CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

Abstract

BackgroundSymbiotic 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 findingsTo 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/SignificanceOur 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.

Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity

“…Microbiome assembly in mosquitoes is influenced by the environment, host and bacterial genetics, and stochastic processes. While the host is instrumental in maintaining microbiome homeostasis [10][11][12][13][14], evidence is emerging that bacterial genetics and microbe-microbe interactions also dictate the prevalence and abundance of microbiota [15][16][17][18]. These processes are important as the microbiome can influence the ability of mosquitoes to transmit pathogens [3,19,20], but potentially other traits related to vectorial capacity.…”

Gut-associated bacteria invade the midgut epithelium of Aedes aegypti and stimulate innate immunity and suppress Zika virus infection in cells

“…Phylogenetic analysis of multilocus sequence typing as described in [76] shows the phylogenetic position of Serratia sp. Alb1 (in red; this study) and C. neteri Alb1 (in blue; [11]). The tree analysis was performed using PhyML under the general time-reversible model with rapid aLRT bootstrap support as implemented as default in seaview (v4.7; [77].…”

“…The 16S rDNA PCR was performed as described previously [75] and the PCR product was Sanger sequenced to identify the bacterial species. To further classify the gut-associated bacteria we completed multilocus sequence typing (MLST) as described previously [11, 76]. The MLST sequences were aligned, concatenated and a maximum likelihood tree under the LG model and rapid bootstrap was constructed using Seaview [77] (Fig.…”

“…It is likely that these processes contribute to the considerable variability seen in the adult microbiome [7–9]. While our understanding of genetic factors that influence host-microbe interactions and microbiome acquisition are expanding [10, 11], we still have a poor knowledge of these interactions at the cellular level. Given the importance of the microbiome on mosquito traits relevant for vectorial capacity and vector competence [12–15], understanding processes that influence microbiome homeostasis is critical for developing microbial-based control strategies [16, 17].…”

CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli

“…Klebsiella pneumoniae and the industrial relevant Enterobacter aerogenes (92,93). The streamlined no-Scar method was recently applied to manipulate the mosquito microbiome symbiont Cedecea neteri, a rare non-model Enterobacteriaceae member with limited characterization (94). With minimal modification to the original method, ompA knockouts were generated to study its contribution in C. neteri biofilm formation and subsequent colonization of the gut.…”