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Jenny Dussán - One of the best experts on this subject based on the ideXlab platform.
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effective gold biosorption by electrospun and electrosprayed bio composites with immobilized Lysinibacillus sphaericus cbam5
Nanomaterials, 2020Co-Authors: Carolina Paezvelez, J L Castromayorga, Jenny DussánAbstract:Electro-hydrodynamic processing, comprising electrospinning and electrospraying techniques, is a novel technology used in the production of nano- and sub-micro-scale materials with specific properties suitable for environmental remediation processes. Polycaprolactone (PCL) micro-fibrous mats and alginate microcapsules were produced using electrospinning and electrospraying techniques respectively, and Lysinibacillus sphaericus CBAM5, a bacterium capable of metal removal by adsorption and accumulation inside the cell, was immobilized in these matrices. The polymeric structure was able to protect and maintain cell viability and the bio-composite materials were used to capture gold from synthetic water samples. The micro-fibrous membranes with immobilized bacteria were able to remove 93% of the gold after 120 h of inclusion in the aqueous medium. Using a filtration system, an efficiency of 64% was obtained for the removal of the precious metal after 10 cycles of filtration (2 h of exposure to the gold solution). In contrast, the microencapsulated L. sphaericus CBAM5 captured 64% of the gold after 4 h of the assay. Thus, both micro-structured matrices were suitable for the immobilization and protection of L. sphaericus CBAM5 and they showed high efficiencies of gold biosorption. Hence, these bio-composite materials could be used to concentrate gold from industrial wastewaters.
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synergistic effect of Lysinibacillus sphaericus and glyphosate on temephos resistant larvae of aedes aegypti
Parasites & Vectors, 2020Co-Authors: Laura Bernal, Jenny DussánAbstract:BACKGROUND: Glyphosate-based herbicides are one of the most commonly used compounds to control perennial weeds around the world. This compound is very persistent in the environment and tends to filter into aquatic ecosystems, affecting non-target species such as mosquito larvae. Aedes aegypti mosquitoes are vectors of multiple arboviruses such as dengue and Zika. Glyphosate can be degraded into non-harmful environmental compounds by Lysinibacillus sphaericus, a spore forming bacterium which can also kill Ae. aegypti larvae. In this study, we assessed the effect of glyphosate concentrations, typically used in Colombia, on the entomopathogenic activity of L. sphaericus against Ae. aegypti larvae. METHODS: Bioassays and toxicity curves were performed to compare the larval mortality between different treatments with and without bacteria and glyphosate (Roundup 747®). Larvae were exposed to both bacteria and glyphosate by adding the compound on chloride-free water. Comparisons were made using both probit regression and ANOVA analysis. RESULTS: ANOVA showed a significant difference in larval mortality when adding glyphosate and L. sphaericus at the same time. Thus, a positive synergic effect on larval mortality was found when L. sphaericus and glyphosate were mixed. According to probit analysis, median lethal dose (LD50) for bacterial mixture was of 10⁶.²³ UFC/ml and for glyphosate was 2.34 g/l. CONCLUSIONS: A positive synergic effect on the mortality of larval Ae. aegypti when exposed to L. sphaericus mixture and glyphosate was found. Molecular studies focusing on the toxin production of L. sphaericus are required to understand more about this synergistic effect.
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glyphosate bioremediation through the sarcosine oxidase pathway mediated by Lysinibacillus sphaericus in soils cultivated with potatoes
Agriculture, 2019Co-Authors: Mario Perez Rodriguez, Carol Melo, Elizabeth Jimenez, Jenny DussánAbstract:Glyphosate-based herbicides (GBH) use has increased drastically over the last decade. This is true especially for potato crops due to their fast harvest cycle and high market demand. In 2015, the World Health Organization (WHO) classified glyphosate and its breakdown product amidomethylphosphonic acid (AMPA) as probably carcinogenic to humans, and it has been reported that these compounds disrupt the ecological and nutritional equilibrium of soils. However, microorganisms with the sarcosine oxidase gene, such as Lysinibacillus sphaericus, can degrade glyphosate through the Carbon-Phosphorus (C-P) pathway without leading to AMPA production. The aim of this study was to evaluate the addition of the plant growth-promoting bacteria (PGPB) L. sphaericus as a bioremediation agent in a potato crop sprayed with a GBH, in conjunction with the nitrogen fixation activity mediated by the bacteria. To that end, a GBH solution was used to treat a potato field, and different treatments (glyphosate (G), bacteria (B), bacteria+glyphosate (BG), and negative control (C)) were evaluated by measuring the glyphosate, AMPA, nitrates, and ammonium concentrations. BG treatment showed a 79% reduction of glyphosate concentration in soil, leading to minimal AMPA production, compared to the 23% reduction observed after G treatment. Furthermore, the ammonium concentrations were significantly higher in samples treated with BG and in C samples (p < 0.005). Therefore, we propose the addition of L. sphaericus as a good bioremediation strategy for soils sprayed with GBH.
