Insecticidal Toxin Complex Proteins from Xenorhabdus nematophilus

Abstract

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ϳ1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ϳ30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.Xenorhabdus and Photorhabdus spp. are two bacterial genera belonging to the family Enterobacteriaceae, known to be associated with entomopathogenic nematodes (1-4) These bacteria represent potential sources for new genes encoding potent insecticidal toxins that could be put into plants as alternatives to Bacillus thuringiensis genes (5). Gene sequence analysis of Xenorhabdus and Photorhabdus bacteria show that these organisms contain a family of related toxin complex (tc) 2 genes located at different loci (6 -9). The toxin complexes are composed of three different classes of protein components, which, according to ffrench-Constant et al. (10,11), can be categorized as class A, B, and C proteins based upon sequence similarity and size. Class A proteins are very large, having a molecular mass of ϳ280 kDa. Class B proteins are ϳ170 kDa, and class C proteins are ϳ110 kDa. There are many different varieties of class A, B, and C proteins in both Gram-negative and Gram-positive bacteria (12-15).From earlier studies, it has been suggested that class A proteins harbor the cytotoxic effects of the Tc toxins, whereas class B and C proteins modulate and enhance the toxicity of class A proteins (16). However, recently, we elucidated the molecular mechanism of the Photorhabdus luminescens Tc complex, which consists of the class A protein TcdA1, the class B protein TcdB2, and the class C protein TccC3 or TccC5 (17). These studies revealed that the class C proteins harbor the biological activity. It was shown that TccC3 and TccC5 are ADP-ribosyltransferases, which target the actin cytoskeleton by modification of actin and Rho GTPases, respectively (17). Moreover, these studies su...

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity

“…1B and 2 and Fig. S3) may indicate sites of surface-bound membrane-derived material, consistent with previous studies linking TcA subunits to host selectivity (12,13,15) and a role for TcA "scaffolds" in cell surface recognition. Density resulting from heterogeneously associated material would be expected to reinforce poorly during averaging, and so the observation that these densities drop below the display threshold of the final structure is consistent with our hypothesis.…”

“…This model (Fig. 6) seems plausible given the implication of TcA-like proteins in cell surface recognition (14,15) and that the pore-forming activity of the P. luminescens TcdA1 protein has been demonstrated (16). Many binary toxins target the actin cytoskeleton, a mode of action for Yen-Tc suggested by the CNF1-like domain of the C1 subunit.…”

“…Interestingly, TcB and TcC components coexpressed with TcA components from other species (or from other Tc clusters) can combine to cause an effect with altered host specificity (12,13), suggesting the TcA component is a determinant of target host range. Further evidence for this is provided by the observation that the TcA components of an insecticidal X. nematophila Tc bind to solubilized insect midgut brush border membranes (14,15). By comparison, TcC-like proteins are thought to represent the main toxin components of the Tc family (7,16).…”

Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

“…Tc proteins of Y. pestis (9), Y. pseudotuberculosis (12,39), P. luminescens (1,40), Y. entomophaga (37), and X. nematophilus (41) have been shown to form large-molecular-weight protein complexes. In general, Tc protein complexes are composed of class A (TcA; Y. pestis YitA and YitB), class B (TcB; Y. pestis YitC), and class C (TcC; Y. pestis YipA and YipB) Tc proteins (9,37,(41)(42)(43)(44)(45)(46). Recent studies on class A Tc proteins from P. luminescens (46), X. nematophilus (41), and Y. entomophaga (43) indicate that class A (TcA) proteins form a multimeric transmembrane pore which interacts with a complex composed of a class B and a class C Tc protein.…”

“…In general, Tc protein complexes are composed of class A (TcA; Y. pestis YitA and YitB), class B (TcB; Y. pestis YitC), and class C (TcC; Y. pestis YipA and YipB) Tc proteins (9,37,(41)(42)(43)(44)(45)(46). Recent studies on class A Tc proteins from P. luminescens (46), X. nematophilus (41), and Y. entomophaga (43) indicate that class A (TcA) proteins form a multimeric transmembrane pore which interacts with a complex composed of a class B and a class C Tc protein. This holotoxin is theorized to bind an as yet unknown receptor on the target cell surface, which induces endocytosis of the Tc protein complex.…”