Abstract
Symbiotic bacteria are pervasive in mosquitoes and their presence can influence development, reproduction, and immunity of their host. It is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, but we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. While CRISPR/Cas9 gene editing is a powerful tool to modify bacterial genomes this approach has yet to be applied to insect symbionts. To demonstrate that gene editing can be completed in non-model bacterial species isolated from insects and to investigate the role of bacterial genes in gut colonization, we mutated the outer membrane protein A (ompA) gene of an Enterobacter symbiont using the CRISPR/Cas9 system. The ΔompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adults, 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 bacterial genetic factors are important for adult gut colonization. Integration of genes (antibiotic resistance and fluorescent markers) into the symbiont genome demonstrated this technology can be exploited to develop novel symbiotic control strategies to interfere with arboviral pathogens such Chikungunya, Zika and Yellow fever viruses transmitted by Aedes mosquitoes. Our results shed insights onto 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.
Importance CRISPR/Cas9 gene editing approaches have revolutionized several biological fields, however despite their applicability for altering bacterial genomes, few studies use this technology in microbes that associated with eukaryotic hosts. Here we use this editing approach to knockout a gene encoding a membrane protein in an Enterobacter isolated from Aedes mosquitoes and show this gene is essential for biofilm formation and promotes bacteria colonization of the gut. A reduced bacterial load of the mutant compared to the wild type or complement strains, was seen in both larval and adult mosquitoes, however this was most evident in adults, likely due differences in the mode of acquisition of microbes at each life stage. Our work extends CRISPR/Cas9 genetic manipulation into a new bacterial species, and in conjunction with other studies, suggests that members within Enterobacteriaceae are amenable to genome engineering by this approach. This study will facilitate the development of novel microbial-based approaches to mitigate mosquito-borne disease.
