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

Hegde, Shivanand, Nilyanimit, Pornjarim, Kozlova, Elena, Anderson, Enyia, Narra, Hema P, Sahni, Sanjeev K, Heinz, Eva ORCID: https://orcid.org/0000-0003-4413-3756 and Hughes, Grant ORCID: https://orcid.org/0000-0002-7567-7185 (2019) 'CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes.'. PLoS Neglected Tropical Diseases, Vol 13, Issue 12, e0007883.

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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.

Item Type:	Article
Subjects:	QU Biochemistry > Genetics > QU 550 Genetic techniques. PCR. Chromosome mapping QX Parasitology > QX 4 General works QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes QX Parasitology > Insects. Other Parasites > QX 525 Aedes
Faculty: Department:	Biological Sciences > Vector Biology Department
Digital Object Identifer (DOI):	https://doi.org/10.1371/journal.pntd.0007883
Depositing User:	Samantha Sheldrake
Date Deposited:	09 Dec 2019 12:16
Last Modified:	09 Dec 2019 12:16
URI:	https://archive.lstmed.ac.uk/id/eprint/13267

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