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the golden activity of Lysinibacillus sphaericus new insights on gold accumulation and possible nanoparticles biosynthesis
Materials, 2018Co-Authors: Maria Camila Bustos, Humberto Ibarra, Jenny DussánAbstract:Power struggles surrounding the increasing economic development of gold mining give rise to severe environmental and social problems. Two new strains of Lysinibacillus sphaericus were isolated from an area of active alluvial gold mining exploitation at El Bagre, Antioquia. The absorption capacity of these strains and some of the L. sphaericus Microbiological Research Center (CIMIC) collection (CBAM5, OT4b.31, III(3)7) were evaluated by spectrophotometry according to a calibration gold curve of HAuCl4− with concentrations between 0 µg/mL and 100 µg/mL. Bioassays with living biomass were carried out with an initial gold concentration of 60 µg/mL. Their sorption capacity was evident, reaching percentages of gold removal between 25% and 85% in the first 2 h and 75% to 95% after 48 h. Biosynthesis of possible gold nanoparticles (AuNPs) in assays with living biomass was also observed. Metal sorption was evaluated using scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS) analysis. The sorption and fabrication capacity exhibited by the evaluated strains of L. sphaericus converts this microorganism into a potential alternative for biomining processes, especially those related to gold extraction.
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Molecular assessment of glyphosate-degradation pathway via sarcosine intermediate in Lysinibacillus sphaericus
Environmental Science and Pollution Research, 2018Co-Authors: Laura E. González-valenzuela, Jenny DussánAbstract:The widespread use of glyphosate has permeated not only small- and large-scale agriculture, but also the fight against drug trafficking and illicit crops. Health, alimentary security, and the rights of peasant and indigenous communities have been compromised in countries with intensive use of glyphosate-based herbicides. In 2015, the International Agency for Research on Cancer classified this substance as probably carcinogenic to humans, leading to the suspension of aerial glyphosate spraying the same year in countries like Colombia, where glyphosate has been extensively used in illicit crop eradication. Notwithstanding, according to a study of the U.S. Geological Survey, traces of glyphosate and its main degradation product, AMPA, remain in soil year after year. This underscores the urgency and importance of assessing new technologies to degrade glyphosate present in soils and waterbodies without leaving persistent byproducts. The aim of this study was to evaluate Lysinibacillus sphaericus ’ glyphosate uptake as a carbon and phosphorous source by a sarcosine-mediated metabolic pathway that releases glycine as final degradation product. To accomplish this, molecular and analytic evidence were collected in vitro from sarcosine oxidase activity, a key enzyme of a degradation pathway which releases byproducts that are easy to incorporate into natural biosynthesis routes.
Panadda Boonserm - One of the best experts on this subject based on the ideXlab platform.
