrical properties of insect larval guts have been studied, but their importance for toxicity of the Cry-type toxins has never been reported in the literature. In the present work, we observed potential-dependent permeabilization of plasma membrane by several polycationic peptides derived from the Cry11Bb protoxin. The peptide BTM-P1d, all D-type amino acid analogue of the earlier reported peptide BTM-P1, demonstrated high membrane-permeabilizing activity in experiments with isolated rat liver mitochondria, RBC (red blood cells) and mitochondria in homogenates of Aedes aegypti larval guts. Two larger peptides, BTM-P2 and BTM-P3, as well as the Cry11Bb protoxin treated with the protease extract of mosquito larval guts showed similar effects. Only protease-resistant BTM-P1d, in comparison with other peptides, displayed A. aegypti larval toxicity. Taking into account the potential-dependent mechanism of membrane permeabilization by studied fragments of the Cry11Bb protoxin and the literature data related to the distribution of membrane and transepithelial potentials in the A. aegypti larval midgut, we suggest an electrical hypothesis of toxicity of the Cry toxins for mosquito larvae. According to this hypothesis, the electrical field distribution is one of the factors determining the midgut region most susceptible for insertion of activated toxins into the plasma membrane to form pores. In addition, potential-dependent penetration of short active toxin fragments into the epithelial cells could induce permeabilization of mitochondria and subsequent apoptosis or necrosis.

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance.

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

Genetic and metabolic biodiversity of Trichoderma from Colombia and adjacent neotropic regions.

The genus Trichoderma has been studied for production of enzymes and other metabolites, as well as for exploitation as effective biological control agents. The biodiversity of Trichoderma has seen relatively limited study over much of the neotropical region. In the current study we assess the biodiversity of 183 isolates from Mexico, Guatemala, Panama, Ecuador, Peru, Brazil and Colombia, using morphological, metabolic and genetic approaches. A comparatively high diversity of species was found, comprising 29 taxa: Trichoderma asperellum (60 isolates), Trichoderma atroviride (3), Trichoderma brevicompactum (5), Trichoderma crassum (3), Trichoderma erinaceum (3), Trichoderma gamsii (2), Trichoderma hamatum (2), Trichoderma harzianum (49), Trichoderma koningiopsis (6), Trichoderma longibrachiatum (3), Trichoderma ovalisporum (1), Trichoderma pubescens (2), Trichoderma rossicum (4), Trichoderma spirale (1), Trichoderma tomentosum (3), Trichoderma virens (8), Trichoderma viridescens (7) and Hypocrea jecorina (3) (anamorph: Trichoderma reesei), along with 11 currently undescribed species. T. asperellum was the prevalent species and was represented by two distinct genotypes with different metabolic profiles and habitat preferences. The second predominant species, T. harzianum, was represented by three distinct genotypes. The addition of 11 currently undescribed species is evidence of the considerable unresolved biodiversity of Trichoderma in neotropical regions. Sequencing of the internal transcribed spacer regions (ITS) of the ribosomal repeat could not differentiate some species, and taken alone gave several misidentifications in part due to the presence of nonorthologous copies of the ITS in some isolates.

Potential-dependent permeabilization of plasma membrane by the peptide BTM-P1 derived from the Cry11Bb1 protoxin.

The peptide BTM-P1, which is derived from the amino acid sequence of the Cry11Bb1 protoxin, is able to permeabilize mitochondrial membranes and reveals antimicrobial activity. In this work we demonstrated that the permeabilizing activity of BTM-P1 for the plasma membrane of rat red blood cells increased in a dose-dependent manner for the concentration range of 1-4 microg/ml. Using osmotic protectants, the radius of pores formed at 4 microg/ml BTM-P1 was determined as 0.8 nm for 5 min hemolysis data, 0.7 nm for 5 min decrease in light dispersion of the cell suspension and 0.5 nm for the light dispersion slope measurements. The permeabilizing activity of 1 microg/ml peptide was increased by valinomycin-induced plasma membrane potential, especially under moderately hypotonic conditions. These results might explain the antimicrobial activity of BTM-P1 and support the hypothesis of potential-dependent and pro-apoptotic character of toxicity of naturally proteolysed Cry11Bb1 protoxin for epithelial cells of mosquito larvae midgut.

B. thuringiensis is a poor colonist of leaf surfaces.

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10(6) cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10(7) cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.