Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

Abstract

Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

FIG 1

Nomenclature system for Vip proteins. The system consists of four ranks based on amino acid sequence identity (9). The primary, secondary, and tertiary ranks distinguish proteins with less than ∼45, 78, and 95% sequence identities, respectively. The quaternary rank distinguishes proteins sharing >95% sequence identity, which can be considered products of “allelic” forms of the same gene but can also have the same sequence that originated from different isolates.

FIG 2

Dendrogram showing the relationships among Vip proteins based on their degree of amino acid identity. Amino acid sequences were aligned by using the Clustal X interface (120). The evolutionary distance was calculated by maximum likelihood analysis, and the tree was constructed by using the MEGA5 program (121). The proteins used in this analysis are as follows: Vip1Aa1 (sequence identification number [Seq. ID no.] 5 in reference 28), Vip1Ab1 (Seq. ID no. 21 in reference 28), Vip1Ac1 (GenBank accession number HM439098), Vip1Ad1 (accession number JQ855505), Vip1Ba1 (accession number AAR40886), Vip1Bb1 (accession number AAR40282), Vip1Ca1 (accession number AAO86514), Vip1Da1 (accession number CAI40767), Vip2Aa1 (RCSB Protein Data Bank accession number 1QS1_A), Vip2Ab1 (Seq. ID no. 20 in reference 28), Vip2Ac1 (accession number AAO86513), Vip2Ad1 (accession number CAI40768), Vip2Ae1 (accession number EF442245), Vip2Af1 (accession number ACH42759), Vip2Ag1 (accession number JQ855506), Vip2Ba1 (accession number AAR40887), Vip2Bb3 (accession number AIA96500), Vip3Aa1 (accession number AAC37036), Vip3Ab1 (accession number AAR40284), Vip3Ac1 (named PS49C; Seq. ID no. 7 in K. Narva and D. Merlo, U.S. patent application 20,040,128,716), Vip3Ad2 (accession number CAI43276), Vip3Ae1 (accession number CAI43277), Vip3Af1 (accession number CAI43275), Vip3Ag2 (accession number ACL97352), Vip3Ah1 (accession number ABH10614), Vip3Ai1 (accession number KC156693), Vip3Aj1 (accession number KF826717), Vip3Ba1 (accession number AAV70653), Vip3Bb2 (accession number ABO30520), Vip3Ca1 (accession number ADZ46178), and Vip4Aa1 (accession number HM044666).

FIG 3

Multiple-sequence alignment of the Vip1 proteins. Sequence identity is indicated by shading, where violet is 100% sequence identity, pale blue is 80 to 100%, yellow is 60 to 80%, and white is <60%. Intervals of 10 amino acids are marked with “*.” SP, signal peptide. Proteins used in this analysis are as follows: Vip1Aa1 (Seq. ID no. 5 in reference 28), Vip1Ab1 (Seq. ID no. 21 in reference 28), Vip1Ac1 (GenBank accession number HM439098), Vip1Ad1 (accession number JQ855505), Vip1Ba1 (accession number AAR40886), Vip1Bb1 (accession number AAR40282), Vip1Ca1 (accession number AAO86514), and Vip1Da1 (accession number CAI40767).

FIG 4

Multiple-sequence alignment of the Vip2 proteins. Sequence identity is indicated by shading, where violet is 100% sequence identity, pale blue is 80 to 100%, yellow is 60 to 80%, and white is <60%. Intervals of 10 amino acids are marked with “*.” SP, signal peptide. The N-terminal domain (N-domain) and C-terminal domain (C-domain) are framed within boxes. The protein sequences used in this analysis are as follows: Vip2Aa1 (RCSB Protein Data Bank accession number 1QS1_A), Vip2Ab1 (Seq. ID no. 20 in reference 28), Vip2Ac1 (GenBank accession number AAO86513), Vip2Ad1 (accession number CAI40768), Vip2Ae1 (accession number EF442245), Vip2Af1 (accession number ACH42759), Vip2Ag1 (accession number JQ855506), Vip2Ba1 (accession number AAR40887), and Vip2Bb3 (accession number AIA96500).

FIG 5

Tridimensional structure of Vip2 showing the two domains in different colors (N-terminal domain in blue and C-terminal domain in orange). (A) Schematic ribbon representation showing the NAD molecule (in blue) bound to the C-terminal domain. (B) Schematic drawing with secondary structure nomenclature. (Reprinted from reference by permission from Macmillan Publishers Ltd.)

FIG 6

Proposed mode of action of the binary Vip1/Vip2 toxin. The Vip1 protoxin is proteolytically processed by midgut proteases. The activated toxin binds to specific receptors either as a monomeric form or after oligomerization. Vip2 then binds to the oligomeric Vip1 protein and enters the cell either by endocytosis of the whole complex or directly through the pore formed by Vip1. Once inside the cytosol, Vip2 catalyzes the transfer of the ADP-ribose group from NAD to the actin monomers, preventing their polymerization.

FIG 7

Multiple-sequence alignment of the Vip3A proteins. Sequence identity is indicated by shading, where violet is 100% sequence identity, pale blue is 80 to 100%, yellow is 60 to 80%, and white is <60%. SP, signal peptide (50); “T,” 65-kDa fragment after proteolysis; “PPS1” and “PPS2,” first and second processing sites, respectively (50). Intervals of 10 amino acids are marked with “*.” The protein sequences used in this analysis are as follows: Vip3Aa1 (GenBank accession number AAC37036), Vip3Ab1 (accession number AAR40284), Vip3Ac1 (named PS49C; Seq. ID no. 7 in Narva and Merlo, U.S. patent application 20,040,128,716), Vip3Ad2 (accession number CAI43276), Vip3Ae1 (accession number CAI43277), Vip3Af1 (accession number CAI43275), Vip3Ag2 (accession number ACL97352), Vip3Ah1 (accession number ABH10614), Vip3Ai1 (accession number KC156693), Vip3Aj1 (accession number KF826717), Vip3Ba1 (accession number AAV70653), Vip3Bb2 (accession number ABO30520), and Vip3Ca1 (accession number ADZ46178).

FIG 8

Conserved Domain Database (CDD) analysis of representative Vip3 proteins. The same sequences as those shown in Fig. 4 were used. CBM, carbohydrate-binding motif; ApbA, ketopantoate reductase motif; Tar, methyl-accepting chemotaxis protein motif; COG1511, motif of a predicted protein membrane of unknown function. TIGR03545 represents a relatively rare but broadly distributed uncharacterized family of proteins, distributed in 1 to 2% of bacterial genomes.

FIG 9

Proposed mode of action of the Vip3 proteins. The full-length protoxin is proteolytically processed by midgut proteases. The 65-kDa fragment binds to specific receptors (with the 22-kDa fragment still bound or not). Pores are then formed, which leads to the death of the cell.

FIG 10

Immunolocalization of Vip3Aa in midgut tissue sections after ingestion by S. frugiperda larvae. (Left) Control larvae. (Right) Larvae that ingested Vip3Aa. Nuclei were stained blue, and the apical and basal membranes were stained red. Binding of Vip3Aa to the apical membrane is shown in green. BM, basal membrane; AM, apical membrane; L, gut lumen. (Reprinted from reference .)

FIG 11

General binding site model for the Cry and Vip proteins in the midgut epithelial membrane of lepidopteran larvae. Cry1Fa and Cry1A proteins, in addition to the shared binding site, may have other sites depending on the insect species considered. Recognition of Vip3Aa sites by Cry1Ia has been found only in S. eridania (of four Spodoptera species tested).