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in vitro analysis of the anticancer activity of Lysinibacillus sphaericus binary toxin in human cancer cell lines
3 Biotech, 2020Co-Authors: Wasutorn Chankamngoen, Boonhiang Promdonkoy, Tavan Janvilisri, Panadda BoonsermAbstract:Binary or Bin toxin produced by Lysinibacillus sphaericus is composed of BinA (42 kDa) and BinB (51 kDa) subunits. These work together to exert maximal toxicity against mosquito larvae via pore formation and induction of apoptosis. The C-terminal domains in both subunits are homologous to those of aerolysin-type β pore-forming toxins, including parasporin-2 (PS2). The latter is one of the Bacillus thuringiensis toxins that exhibits specific cytotoxicity against human cancer cells. The present study investigates the possible anticancer activity of Bin toxin using PS2 as a control. We demonstrate that treatment with a high concentration of trypsin-activated Bin inhibits cell proliferation in human cancer cells A549, Caco-2, HepG2, HK-1 and KKU-M055. In the most susceptible cells, HK-1, Bin toxin exposure led to morphological alterations, decreased migration, decreased adhesion activity and apoptosis induction. Although these effects necessitated high concentrations, they suggest that Bin toxin may be optimized as a novel potential cancer-therapeutic agent.
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an aromatic cluster in Lysinibacillus sphaericus binb involved in toxicity and proper in membrane folding
Archives of Biochemistry and Biophysics, 2018Co-Authors: Sivadatch Chooduang, Wahyu Surya, Jaume Torres, Panadda BoonsermAbstract:Abstract The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. Based on structural alignments with other toxins, an aromatic cluster in the C-terminal domain of BinB (termed here BC) has been proposed to be important for toxicity. We tested this experimentally using BinB mutants bearing single mutations in this aromatic cluster. Consistent with the hypothesis, two of these mutations, F311A and F315A, were not toxic to Culex quinquefasciatus larvae and were unable to permeabilize liposomes or elicit ion channel activity, in contrast to wild-type BinB. Despite these effects, none of these mutations altered significantly the interaction between the activated forms of the two subunits in solution. These results indicate that these aromatic residues on the C-terminal domain of BinB are critical for toxin insertion in membranes. The latter can be by direct contact of these residues with the membrane surface, or by facilitating the formation a membrane-inserting oligomer.
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molecular analysis of culex quinquefasciatus larvae responses to Lysinibacillus sphaericus bin toxin
PLOS ONE, 2017Co-Authors: Chontida Tangsongcharoen, Boonhiang Promdonkoy, Natapong Jupatanakul, George Dimopoulos, Panadda BoonsermAbstract:Lysinibacillus sphaericus produces the mosquito larvicidal binary toxin consisting of BinA and BinB, which are both required for toxicity against Culex and Anopheles larvae. The molecular mechanisms behind Bin toxin-induced damage remain unexplored. We used whole-genome microarray-based transcriptome analysis to better understand how Culex larvae respond to Bin toxin treatment at the molecular level. Our analyses of Culex quinquefasciatus larvae transcriptome changes at 6, 12, and 18 h after Bin toxin treatment revealed a wide range of transcript signatures, including genes linked to the cytoskeleton, metabolism, immunity, and cellular stress, with a greater number of down-regulated genes than up-regulated genes. Bin toxin appears to mainly repress the expression of genes involved in metabolism, the mitochondrial electron transport chain, and the protein transporter of the outer/inner mitochondrial membrane. The induced genes encode proteins linked to mitochondrial-mediated apoptosis and cellular detoxification including autophagic processes and lysosomal compartments. This study is, to our knowledge, the first microarray analysis of Bin toxin-induced transcriptional responses in Culex larvae, providing a basis for an in-depth understanding of the molecular nature of Bin toxin-induced damage.
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binary toxin subunits of Lysinibacillus sphaericus are monomeric and form heterodimers after in vitro activation
PLOS ONE, 2016Co-Authors: Wahyu Surya, Sivadatch Chooduang, Jaume Torres, Yeu Khai Choong, Panadda BoonsermAbstract:The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.
