Modulation of Kinase Activities In Vitro by Hepatitis C Virus Protease NS3/NS4A Mediated-Cleavage of Key Immune Modulator Kinases.

Hepatitis C Virus NS3/NS4A, a serine protease complex, has been found to interact with many host proteins and cause various adverse effects on cellular function and immune response. For example, the cleavage of important immune factors by NS3/NS4A has been suggested as a mechanism for the hepatitis C virus to evade innate immunity. The spectrum of susceptible substrates for NS3/NS4A cleavage certainly includes important immune modulator kinases such as IKKα, IKKß, IKKε, and TBK1, as demonstrated in this paper. We show that the kinase activities of these four host kinases were transformed in unexpected ways by NS3/NS4A. Treatment with NS3/NS4A caused a significant reduction in the kinase activities of both IKKα and IKKß, suggesting that HCV might use its NS3/NS4A protease activity to deactivate the NF-κB-associated innate immune responses. In contrast, the kinase activities of both IKKε and TBK1 were enhanced after NS3/NS4A treatment, and more strikingly, the enhancement was more than 10-fold within 20 min of treatment. Our mass spectroscopic results suggested that the cleavage after Cys89 in the kinase domain of IKKε by NS3/NS4A led to their higher kinase activities, and three potential mechanisms were discussed. The observed kinase activity enhancement might facilitate the activation of both IKKε- and TBK1-dependent cellular antiviral pathways, likely contributing to spontaneous clearance of the virus and observed acute HCV infection. After longer than 20 min cleavage, both IKKε- and TBK1 gradually lost their kinase activities and the relevant antiviral pathways were expected to be inactivated, facilitating the establishment of chronic HCV infection.

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism.

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb toxicities to coleopteran larvae by a toxin-binding fragment of an insect cadherin.

The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis are used in biopesticides and transgenic crops to control larvae of leaf-feeding beetles and rootworms. Cadherins localized in the midgut epithelium are identified as receptors for Cry toxins in lepidopteran and dipteran larvae. Previously, we discovered that a peptide of a toxin-binding cadherin expressed in Escherichia coli functions as a synergist for Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae. Here we report that the fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm binds toxin and enhances Cry3 toxicity to larvae of naturally susceptible species. The cadherin fragment (CR8 to CR10 [CR8-10]) of western corn rootworm Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. By an enzyme-linked immunosorbent microplate assay, we demonstrated that the CR8-10 peptide binds alpha-chymotrypsin-treated Cry3Aa and Cry3Bb toxins at high affinity (11.8 nM and 1.4 nM, respectively). Coleopteran larvae ingesting CR8-10 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxin. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle Leptinotarsa decemlineata, southern corn rootworm Diabrotica undecimpunctata howardi, and western corn rootworm. The extent of Cry3 toxin enhancement, which ranged from 3- to 13-fold, may have practical applications for insect control. Cry3-containing biopesticides that include a cadherin fragment could be more efficacious. And Bt corn (i.e., corn treated with B. thuringiensis to make it resistant to pests) coexpressing Cry3Bb and CR8-10 could increase the functional dose level of the insect toxic activity, reducing the overall resistance risk.

Cadherin fragments from Anopheles gambiae synergize Bacillus thuringiensis Cry4Ba's toxicity against Aedes aegypti larvae.

A peptide from cadherin AgCad1 of Anopheles gambiae larvae was reported as a synergist of Bacillus thuringiensis subsp. israelensis Cry4Ba's toxicity to the Anopheles mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba's toxicity to Aedes aegypti larvae, but a Diabrotica virgifera virgifera cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba's toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval A. aegypti brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against A. aegypti.

Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity.

A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. 125I-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. AgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.

Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus.

BACKGROUND: Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. RESULTS: A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. CONCLUSION: An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba.

Proteomic identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba binding proteins in midgut membranes from Aedes (Stegomyia) aegypti Linnaeus (Diptera, Culicidae) larvae.

