Sarjeet S. Gill

Bacillus thuringiensis: A story of a successful bioinsecticide.

Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture.

Identification, Isolation, and Cloning of a Bacillus thuringiensis CryIAc Toxin-binding Protein from the Midgut of the Lepidopteran Insect Heliothis virescens

Bacillus thuringiensis toxins are insecticidal to a variety of insect species. The selectivity of the toxins produced by these bacteria is dependent on both the toxin structure and the receptor sites that are present in different insect species. One of these toxins, CryIAc, is highly insecticidal to the noctuid pest Heliothis virescens. Using toxin overlay assay, a 120-kDa glycoprotein was identified as a toxin-binding protein. This protein was partially purified, its N-terminal sequence was determined, and the full-length cDNA encoding this protein was isolated from a H. virescens midgut library. The B. thuringiensis toxin-binding protein, BTBP, has high homology to aminopeptidase N from eukaryotes and prokaryotes.

Steroid Induction of a Peptide Hormone Gene Leads to Orchestration of a Defined Behavioral Sequence

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence.

A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae

A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI-ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI-ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI-ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI-ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.

A novel type of RNase III family proteins in eukaryotes.

The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. We isolated the drosha gene, a new member of this family in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila drosha throughout its entire protein sequence. A potential drosha homologue is also found among the deposited human cDNA sequences. A comparison of these drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family.

P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti.

SUMMARY This study describes the expression patterns of P-type Na+/K+-ATPase and V-type H+-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene, both ATPases are expressed in all of the osmoregulatory tissues of larvae (midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach, Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na+/K+-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na+/K+-ATPase, apical V-type H+-ATPase) and ion-transporting (V-type H+-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations, whereas the adult stomach has uniform expression of basolateral P-type Na+/K+-ATPase and apical V-type H+-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H+-ATPase (apically and cytoplasmically) whereas P-type Na+/K+-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na+/K+-ATPase (basal) and V-type H+-ATPase (apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na+/K+-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na+/K+-ATPase and V-type H+-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.

Blood meal induces global changes in midgut gene expression in the disease vector, Aedes aegypti

Blood feeding is an essential developmental process for many arthropods and plays a significant role in disease transmission. Understanding physiological responses in the midgut is important because it is the primary site of blood meal digestion and pathogenic infection. Processes that occur in the midgut in response to a blood meal have been studied but are poorly understood. Here, we use cDNA microarrays to examine midgut gene expression on a global level in response to blood feeding to assist in unraveling these processes. We have developed Aedes aegypti microarrays consisting of clones obtained from an expressed sequence tag project. Individual clones were amplified by polymerase chain reaction and printed onto glass slides. These microarrays were used to study the effects of a blood meal on midgut gene expression over a 72-h time course. As a result, a number of genes involved in processes such as nutrient uptake and metabolism, cellular stress responses, ion balance, and PM formation, as well as a number of unknown genes were induced or repressed in response to a blood meal based on this microarray data.

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R1) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R1 promotes cleavage of the amino-terminal end, including helix α-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7–12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a “ping pong” binding mechanism with both receptors is involved in toxin action.

Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster

Ecdysis‐triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH‐like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.

In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases

Proteases with trypsin-, chymotrypsin- and thermolysin-like specificity were detected in Culex quinquefasciatus larval midguts. Their activities were monitored by N-terminal amino acid sequence analysis of the Bacillus thuringiensis subsp. israelensis CryIVD toxin proteolytic fragments. These proteases are located in the larval midgut and in different fractions obtained during the preparation of brush border membrane vesicles. The activity of the midgut proteases increased with an increase in pH. Both the chymotrypsin- and thermolysin-like activities are involved in the processing of solubilized CryIVD toxin, whereas an additional trypsin-like protease is necessary for the CryIVD parasporal inclusion processing. The solubilized CryIVD toxin was first cleaved between Thr347 and Phe348 and between Phe348 and Tyr349, generating a 40-kDa N-terminal fragment and a 32.5-kDa C-terminal fragment. The C-terminal domain was resistant to further processing, with only a small amount of a 31-kDa product appearing due to the action of a thermolysin-like protease. However, the N-terminal domain was very unstable, and was further degraded to about 30 kDa. Unlike the solubilized CryIVD toxin, the processing of the CryIVD parasporal inclusion was very slow at neutral pH. Three protease-resistant products were detected at pHs higher than 9.5 with an overnight incubation at 37 degrees C. The 30- and 28.5-kDa C-terminal peptides are proteolytic products of trypsin- and chymotrypsin-like proteases, respectively; while the 28-kDa N-terminal peptide has 27 amino acids deleted from the N-terminal end by a thermolysin-like protease.