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interaction of Lysinibacillus sphaericus binary toxin with mosquito larval gut cells binding and internalization
Journal of Invertebrate Pathology, 2015Co-Authors: Hataikarn Lekakarn, Boonhiang Promdonkoy, Panadda BoonsermAbstract:The binary toxin produced by Lysinibacillus sphaericus is composed of BinA and BinB subunits. Together, but not separately, the two subunits are highly toxic to Culex quinquefasciatus larvae, but show no toxicity to Aedes aegypti. The molecular mechanism underlying intoxication has not been clearly elucidated. The present study compares the binding and the internalization of binary toxin into the midgut epithelial cells of susceptible C. quinquefasciatus mosquito larvae with those of Bin-refractory A. aegypti. The guts from larvae fed with fluorescently labeled toxin were dissected and analyzed using a confocal laser scanning microscope. When fed with a mixture of both components, co-localization of BinA and BinB was detected both on the cell surface and in the cytoplasm of Culex larval gut cells. However, administration of BinA alone resulted in localization only on the cell membrane, whereas BinB alone was detected both on the cell membrane and inside the cytoplasm. In contrast, when a mixture of both components, or each individual component, was fed to Aedes larvae, BinA and BinB were unable to reach the cytoplasm and were localized only on the cell membrane. These results are consistent with the suggestion that the internalization of BinA is essential for toxicity, and that BinB is required for this internalization into susceptible larval gut cells.
Zhiming Yuan - One of the best experts on this subject based on the ideXlab platform.
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vb_lspm 01 a novel myovirus displaying pseudolysogeny in Lysinibacillus sphaericus c3 41
Applied Microbiology and Biotechnology, 2018Co-Authors: Peiling Geng, Xiaofu Wan, Jiao Cheng, Zhiming YuanAbstract:Lysinibacillus sphaericus has great application potential not only in the biocontrol of mosquitoes but also in the bioremediation of toxic metals. Phages contribute to the genetic diversity and niche adaptation of bacteria, playing essential roles in their life cycle, but may also cause economic damage for industrially important bacteria through phage contamination during fermentation. In this study, the L. sphaericus phage vB_LspM-01, which belongs to the Myoviridae family, was isolated and characterized. Results showed that vB_LspM-01 could specifically infect most tested L. sphaericus isolates but was not active against isolates belonging to other species. Furthermore, phage-born endolysin exhibited a broader antimicrobial spectrum than the host range of the phage. The vB_LspM-01 genome had no obvious similarity with that of its host, and ca. 22.6% of putative ORFs could not get a match with the public databases. Phylogenic analysis based on the putative terminase large subunit showed high similarity with the phages identified with pac-type headful packaging. The vB_LspM-01 encoding genes were only detected in a tiny percentage of L. sphaericus C3-41 individual cells in the wild population, whereas they showed much higher frequency in the resistant population grown within the plaques; however, the phage genes could not be stably inherited during host cell division. Additionally, the vB_LspM-01 encoding genes were only detected in the host population during the logarithmic growth phase. The mitomycin C induction helped the propagation and lysogeny-lysis switch of vB_LspM-01. The study demonstrated that vB_LspM-01 can be present in a pseudolysogenic state in L. sphaericus C3-41 populations.
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regulator degu is required for multicellular behavior in Lysinibacillus sphaericus
Research in Microbiology, 2018Co-Authors: Yimin Hu, Zhiming Yuan, Shen Tian-yi, Yong Ge, Xiaomin HuAbstract:Abstract DegS and DegU make up a two component system belonging to a class of signal transduction systems that play important roles in a broad range of bacterial responses to the environment. However, little study has been done to explore the physiological functions of DegS-DegU in mosquitocidal Lysinibacillus sphaericus . In this study, it was found that deletion of degU or degS-degU inhibited the swarming motility, biofilm formation, sporulation and binary toxin production through regulating the related genes, and phosphorylation was necessary for the functions of DegU. Based on the findings, a regulation network mediated by DegU was delineated. Both DegU-pi and Spo0A-pi positively regulates genes which are linked with the transition from stage Ⅱ to the end of the sporulation process and also influences the production of binary toxins via regulation on sigE . Both DegU-pi and Spo0A-pi negatively regulate abrB/sinR and influence the biofilm formation. DegU-pi can positively regulate the motility via the regulation on sigD. Whether the regulations are directly or indirectly need to be explored. Moreover, Spo0A-pi may indirectly regulate the swarming motility through negatively regulating DegU. It was concluded that DegU is a global transcriptional regulator on cell swarming motility, biofilm formation, sporulation and virulence in L. sphaericus .
