Hope Brooks

ENGL 202C 20 February 2013

Bacillus thuringiensis Mode of Action in Lepidopteran Pests

INTRODUCTION
Bacillus thuringiensis (Bt) is a soil-dwelling bacterium that produces species-specific insecticidal proteins during spore-production. Since its discovery by Shigetane Ishiwatari in 1901, Bt strains toxic to Lepidoptera (moths and butterflies) larvae, Diptera (flies), Coleoptera (beetles), Hemiptera (true bugs), and nematodes have been found. Today, Bt is used extensively as a biological pesticide in organic agriculture, in urban aerial pest control programs, and, increasingly, in Figure 1: Schematic of Bts mode of action genetically modified crops. To better understand how Bt kills lepidopteran larvae, the process can be broken down into four steps (shown in Figure 1): ingestion by the insect, protein binding to midgut epithelial cell receptors, breakdown of the midgut, and death of the insect by septicemia3.

INGESTION BY THE INSECT

When a larvae ingests Bt endotoxins, a class of crystalline (Cry) and cytolytic (Cyt) insecticidal proteins, are released (shown in Figures 2 and 3)2. Cry and Cyt proteins are classified as poreforming bacterial toxins (PFT) which are water-soluble and able to change shape in order to enter or cross their hosts cell membrane1. Initially, Cry and Cyt proteins are bound in an insoluble 130-140 kDa protein2. In the highly alkaline environment of the larvaes midgut, the Cry- and Cytcontaining proteins are dissolved and cleaved by proteases, enzymes which break

Figure 2: Scanning electron image of Cry proteins

peptide bonds during protein catabolism. The resulting 60-70 kDa protease-resistant Cry and Cyt proteins are activated by cleaving part of the proteins N-terminus, the end of the protein terminated by a free amine (-NH2) group. In addition, Cry proteins require cleaving of the Cterminus, the end of the protein terminated by a free carboxyl (-COOH) group, in order to initiate protein activation1.

Figure 3: Cry and Cyt proteins produced by Bacillus thuringiensis

PROTEIN BINDING TO MIDGUT EPITHELIAL CELL RECEPTORS

Upon activation, Cry and Cyt proteins bind to receptors in the tissues lining the larvaes midgut (shown in Figure 4). Four receptor molecules are active in binding Cry proteins, including a cadherinlike protein (CADR), a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase-N (APN), a GPI-anchored alkaline phosphatase (ALP), and a glycoconjugate (shown in Figure 5). Cadherins are calciumdependent protein receptors which cross the cell membrane and are used in cell Figure 4: Cry and Cyt proteins binding to receptors in the larvaes midgut adhesion, migration, cytoskeletal organization, and morphogenesis. Cry proteins bind to cadherins using a region termed domain II (shown in Figure 6). APNs are a broad class of proteins which aid in the digestion of amino

acids from protein catabolism. Cry proteins bind to APNs using a region termed domain III (shown in Figure 6). ALPs, another class of protein receptors, aid in removing phosphate (PO43-) groups from many classes of molecules under alkaline conditions. Crys mode of binding to ALPs has not been adequately described. Glycoconjugates are a class of carbohydrates which are covalently linked to other molecules like proteins. As for ALPs, Crys mode of binding to glycoconjugates has not been adequately described1.

Figure 5: Representation of four receptor molecules active in binding Cry proteins

Figure 6: Diagram of the three domains in Cry proteins

BREAKDOWN OF THE MIDGUT

After the Cry and Cyt proteins bind to the four receptor molecules, the larvaes midgut begins to breakdown. The breakdown of the midgut can be divided into two steps: initial modification of Cry protein structure and formation of lytic pores in the midgut1.

Figure 7: Representation of the process by which Bt breaks down the midgut

INITIAL MODIFICATION OF CRY PROTEIN STRUCTURE

PFTs are initially found as soluble oligomers, molecules containing a limited number of monomer units. Yet, PFT protein structures must be modified from their soluble oligomer state to facilitate their insertion into the midgut epithelial membrane and increase their toxicity. Within the larvaes midgut, proteases modify the structure of Cry proteins by cleaving their Nterminals1.

FORMATION OF LYTIC PORES IN THE MIDGUT

Following modification, Cry and Cyt proteins are inserted into the midgut epithelial membrane. Initially, Cyt proteins directly interact with membrane lipids and insert themselves into the midgut epithelial membrane. Cry proteins associate with the Bt-R1 receptor molecules in the midgut epithelial membrane. Upon association with the Bt-R1 receptor molecules, the Cry proteins form pre-pore oligometric structures which bind to APN. The pre-pore oligometric structures have a higher affinity to APN than unassociated Cry proteins because of Cry proteins unfolding in the basic pH of the insect midgut and interacting with Bt-R1. After binding to APN, the pre-pore structure forms molten globules which are inserted into lipid rafts, regions of glycolipids and protein receptors found in the midgut epithelial membrane. Then, cells of the midgut epithelial membrane swell and lytic pores begin to form1.

DEATH OF THE INSECT BY SEPTICEMIA

Over time, Bt proliferates in the insect and produces Cry and Cyt proteins. At the organism level, increased concentrations of toxic proteins lead to septicemia, a systemic inflammatory response to infection by pathogens. At the cellular level, the larvaes cells experience osmotic shock, a sudden change in surrounding solute concentration following pore formation. Osmotic shock and swelling midgut epithelial cells induce cell death in the larvaes midgut epithelial membrane. Overall, the increasing concentrations of Bt and its protein metabolites, osmotic shock, and cellular swelling cause the insect to die1.

SUMMARY
Today, Bt is widely used as a species-specific biological control agent for many orders of insect pests, including Lepidoptera and Coleoptera. Its mode of action in lepidopteran larvae can be broken into four steps. Initially, Bt is ingested by the larvae. The Cry and Cyt proteins produced by Bt bind to the midgut epithelial cell receptors, initiating the breakdown of the larvaes midgut. Ultimately, the accumulation of Bt and its protein metabolites causes the larvae to die of septicemia.

WORKS CITED
1

Bravo, A., Gill, S., Soberon, M. (2006). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423435. Retrieved from http://owl.english.purdue.edu/owl/resource/560/10/. Deacon, J. The Microbial World: Bacillus thuringiensis. Retrieved from http://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htm. University of California San Diego. Bacillus thuringiensis. Retrieved from http://www.bt.ucsd.edu/bt_history.html.

Figure 1: Shelton, A. (1993). Biological Control: A Guide to Natural Enemies in North America. Retrieved from http://www.biocontrol.entomology.cornell.edu/pathogens/bacteria.html. Figure 2: Life Sciences Foundation. Retrieved from http://www.lifesciencesfoundation.org/printer_events-Bacillus_thuringiensis.html. Figure 3 & 5: Bravo, A., Gill, S., Soberon, M. (2006). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423435. Retrieved from http://owl.english.purdue.edu/owl/resource/560/10/. Image 4: United States Department of Agriculture Forest Service. (2010). Managing Invasive Forest Insect Pests with Bacillus thuringiensis. Retrieved from http://www.nrs.fs.fed.us/disturbance/invasive_species/bt/. Figure 6: Deacon, J. The Microbial World: Bacillus thuringiensis. Retrieved from http://archive.bio.ed.ac.uk/jdeacon/microbes/bt.htm. Figure 7: Bravo, A., Soberon, M. (2008). Avoiding Insect Resistance to Cry Toxins from Bacillus thuringiensis. Retrieved from http://www.isb.vt.edu/articles/may0803.htm.