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

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.

Fig 1. Midgut infection of C. neteri and E. coli in mono-associations of Aedes mosquitoes.

C. neteri forms a biofilm in the gut of 3–4 day old Ae. aegypti adult mosquitoes (left) while no bacteria were observed in the gut of mosquitoes reared with E. coli under gnotobiotic conditions (right). Bacteria possessed the pRAM-mCherry plasmid, which expresses the mCherry fluorescent protein and conferred resistance to kanamycin. Blue–host nuclei stained by DAPI. Green–host actin cytoskeleton stained with phalloidin. The scale bar is 70 μm.

Fig 2. CRISPR/Cas9 genome editing in bacteria.

A schematic of the editing approach and screening of putative mutants in (A) E. coli and (B) C. neteri. A ~1kb fragment of E. coli BL21(DE3) was deleted using no-SCAR protocol. The 250 bp of the left arm (LA) and right arm (RA) was assembled to generate the 500 bp donor DNA. The transformants were screened via colony PCR with primers binding in regions flanking the deletion. Similar to the strategy employed in E. coli, the knockout of the ompA gene from C. neteri isolated from the mosquito gut was created by deleting the 598 bp fragment. The grey area indicates the PAM site in the ompA gene and arrow shows the cleavage site in the genome. (C) The sequence of the ompA mutation in E. coli and C. neteri was confirmed by Sanger sequencing. The sequence above the gene within the dotted line has been deleted. The chromatogram shows the 10 bp flanking the deletion.

Fig 3. In vitro characterization of the ompA mutation.

(A) The C. neteri ΔompA mutant had a similar growth rate compared to both the WT and the ΔompA/ompA complement in liquid LB media. Five technical replicates were used to create growth curves. (B) The stability of mutant was evaluated in vitro by continuous subculturing in LB media. Genomic DNA from alternative subcultures was used as template for PCR using primers that amplified across the deletion. The stability assay was repeated twice. Two separate gel images were merged to create figure 3B (passage 8 was run on a separate gel to passages 0–6). (C) Biofilm formation was assessed using the CV biofilm assay for the WT, ΔompA mutant and the ΔompA/ompA complement. Two biological replicates were completed. (D) Quantification of the relative biofilm formation normalized by the number of bacteria per well (N = 3). Error bars represent standard error. The assay was repeated twice.

Fig 4. The ΔompA mutant poorly infected mosquitoes.

Infection of C. neteri strains (WT, ΔompA mutant and ΔompA/ompA complement; the former two possessed the pRAM-Cherry plasmid while the latter possessed the pRAM-Cherry-Ent-OmpA plasmid) reared in a mono-association using a gnotobiotic rearing approach for larvae (A and C) and adults (B and D). L4 larvae and 3–4 days post emergence adults were screened for bacterial load by plating on selective LB media with kanamycin to quantify the bacteria. The prevalence of infection (number of mosquitoes infected) between the treatments was calculated comparing the number of infected to uninfected larvae (A) or adults (B). Density of bacteria (CFU/mosquito) in larvae (C) and adults (D). The assay was repeated twice. Results display pooled data from each independent replicate. Box and whiskers show the median, the 25^th and 75^th percentiles and the minimum and maximum values.

Fig 5. The ΔompA mutant does not affect growth rates or development of mosquitoes.

The growth rate (time to pupation) (A) and development (percentage of L1 larvae to reach adulthood) (B) was observed in mosquitoes infected with C. neteri strains (WT, ΔompA mutant and ΔompA/ompA complement) reared in a mono-association. The experiment was done twice with a minimum of 15 individuals. Sample size for panel A indicates number of individuals, while for B indicates the number of replicate flasks. Each flask has 20 mosquitoes.

Fig 6. Integration of mCherry and gentamicin into the C. neteri genome.

Sanger sequence across the integration site, stability of the inserted gene and in vitro expression of the inserted gene for the ΔompA::mCherry (A-C) and the ΔompA:: gentamicin (B-D) strains. The chromatogram shows the sequence spanning the inserted sites. Strains were continually subcultured for 10 passages and PCR was done to examine the stability of the insert (C; ΔompA::mCherry plus WT, D; ΔompA::gentamicin passaged with (ab+) or without (ab-) gentamicin plus WT). mCherry fluorescence (E) or ability to grow on selective media containing gentamicin (F) confirmed the expression of the transgene in vitro. Mosquitoes were inoculated with the C. neteri strains to confirm expression of the transgene in vivo. Dissected midgut infected with ΔompA::mCherry (left) or negative control (right; WT bacteria without expression plasmid) (G). Midguts were stained with phalloidin (green) and DAPI (blue). The scale bar is 30 μM. The WT and ΔompA::gentamicin C. neteri strains were fed to adult mosquitoes for 3 days in a sugar meal before gentamicin was administered to mosquitoes in sugar without bacteria (H). Mosquitoes were collected every second day and CFUs assessed. Pairwise comparisons were conducted at each time point using a T test.