The Independent Biological Activity of Bacillus thuringiensis Cry23Aa Protein Against Cylas puncticollis.

The Cry23Aa/Cry37Aa proteins from Bacillus thuringiensis (Bt) have been described toxic to Cylas puncticollis larvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity toward C. puncticollis larvae has been assessed. The results showed that both proteins were toxic for C. puncticollis larvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity against C. puncticollis larvae. Also, the binding behavior of Cry23Aa protein to midgut receptors of C. puncticollis larvae has been determined. According to our results, Cry23Aa binds to C. puncticollis brush border membrane vesicles (BBMV) specifically and independently of Cry37Aa. Due to the lack of common binding sites, Cry23Aa can be pyramided with Cry3Aa protein for better management of C. puncticollis.

Proteomic composition of Nipah virus-like particles.

Virions are often described as virus-only entities with no cellular components with the exception of the lipids in their membranes. However, advances in proteomics are revealing substantial amounts of host proteins in the viral particles. In the case of Nipah virus (NiV), the viral components in the virion have been known for some time. Nonetheless, no information has been obtained regarding the cellular proteins in the viral particles. To address this question, we produced Virus-Like Particles (VLPs) for NiV by expressing the F, G and M proteins in human-derived cells. Next, the proteomic content in these VLPs was analyzed by LC-MS/MS. We identified 67 human proteins including soluble and membrane-bound proteins involved in vesicle sorting and transport. Interestingly, many of them have been reported to interact with other viruses. Finally, thanks to the semi-quantitative nature of our data we were able to estimate the ratio among F, G and M proteins and also the ratio between cellular and viral proteins in the VLPs. We believe our data contribute to the better understanding of NiV life cycle and might facilitate future attempts for developing antiviral agents and the design of further experimental studies for this deadly infection. BIOLOGICAL SIGNIFICANCE: Traditionally viral particles have been described as pure entities carrying only viral-derived proteins. Advances in proteomics are changing this simplified view. Host proteins have been identified in many viruses (especially in enveloped viruses). These cell-derived proteins participate in multiple steps in the viral life cycle and might be as important for the survival of the virus as any other viral-encoded protein. In this work, we analyze utilizing LC-MS/MS the cellular proteins incorporated or bound to the virions of Nipah virus (NiV), an emerging, highly pathogenic, zoonotic virus from the Paramyxoviridiae family. Furthermore, we analyzed the ratio between cellular and viral proteins and among the viral F, G and M proteins in the viral particles. The characterization of the Nipah virus-human interactions occurring in the virion might facilitate the development of new therapeutic and prophylactic therapies for this viral illness.

Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM).

Unshared binding sites for Bacillus thuringiensis Cry3Aa and Cry3Ca proteins in the weevil Cylas puncticollis (Brentidae).

Bacillus thuringiensis Cry3Aa and Cry3Ca proteins have been reported to be toxic against the African sweetpotato pest Cylas puncticollis. In the present work, the binding sites of these proteins in C. puncticollis brush border vesicles suggest the occurrence of different binding sites, but only one of them is shared. Our results suggest that pest resistance mediated by alteration of the shared Cry-receptor binding site might not render both Cry proteins ineffective.

Shared binding sites for the Bacillus thuringiensis proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African sweet potato pest Cylas puncticollis (Brentidae).

Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu(47) for the 70-kDa form or Ile(88) for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.