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differential expression of duplicated binary toxin genes bina binb in Lysinibacillus sphaericus c3 41
Letters in Applied Microbiology, 2017Co-Authors: Xingchen Xiang, Zhiming YuanAbstract:Due to the self-produced binary toxins BinA/BinB, some Lysinibacillus sphaericus isolates exhibit high toxicity against mosquito larvae and are utilized in mosquito control programs. Previous whole genome sequencing revealed the presence of a large plasmid pBsph in L. sphaericus C3-41, which contains a 30.5-kb duplication of the genome including the binary toxin genes binA/binB. This was confirmed by Southern blot and qPCR experiments in this study. Mutants of L. sphaericus C3-41, bearing disruptions of the chromosomal-bin (C3-41ΔCab) or the plasmid-bin (C3-41ΔPab) and a plasmid cured strain G725, were generated. It was observed that the three mutants, especially that of C3-41ΔPab and G725, had lower mRNA level from the early to middle sporulation phase and expressed less binary toxin during the whole sporulation phase. The mosquitocidal activity of the wild strain against 4th instar Culex quinquefasciatus larvae determined by LC50 displayed about 4, 15 and 35 times higher than that of C3-41ΔCab, C3-41ΔPab and G725, respectively. These results suggest that both chromosomal- and plasmid-borne bin genes, especially the latter, contribute to the full toxicity of the wild strain. The study provides an important clue for the evolution and application of mosquitocidal L. sphaericus. This article is protected by copyright. All rights reserved.
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collagen like glycoprotein bcls is involved in the formation of filamentous structures of the Lysinibacillus sphaericus exosporium
Applied and Environmental Microbiology, 2014Co-Authors: Ni Zhao, Tingyu Shi, Zhiming YuanAbstract:Lysinibacillus sphaericus produces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. Unlike Bacillus cereus group strains, the exosporium of L. sphaericus is usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) from L. sphaericus C3-41 was characterized. Thin-section electron microscopy revealed that deletion of bclS resulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores. In vivo visualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that the bclS mutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in the bclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature.
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ccpa mediated enhancement of sugar and amino acid metabolism in Lysinibacillus sphaericus by nmr based metabolomics
Journal of Proteome Research, 2012Co-Authors: Chongyang Huang, Dasheng Zheng, Yulan Wang, Zhiming YuanAbstract:Lysinibacillus sphaericus is a bacterium incapable of metabolizing sugars with the sole exception of N-acetylglucosamine. To unravel the regulatory role of catabolite control protein A (CcpA) in the sugar metabolism of L. sphaericus, a ccpA deficient mutant was constructed by homologous recombination. The mutant showed growth deficiency and a low efficiency of carbon and energy utilization. NMR spectroscopy in combination with multivariate data analysis revealed that the metabolome of L. sphaericus was dominated by 25 metabolites mainly including amino acids, carbohydrate derivatives and organic acids, and that the mutation of the ccpA gene caused significant reduction of leucine, valine, alanine, threonine, glutamate, lysine, d-ornithine, tyrosine, uridine 5′-diphospho-N-acetlyglucosamine formate, fumarate, phenylalanine, aspartate, asparagine, and acetate but elevation of ribose-5-phosphate, and uracil. Furthermore, the networks of CcpA-mediated regulation based on the metabolome were constructed by arr...
Vinay Kumar - One of the best experts on this subject based on the ideXlab platform.
