Joel J. Sheets

Photorhabdus luminescens Toxins ADP-Ribosylate Actin and RhoA to Force Actin Clustering

Tripartite Toxin Luminescent bacterial symbionts of nematode worms that attack insects have long stirred interest in their possibilities for biological control. The bacteria produce a family of toxins composed of at least three subunits that resemble a widely occurring class of bacterial toxins also produced by human pathogens. Lang et al. (p. 1139) have elucidated the mode of action and structural interactions of some of these tripartite protein toxins and found that they poison the cells actin cytoskeleton by catalyzing unusual reactions. One toxin mediated adenosine diphosphate (ADP)–ribosylation at threonine-148 to cause actin polymerization, another ADP-ribosylated Rho protein at glutamine-63, and both synergized to cause actin clustering and cell paralysis. A bacterial toxin targets and modifies the actin cytoskeleton in insect larvae. The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)–ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins.

Kinetics of Uptake, Clearance, Transfer, and Metabolism of Hexaflumuron by Eastern Subterranean Termites (Isoptera: Rhinotermitidae)

Abstract The rates of uptake, clearance, insect-to-insect transfer, and metabolism of [14C]hexaflumuron [N-(((3,5-dichloro-4-(1,1,2,2-tetrafluroethoxy)phenyl)- amino)carbonyl)-2,6-diflurobenzamide] were measured in eastern subterranean termite workers, Reticulitermes flavipes (Kollar), fed cellulose diets containing either 0.1 or 0.5% (wt:wt) hexaflumuron. The rate of uptake, level of maximum uptake, and amount of insect-to-insect transfer were concentration dependent. The clearance rate constant for hexaflumuron was independent of concentration, with a mean value of 3.2 × 10−3 /h. This corresponds to a mean half-life for hexaflumuron inside termites of 9 d. No evidence of metabolism of hexaflumuron to additional products was detected when extracting and examining the radioactivity contained in the fecal and regurgitated material within the termite holding apparatus 40 d after exposure to the chemical. Hexaflumuron was efficiently transferred from treated to untreated termites, through trophallaxis, resulting in spread of the toxicant throughout the insect population. The combination of uptake and efficient transfer of hexaflumuron between treated and untreated termites ensures broad distribution of the material even to insects not directly exposed to the toxicant. The distribution of hexaflumuron by termite workers, along with their minimal ability to metabolize the compound to other metabolites, and their slow ability to clear the material from the termite population results in death of the entire group of termites contained within the holding apparatus.

Insecticidal Toxin Complex Proteins from Xenorhabdus nematophilus STRUCTURE AND PORE FORMATION

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 ∼1,120 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 ∼30-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.

Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

ABSTRACT Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae.

Targeting of the actin cytoskeleton by insecticidal toxins from Photorhabdus luminescens

Photorhabdus luminescens produces several types of protein toxins, which are essential for participation in a trilateral symbiosis with nematodes and insects. The nematodes, carrying the bacteria, invade insect larvae and release the bacteria, which kill the insects with their toxins. Recently, the molecular mechanisms of the toxin complexes PTC3 and PTC5 have been elucidated. The biologically active components of the toxin complexes are ADP-ribosyltransferases, which modify actin and Rho GTPases, respectively. The actions of the toxins are described and compared with other bacterial protein toxins acting on the cytoskeleton.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens

ABSTRACT Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens. Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Insecticidal activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) and other lepidopteran pests.

ABSTRACT Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.

In vitro metabolism of the N-alkyl-N-(5-isothiazolyl)-and N-(alkylisothiazolin-5-ylidene)phenylacetamides. Evidence of proinsecticidal activity

In vitro studies have demonstrated that N-(4-chloro-3-methyl-5-isothiazolyl)-N-methyl-2-[p-((α,α,α-trifluoro-p-tolyl)oxy]phenyl]acetamide (2a) undergoes NADPH-dependent metabolism, which is catalyzed by monooxygenase enzymes, in rat liver microsomes. The primary metabolite in rat was found to arise from ring-methyl hydroxylation, while N-demethylation to give N-(4-chloro-3-methyl-5-isothiazolyl)-2-[p-[(a,a,a-trifluoro-p-tolyl)oxy]phenyl]acetamide (1) was also observed to occur, but at a slower rate. In microsomal proteins prepared from tobacco budworm midgut tissues, the reverse was observed, as 1 is the predominant metabolite, while ring-methyl hydroxylation occurs at a slower rate. The overall rate of metabolism in trout liver microsomes was found to be 50-fold slower than in rat and afforded 1 as the predominant metabolite. Metabolism studies conducted on the N-alkyl-N-(5-isothiazolyl)- and N-(alkyl-isothiazolin-5-ylidene)phenylacetamides (2 and 3) have shown that the ring-alkylated isomers 3 were converted to 1 more rapidly than isomers 2 in all three species. In general, the rate of conversion to 1, or bioactivation, increased with increasing radical or carbocation stability of the alkyl group in rat and trout liver. In tobacco budworm, however, bioactivation was highest in the ethyl and n-propyl analogues. The ratio of bioactivation in tobacco budworm to that in trout, used as a predictor of selectivity, was observed to be highest with the methyl group.

RONALD ESTABROOK'S EARLY GUIDANCE OF A POSTDOCTORAL FELLOW CONCERNING THE INTRICACIES OF STEROID METABOLISM BY CYTOCHROMES P450

Ronald Estabrook made his initial impact studying cytochrome P450 by demonstrating the oxidative metabolism function of this unique class of enzymes, which had an unusual spectral peak at 450 nm when reduced and in the presence of carbon monoxide. Utilizing a photochemical action spectrum, he demonstrated that a cytochrome P450 was responsible for steroid 21 hydroxylation catalyzed by microsomes prepared from adrenal cortex tissue. As a young postdoctoral student, I was given the unique opportunity to learn from a true pioneer in this field. Ron had a surprisingly small laboratory at that time that allowed me to closely interact with a great scientist to learn about the important role cytochrome P450 proteins play in a wide variety of different organisms catalyzing oxidative metabolism reactions essential to life and to provide organisms, with the means to defend against xenobiotics.