On September 28, 2022, in a latest study published on "Nature" , a research team led by Professor Dong Min and others from , Harvard Medical School, used CRISPR/Cas9 screening technology for the first time on insect cell to find a receptor for bacterial proteotoxin (Tc toxin) specifically targeting insects. This work reveals the mechanism of action of toxins and the specificity of different insects at the molecular level, not only establishing a deep understanding of the function of this toxin in nature, but also helps to further develop efficient and specific bioinsecticides in the future to control agricultural pests and other diseases-borne insects.
Bacterial proteotoxin is the main weapon of many pathogenic bacteria, such as diphtheria toxin, anthrax toxin, Botox toxin , etc. In recent years, the whole genome CRISPR/Cas9 screening technology has been widely used on mammalian cells , becoming the main experimental method for studying these toxins and pathogens targeting humans and animals. Insects are the most diverse animals on the earth and play a crucial role in the entire ecological chain. In nature, there are many bacterial proteotoxins that specifically target insects, and some have been widely used in agricultural pest control. The existing CRISPR/Cas9 screening technology relies on a lentiviral transfection system targeting mammalian cells and is not applicable to insect cells. It greatly limits people's research on the mechanism of action, resistance, host specificity and the development of new insecticidal toxins against insects. This work establishes a method for non-viral, genome-wide CRISPR/Cas9 screening toxin receptors on insect cells, providing a pioneering example for the application of the key genome-wide screening technology to study a wide variety of insect-targeting toxins, pathogens, viruses, numerous arboreal pathogens and viruses, as well as insect cell biology issues.
The authors mainly study a bacterial toxin Tc toxin derived from the insect pathogen nematode intestinal symbiotic bacteria. This toxin has an extraordinary ecological role: this type of bacteria parasitizes in specific nematodes in the soil. These nematodes specifically search for and invade the insect larvae in the soil, and then release these bacteria into the insect's body cavity. These bacteria then use the secreted toxins to inhibit the insect's immune response, and then the bacteria reproduce in large quantities. The nematodes use bacteria as food to reproduce the next generation, and ultimately kill the insects. In 1998, the RH french-Constant group first reported that active protein toxin (Tc Toxin) was isolated from the insect pathogen nematode symbiotic bacteria, Photorhabdus luminescens. In order to better utilize and develop an important biological control resource of insect pathogen nematodes, people have always been interested in the study of the molecular mechanisms related to Tc toxins, and have successively found thousands of members of the Tc toxin family in bacteria of different bacteria, but they know very little about their insect host factors.
Dongmin's laboratory found that the representative of the Tc toxin family (pTc) is less toxic to multiple human cell lines, and is more toxic to fruit fly S2R+ cytotoxic. The 5 pM toxin can make 50% of the fruit fly cells present a toxic phenotype, which implies that there is a receptor highly specific to the Tc toxin on the surface of fruit fly S2R+ cells. At the same time, Professor Norbert Perrimon's laboratory of Harvard Medical School developed a library used for whole-gene CRISPR/Cas9 screening in Drosophila S2R+ cells in 2018, using integrase-mediated site-directed integration of gRNA genomes, without the need for lentivirus transfection. The authors found that pTc toxin can inhibit S2R+ cell division by modifying actin in cells, but the genome of poisoned S2R+ cells can still maintain replication, which in turn leads to the increasing volume of poisoned S2R+ cells over time. It is precisely using this feature that in the actual screening, the author cleverly used a 30-micron pore cell screen to isolate poisoned cells and cells that are resistant to toxins. Through several rounds of enrichment and high-throughput sequencing of , a single transmembrane Mucin family protein, Visgun, was finally identified as a pTc toxin-specific receptor.
Figure 1: Tc toxin Whole genome screening
Then the researchers further revealed that Aedes aegypti mosquito , Malignus mosquito and Coleoptera can all recognize pTc toxins efficiently, while Fallia tumour and tumour Visgun homologous proteins derived from Lepidoptera and Visgun homologous proteins cannot be used as receptors. By comparing their protein sequences, it is suggested that highly enriched O-glycan modification sites are a necessary condition for whether Visgun homologous proteins in different insect species can be used as pTc toxin receptors. Interestingly, the human homologous protein CD164, just lacks this feature, and these findings explain the choice specificity of Tc toxins for hosts at the molecular level.
Finally, the author used fruit fly as the insect host model and found that Visgun protein was mainly expressed in blood cells, and these cells are the main immune system of insects. Knocking out of Visgun protein can reduce the sensitivity of primary blood cells to pTc toxins, and these blood cells still maintain the ability to phagocytize bacteria after being pretreated by pTc toxin. After being infected with luminescent vasculitis, Visgun protein knockout flies also showed a lower lethality and pathogen load, which revealed that in the early stages of pathogen infection, after low doses of Tc toxin are secreted into the insect body cavity, they can enter insect blood cells through highly specific Visgun receptors, creating conditions for further colonization of pathogenic bacteria and ultimately killing the host by destroying the insect immune system defense line.
Figure 2: Knocking out Visgun protein reduces sensitivity of Drosophila blood cells to Tc toxins
Paper link:
https://doi.org/10.1038/s41586-022-05250-7
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