We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes.
- Authors
Hegde, Shivanand; Nilyanimit, Pornjarim; Kozlova, Elena; Anderson, Enyia R.; Narra, Hema P.; Sahni, Sanjeev K.; Heinz, Eva; Hughes, Grant L.
- Abstract
Background: Symbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts. Methodology/Principal findings: To investigate the role of bacterial genetic factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of a Cedecea neteri symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adult mosquitoes, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into the symbionts genome and demonstrated that these genes were functional in vitro and in vivo. Conclusions/Significance: Our results shed insights into the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes. Author summary: Microbiota profoundly affect their host but few studies have investigated the role of bacterial genetics in host-microbe interactions in mosquitoes. Here we applied the CRISPR/Cas9 gene editing system to knockout a membrane protein in Cedecea neteri, which is a dominant member of the mosquito microbiome. The mutant strain had an impaired capacity to form biofilms, infected larvae and adults at lower titers, and had a reduced prevalence in adults. The lower prevalence in adults, but not larvae, likely reflects the difference in the modes of bacterial acquisition from the larval water of these two life stages. Importantly from an applied perspective, we also demonstrated that this editing technology can be harnessed for site-specific integration of genes into the bacterial chromosome. In proof-of-principle studies we integrated either a fluorescent protein or gene conferring antibiotic resistance into the bacterial genome and showed these transgenes were functional in mosquitoes. The specificity, flexibility, and simplicity of this editing approach in non-model bacteria will be useful for developing novel symbiotic control strategies to mitigate the burden of arthropod-borne disease.
- Subjects
AEDES aegypti; DELETION mutation; MOSQUITO vectors; BACTERIAL genetics; MOSQUITOES; DRUG resistance in bacteria; BACTERIAL chromosomes
- Publication
PLoS Neglected Tropical Diseases, 2019, Vol 13, Issue 12, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0007883