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

Shivanand Hegde, Pornjarim Nilyanimit, Elena Kozlova, Enyia R. Anderson, Hema P. Narra, Sanjeev K. Sahni, Eva Heinz, Grant L. Hughes

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)e0007883
JournalPLoS neglected tropical diseases
Volume13
Issue number12
DOIs
StatePublished - Dec 1 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
Aedes
Gene Deletion
Biofilms
Culicidae
Genes
Yellow fever virus
Germ-Free Life
Bacterial Genomes
Dengue
OMPA outer membrane proteins
Microbiota
Microbial Drug Resistance
Larva
Insects
Genome
Technology
Bacteria
Phenotype

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

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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 R.; Narra, Hema P.; Sahni, Sanjeev K.; Heinz, Eva; Hughes, Grant L.

In: PLoS neglected tropical diseases, Vol. 13, No. 12, 01.12.2019, p. e0007883.

Research output: Contribution to journalArticle

Hegde, Shivanand ; Nilyanimit, Pornjarim ; Kozlova, Elena ; Anderson, Enyia R. ; Narra, Hema P. ; Sahni, Sanjeev K. ; Heinz, Eva ; Hughes, Grant L. / CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes. In: PLoS neglected tropical diseases. 2019 ; Vol. 13, No. 12. pp. 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.",
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T1 - CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

AU - Hegde, Shivanand

AU - Nilyanimit, Pornjarim

AU - Kozlova, Elena

AU - Anderson, Enyia R.

AU - Narra, Hema P.

AU - Sahni, Sanjeev K.

AU - Heinz, Eva

AU - Hughes, Grant L.

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AB - 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.

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