The Parasporal Body of Bacillus thuringiensis subsp. israelensis: A Unique Phage Capsid-Associated Prokaryotic Insecticidal Organelle

SIMPLE SUMMARY: Bacillus thuringiensis subsp. israelensis (Bti) is widely used for controlling disease-carrying mosquitoes. It achieves this by synthesizing four proteins (Cry4Aa1, Cry4Ba1, Cry11Aa1, and Cyt1Aa1), encoded by its pBtoxis plasmid, which target and kill mosquito larvae. These proteins are synthesized during sporulation and stored in three distinct compartments within the parasporal body (PB). As the spore and parasporal body originate from the same cell, they are in close proximity. When larvae ingest them, the four larvicidal proteins dissolve in the midgut lumen and bind to midgut cells, leading to the death of the larva. This creates a nutrient-rich environment for the germinated Bti spores to initiate vegetative growth. The compartments, along with the entire parasporal body, are encased in a net-like multilamellar fibrous matrix (MFM) that contains unique proteins, including Bt152 and Bt075, which are also encoded by pBtoxis. Bt075 shares similarities with encapsulin shell proteins, a class of proteins that encases various cargos. Our findings suggest the larvicidal proteins are packaged into their respective compartments of the parasporal body during translation. Although the parasporal body is larger and more complex than the compartments formed by encapsulins, they both create distinct encased compartments within the bacterial cell, thereby functioning as bacterial organelles. ABSTRACT: The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti’s pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.