Biomedical subjects

Carolina Rausell

Publications and source records attributed to Carolina Rausell.

6 recordsLinked to original sources

PubMed2006

Structural changes of the Cry1Ac oligomeric pre-pore from bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion.

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.

Acetylglucosamine↗

PubMed2006

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins.

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.

Animals↗

PubMed2004

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.

Anilino Naphthalenesulfonates↗

PubMed2004

Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate.

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.

Acrylamide↗

PubMed2003

The genome sequence of Blochmannia floridanus: comparative analysis of reduced genomes.

Bacterial symbioses are widespread among insects, probably being one of the key factors of their evolutionary success. We present the complete genome sequence of Blochmannia floridanus, the primary endosymbiont of carpenter ants. Although these ants feed on a complex diet, this symbiosis very likely has a nutritional basis: Blochmannia is able to supply nitrogen and sulfur compounds to the host while it takes advantage of the host metabolic machinery. Remarkably, these bacteria lack all known genes involved in replication initiation (dnaA, priA, and recA). The phylogenetic analysis of a set of conserved protein-coding genes shows that Bl. floridanus is phylogenetically related to Buchnera aphidicola and Wigglesworthia glossinidia, the other endosymbiotic bacteria whose complete genomes have been sequenced so far. Comparative analysis of the five known genomes from insect endosymbiotic bacteria reveals they share only 313 genes, a number that may be close to the minimum gene set necessary to sustain endosymbiotic life.

Animals↗

PubMed2003

Reductive genome evolution in Buchnera aphidicola.

We have sequenced the genome of the intracellular symbiont Buchnera aphidicola from the aphid Baizongia pistacea. This strain diverged 80-150 million years ago from the common ancestor of two previously sequenced Buchnera strains. Here, a field-collected, nonclonal sample of insects was used as source material for laboratory procedures. As a consequence, the genome assembly unveiled intrapopulational variation, consisting of approximately 1,200 polymorphic sites. Comparison of the 618-kb (kbp) genome with the two other Buchnera genomes revealed a nearly perfect gene-order conservation, indicating that the onset of genomic stasis coincided closely with establishment of the symbiosis with aphids, approximately 200 million years ago. Extensive genome reduction also predates the synchronous diversification of Buchnera and its host; but, at a slower rate, gene loss continues among the extant lineages. A computational study of protein folding predicts that proteins in Buchnera, as well as proteins of other intracellular bacteria, are generally characterized by smaller folding efficiency compared with proteins of free living bacteria. These and other degenerative genomic features are discussed in light of compensatory processes and theoretical predictions on the long-term evolutionary fate of symbionts like Buchnera.

Base Sequence↗