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receptor protein of Lysinibacillus sphaericus mosquito larvicidal toxin displays amylomaltase activity
Insect Biochemistry and Molecular Biology, 2018Co-Authors: Mahima Sharma, Gagan D Gupta, Vinay KumarAbstract:Abstract The activated binary toxin (BinAB) from Lysinibacillus sphaericus binds to surface receptor protein (Cqm1) on the midgut cell membrane and kills Culex quinquefasciatus larvae on internalization. Cqm1 is attached to cells via a glycosyl-phosphatidylinositol (GPI) anchor. It has been classified as a member of glycoside hydrolase family 13 of the CAZy database. Here, we report characterization of the ordered domain (residues 23–560) of Cqm1. Gene expressing Cqm1 of BinAB susceptible mosquito was chemically synthesized and the protein was purified using E. coli expression system. Values for the Michaelis-Menten kinetics parameters towards 4-nitrophenyl α-D-glucopyranoside (α-pNPG) substrate were estimated to be 0.44 mM ( K m) and 1.9 s −1 ( k cat). Thin layer chromatography experiments established Cqm1 as α-glucosidase competent to cleave α-1,4-glycosidic bonds of maltose and maltotriose with high glycosyltransferase activity to form glucose-oligomers. The observed hydrolysis and synthesis of glucose-oligomers is consistent with open and accessible active-site in the structural model. The protein also hydrolyses glycogen and sucrose. These activities suggest that Cqm1 may be involved in carbohydrate metabolism in mosquitoes. Further, toxic BinA component does not inhibit α-glucosidase activity of Cqm1, while BinB reduced the activity by nearly 50%. The surface plasmon resonance study reveals strong binding of BinB with Cqm1 ( K d, 9.8 nM). BinA interaction with Cqm1 however, is 1000-fold weaker. Notably the estimated K d values match well with dissociation constants reported earlier with larvae brush border membrane fractions. The Cqm1 protein forms a stable dimer that is consistent with its apical localization in lipid rafts. Its melting temperature ( T m ) as observed by thermofluor-shift assay is 51.5 °C and Ca 2+ provides structural stability to the protein.
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polyethylene glycol conjugation enhances mosquito larvicidal activity of Lysinibacillus sphaericus bina protein
Biophysical Journal, 2017Co-Authors: Mahima Sharma, Ramesh S Hire, Ashok B Hadapad, Gagan D Gupta, Vinay KumarAbstract:Mosquitoes are known to spread human diseases like West Nile fever, dengue, malaria, zika etc. accounting for millions of deaths worldwide. Lysinibacillus sphaericus, a gram positive, spore producing commensal soil bacterium, has been used worldwide for controlling mosquito population like Culex and Anopheles, and is regarded as safe against non-target organisms. Binary toxin, composed of BinA (41.9 kDa) and BinB (51.4 kDa) component proteins, is responsible for the high larvicidal activity of several L. sphaericus strains. The two proteins exert high toxicity when administered together. BinA alone displays larvicidal activity, in the absence of BinB, albeit at reduced levels. But instability, shorter half-lives and rapid proteolytic digestion can limit their use as an effective insecticide. We for the first time demonstrate the beneficial effect of PEGylation (covalent attachment of polyethylene glycol) on mosquito-larvicidal activity of BinA protein. PEG-protein conjugates were synthesized using PEG-isocyanate polymer. The resulting bio-conjugates were purified to homogeneity by column chromatography methods. These were characterized by various biophysical methods like MALDI-TOF, DLS, DSF and CD. Two different isoforms of PEG-BinA conjugates are expected from biophysical analysis, which appear to be mono-PEGylated but may differ in the site of PEG attachment to BinA protein. The PEGylated proteins displayed preservation of protein's native structure and exhibited improved thermal stability by about 3-5 oC. The PEGylated proteins were also checked for stability against complex proteolytic environment. Regardless of the site of modification, the two isoforms showed a remarkable 7-fold improvement in the larvicidal activity of BinA protein against 3rd instar Culex larvae, compared to the unmodified protein. The PEGylation of recombinantly produced BinA can be achieved easily. It promises a judicious approach towards mosquito population control.
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pegylation enhances mosquito larvicidal activity of Lysinibacillus sphaericus binary toxin
Bioconjugate Chemistry, 2017Co-Authors: Mahima Sharma, Ramesh S Hire, Ashok B Hadapad, Gagan D Gupta, Vinay KumarAbstract:Toxic strains of Lysinibacillus sphaericus have been used in the field for larval control of mosquito vector diseases. The high toxicity of L. sphaericus is attributed to the binary (BinAB) toxin produced as parasporal crystalline inclusions during the early stages of sporulation. BinA and BinB, the primary components of these spore-crystals, exert high toxicity when administered together. However, instability, short half-lives, and rapid proteolytic digestion can limit their use as an effective insecticide. BinA alone displays larvicidal toxicity, in the absence of BinB, albeit with much reduced activity. Here for the first time, we demonstrate the beneficial effect of PEGylation (covalent attachment of polyethylene glycol) on mosquito-larvicidal activity of BinA. Polymer conjugation was achieved using 750 Da polyethylene glycol (PEG) at two different pH values (pH 7.2 and 8.5). Two different isoforms of the biopolymers, purified to homogeneity, were highly water-soluble and resistant to trypsin and prot...