Novel Bacillus thuringiensis subsp. israelensis (Bti) Cry4Ba toxin-binding proteins have been identified in gut brush border membranes of the Aedes (Stegomyia) aegypti mosquito larvae by combining 2-dimensional gel electrophoresis (2DE) and ligand blotting followed by protein identification using mass spectrometry and database searching. Three alkaline phosphatase isoforms and aminopeptidase were identified. Other Cry4Ba binding proteins identified include the putative lipid raft proteins flotillin and prohibitin, V-ATPase B subunit and actin. These identified proteins might play important roles in mediating the toxicity of Cry4Ba due to their location in the gut brush border membrane. Cadherin-type protein was not identified, although previously, we identified a midgut cadherin AgCad1 as a putative Cry4Ba receptor in Anopheles gambiae mosquito larvae [Hua, G., Zhang, R., Abdullah, M.A., Adang, M.J., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47, 5101-5110]. Other identified proteins in this study that might have lesser roles include mitochondrial proteins such as ATP synthase subunits, mitochondrial processing peptidase and porin; which are likely contaminants from mitochondria and are not brush border membrane components. Trypsin-like serine protease was also identified as a protein that binds Cry4Ba. Identification of these toxin-binding proteins will lead to a better understanding of the mode of action of this toxin in mosquito.

Manduca sexta (Lepidoptera: Sphingidae) cadherin fragments function as synergists for Cry1A and Cry1C Bacillus thuringiensis toxins against noctuid moths Helicoverpa zea, Agrotis ipsilon and Spodoptera exigua.

BACKGROUND: Specific Bacillus thuringiensis Berliner (Bt) toxins are effective against a narrow spectrum of species. While specificity is an advantage for limiting adverse effects on non-target organisms, it is also the primary drawback of Bt's application for controlling multiple pest species in agriculture, forestry and other areas. Recently, it was reported that a small toxin-binding fragment of Manduca sexta (Joh.) cadherin acts as a synergist of Bt toxins to M. sexta, Heliothis virescens F. and Helicoverpa zea (Boddie). These insects are quite susceptible to the Cry1A toxins. The first aim of the present study was to determine if longer-sized fragments of M. sexta cadherin differed in the level of toxin enhancement. The second aim was to examine enhancement of Bt toxins against relatively Bt-tolerant insects Agrotis ipsilon (Hufn.) and Spodoptera exigua (Hübner). RESULTS: Cadherin fragments longer than previously reported had improved synergistic properties. Significant enhancement of Bt Cry1A toxins against A. ipsilon and S. exigua was found. A cadherin fragment also increased Cry1C toxicity to S. exigua. CONCLUSIONS: The commercial development of this synergist has the potential to widen the spectrum of Bt toxicity to other important agricultural lepidopteran insect pests and thus increase its usefulness in agriculture.

Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin.

The insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are broadly used to control insect pests with agricultural importance. The cadherin Bt-R(1) is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R(1) region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. Here, we report that a peptide containing this region expressed in Escherichia coli functions as a synergist of Cry1A toxicity against lepidopteran larvae. Far-UV circular dichroism and (1)H-NMR spectroscopy confirmed that our purified CR12-MPED peptide mainly consisted of beta-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (K(d) = 32 nM) and insect midgut microvilli but did not alter Cry1Ab or Cry1Ac binding localization in the midgut. By BIAcore analysis we demonstrate that Cry1Ab binds CR12-MPED at high (9 nM)- and low (1 microM)-affinity sites. CR12-MPED-mediated Cry1A toxicity enhancement was significantly reduced when the high-affinity Cry1A-binding epitope ((1416)GVLTLNIQ(1423)) within the peptide was altered. Because the mixtures of low Bt toxin dose and CR12-MPED peptide effectively control target insect pests, our discovery has important implications related to the use of this peptide to enhance insecticidal activity of Bt toxin-based biopesticides and transgenic Bt crops.

Enhancement of Cry19Aa mosquitocidal activity against Aedes aegypti by mutations in the putative loop regions of domain II.

Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex.

Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering.

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.