Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence
| Autor: | B. K. L. Sim, Abraham G. Eappen, Stephen L. Hoffman, Robert A. Harrell, Peter F. Billingsley, M. Hosseini, W. Reid, E. Inbar, Robert Alford, Sumana Chakravarty, Tao Li |
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| Rok vydání: | 2021 |
| Předmět: |
Male
Plasmodium Physiology Eggs Artificial Gene Amplification and Extension Disease Vectors Mosquitoes Polymerase Chain Reaction Synthetic Genome Editing Genome Engineering Medical Conditions Reproductive Physiology Medicine and Health Sciences Biology (General) Reproduction Crispr Anopheles Eukaryota Fecundity Body Fluids Insects Infectious Diseases Blood Insect Proteins Engineering and Technology Female Synthetic Biology Anatomy Research Article Arthropoda QH301-705.5 Immunology Bioengineering Mosquito Vectors Biology Research and Analysis Methods Microbiology Population Metrics Virology Parasite Groups parasitic diseases Parasitic Diseases Genetics Animals Microbiome Molecular Biology Techniques Molecular Biology Gene Anopheles stephensi Bacteria Population Biology fungi Organisms Oocysts Biology and Life Sciences Plasmodium falciparum RC581-607 Synthetic Genomics biology.organism_classification Invertebrates PfSPZ vaccine Malaria Insect Vectors Species Interactions Parasitology Immunologic diseases. Allergy CRISPR-Cas Systems Digestive System Zoology Entomology Apicomplexa |
| Zdroj: | PLoS Pathogens PLoS Pathogens, Vol 17, Iss 11, p e1009770 (2021) |
| ISSN: | 1553-7374 |
| DOI: | 10.1371/journal.ppat.1009770 |
| Popis: | PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1’s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males. Author summary The Leucin-Rich Protein LRIM1 is an important component of the complement-like cascade in Anopheles mosquitoes and was shown previously to have anti-parasitic activity, supporting melanization and phagocytosis of Plasmodium ookinetes. To improve the manufacturing of Sanaria’s PfSPZ vaccine, by increasing Pf infection intensity in the mosquitoes, we have generated an LRIM1 knockout line (Δaslrim1) using CRISPR-Cas9. Both bacterial load and the bacterial community composition altered significantly in Δaslrim1; these alterations were associated with reduced mosquito survival and, contrary to our expectations, dramatic reduction in Pf infection intensity. Reproductive capacity was reduced significantly in both females (fecundity) and males (fertility) of the Δaslrim1 line. Δaslrim1 mosquitoes cannot be used in the manufacturing of PfSPZ vaccine, but the data reveal novel and unexpected roles of LRIM1 in different aspects of the mosquitoes’ vectorial capacity, such as survival and reproduction. Importantly, our findings demonstrate the importance of LRIM1 in controlling the microflora in the mosquitoes and thereby allowing successful infection of the parasites. |
| Databáze: | OpenAIRE |
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