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PEGylation Enhances Mosquito-Larvicidal Activity of Lysinibacillus sphaericus Binary Toxin
2017Co-Authors: Mahima Sharma, Ramesh S Hire, Ashok B Hadapad, Gagan D Gupta, Vinay KumarAbstract:Toxic strains of Lysinibacillus sphaericus have been used in the field for larval control of mosquito vector diseases. The high toxicity of L. sphaericus is attributed to the binary (BinAB) toxin produced as parasporal crystalline inclusions during the early stages of sporulation. BinA and BinB, the primary components of these spore-crystals, exert high toxicity when administered together. However, instability, short half-lives, and rapid proteolytic digestion can limit their use as an effective insecticide. BinA alone displays larvicidal toxicity, in the absence of BinB, albeit with much reduced activity. Here for the first time, we demonstrate the beneficial effect of PEGylation (covalent attachment of polyethylene glycol) on mosquito-larvicidal activity of BinA. Polymer conjugation was achieved using 750 Da polyethylene glycol (PEG) at two different pH values (pH 7.2 and 8.5). Two different isoforms of the biopolymers, purified to homogeneity, were highly water-soluble and resistant to trypsin and proteinase K. The mono-PEGylated BinA isoforms also displayed preservation of the toxin structure with improved thermal stability by about 3–5 °C, as evident from thermal denaturation studies by circular dichroism and differential scanning fluorimetry. Notably, PEGylation enhanced BinA toxicity by nearly 6-fold. The PEGylated BinA isoforms alone displayed high larvicidal activity (LC50 value of ∼3.4 ng/mL) against the third instar Culex larvae, which compares favorably against LC50 reported for the combination of BinA and BinB proteins. Since BinA can be synthesized easily through recombinant technology and easily PEGylated, the conjugated biopolymers offer a promising opportunity for mosquito control programs
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an oligomeric complex of bina binb is not formed in situ in mosquito larvicidal Lysinibacillus sphaericus ispc 8
Journal of Invertebrate Pathology, 2014Co-Authors: Ramesh S Hire, Mahima Sharma, Ashok B Hadapad, Vinay KumarAbstract:Abstract Binary toxin of Lysinibacillus sphaericus is composed of two polypeptides; receptor binding BinB and toxic BinA. Both the polypeptides are required for maximal toxicity. It has been suggested that binary toxin exerts toxicity as a heterotetramer constituted by two copies of each of the component polypeptides. It has also been observed that oligomers consisting of two copies of BinA and BinB are pre-formed in L. sphaericus spore–crystals. However, recombinant proteins from Escherichia coli expression system elute individually as monomers. We purified the likely oligomeric complex from the spore–crystals of highly toxic L. sphaericus ISPC-8 strain and probed it with proteomic tools. The analysis showed that the high molecular mass complex in the toxic spore–crystals is composed of only surface layer protein (SlpC). The purified SlpC from the local isolate exists as a dimer and also showed poor mosquito-larvicidal activity.
Mauro Tonolla - One of the best experts on this subject based on the ideXlab platform.
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Dynamics of Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus Spores in Urban Catch Basins after Simultaneous Application against Mosquito Larvae
2016Co-Authors: Valeria Guidi, Angelika Lehner, Mauro TonollaAbstract:Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (60.08) and 4.13 (60.09) log ml21 in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 105 g21 were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days). Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clona
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dynamics of bacillus thuringiensis var israelensis and Lysinibacillus sphaericus spores in urban catch basins after simultaneous application against mosquito larvae
PLOS ONE, 2013Co-Authors: Valeria Guidi, Angelika Lehner, Peter Luthy, Mauro TonollaAbstract:Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (± 0.08) and 4.13 (± 0.09) log ml(-1) in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 10(5) g(-1) were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days). Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clonal population as strain 2362 from catch basins never treated with Lsph-based products indicates that the use of a combination of Bti and Lsph for the control of mosquitoes does not introduce non-indigenous microorganisms in this area.
