Role of tryptophan residues in toxicity of Cry1Ab toxin from Bacillus thuringiensis

Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.     

Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Sporothrix schenckii Complex in Medellin,Colombia

Background

Sporotrichosis is a subcutaneous mycosis that affects humans and other animals. Infection prevails in tropical and subtropical countries. Until a few years ago, it was considered that two varieties of Sporothrix schenckii caused this mycosis, but by applying molecular taxonomic markers, it has been demonstrated that there are several cryptic species within S. schenckii complex which varies in susceptibility, virulence, and geographic distribution.

Objective

This study aimed to identify the clinical isolates of Sporothrix spp. from patients with sporotrichosis in Medellin, Colombia, using two markers and to evaluate the in vitro susceptibility to itraconazole.

Methods

Thirty-four clinical isolates of Sporothrix spp. from Colombia, three from Mexico, and one from Guatemala were identified through sequencing of the noncoding region ITS-1?+?5.8SDNAr?+?ITS-2 and of the fragment containing exons 3 and 4 of the β-tubulin gene. Clinical isolate sequences were compared with GenBank reference sequences using the BLASTN tool, and then, phylogenetic analysis was performed. Besides, the in vitro susceptibility to itraconazole was evaluated by determining the minimum inhibitory concentrations according to the CLSI M38-A2 method.

Results

Clinical isolates were identified by morphology as Sporothrix spp. Using the molecular markers, ITS and β-tubulin, isolates were identified as S. schenckii sensu stricto (25) and Sporothrix globosa (13). Susceptibility to itraconazole was variable among clinical isolates.

Conclusion

This is the first scientific publication that identifies species that cause sporotrichosis in Colombia, along with the antifungal susceptibility to itraconazole.

     

Effects of disulfide bridges in domain I of Bacillus thuringiensis Cry1Aa delta-endotoxin on ion-channel formation in biological membranes

The delta-endotoxin family of toxic proteins represents the major component of the insecticidal capability of the bacterium Bacillus thuringiensis. Domain I of the toxins, which is largely alpha-helical, has been proposed to unfold at protein entry into the membrane of a target insect, following models known as the penknife and umbrella models. We extended the analysis of a previous work in which four disulfide bridges were constructed in domain I of the Cry1Aa delta-endotoxin that putatively prevented unfolding during membrane partitioning. Using bioassays and voltage clamping of whole insect midgut instead of artificial lipid bilayers, it was found that, while toxicity and inhibition of the short-circuit current were reduced, only one of the disulfide bridges eliminated the activity of the toxins in the insect midgut membrane, and in that case, the loss of toxicity was due to the single amino acid substitution, R99C. It is proposed that at least alpha helices 4, 5, 6, and 7 and domain II partition in the midgut membranes of target insects, in support of an insertion model in which the whole protein translocates into the midgut membrane.     

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.     

Environmental assessment of energy-driven biorefineries: the case of the coffee cut-stems (CCS) in Colombia

The International Journal of Life Cycle Assessment - Coffee cut-stems (CCS) are typically left in the field after coffee harvesting as fertilizer or used partially for cooking and drying of coffee...     

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Panbiogeographical analysis of the genus Bomarea (Alstroemeriaceae)

Aim  A panbiogeographical analysis of the genus Bomarea was undertaken in order to determine generalized tracks and biogeographical nodes, and to evaluate the current distribution of the genus based on the available tectonic information and the biogeographical regionalization of Latin America.
Location  The Neotropical region from northern Mexico to northern Argentina, and the Nearctic and Andean regions.
Method  A total of 2205 records of 101 species were analysed, representing 95% of the species assigned to Bomarea . Localities were represented on maps and their individual tracks were drawn. Based on their comparison, generalized tracks were detected and mapped. Nodes were identified in the areas where different generalized tracks were superimposed.
Results  Five generalized tracks were recovered. One is located in the Eastern Central America and Western Panamanian Isthmus provinces (Caribbean subregion, Neotropical region), which was supported by three species of Central American distribution. The four remaining generalized tracks were located in South America, in the North Andean Paramo, Cauca and Puna biogeographical provinces. These tracks were supported by species of Bomarea with an Andean distribution. Biogeographical nodes were established in the Central Andean region of Colombia, central Ecuador and central Peru.
Main conclusions  The nodes obtained for Bomarea support a hybrid origin for the Andean region, which presents diverse components from both northern and southern South America. Likewise, the track recovered between Colombia and Ecuador includes Andean and Neotropical areas, providing further support for this hypothesis. The nodes obtained are coherent with vicariant elements evident for Bomarea. Species of three clades proposed for Bomarea are distributed in specific generalized tracks.