CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut symbiont impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

Shivanand Hegde, Pornjarim Nilyanimit, Elena Kozlova, Hema P Narra, Sanjeev K Sahni, Grant L Hughes

Preprint posted on 14 August 2018

Article now published in PLOS Neglected Tropical Diseases at

Gut microbiota influence mosquito vectorial capacity: a study identifies the role of Enterobacter ompA gene in biofilm formation and gut colonization of A. aegypti.

Selected by Snehal Kadam

Categories: genetics, microbiology

Context and background: Mosquitoes are home to many microbes, including various human pathogens and these mosquitoes act as vectors for their transmission. Aedes aegypti mosquitoes are the vector for major illnesses caused by dengue, yellow fever and Zika viruses. However, in recent years, studies have identified a role for gut bacterial microbiota in influencing vector capacity. For example, studies have shown that the absence of the endogenous bacterial microbes can cause a two-fold increase in dengue virus load within the Aedes mosquito gut. Thus it is important to understand the Aedes gut colonization by these bacteria. Like various host-pathogen interactions, bacterial genes have been shown to be important in gut colonization in invertebrates. In Sodalis glossinidius, a phylogenetic relative of A. aegypti, the ompA gene was shown to be important for biofilm formation as well as colonization of the gut of Tsetse flies. The flies had lower mortality when they harboured Sodalis strains expressing the ompA gene from a pathogen E.coli strain. These findings, along with the studies suggesting an influence of the bacterial microbiome on vectorial capacity, argue for an important role of bacterial genetic factors in biofilm formation, gut colonization and thus a role in virulence.

Using the CRISPR-Cas9 system in the host-associated microbe Enterobacter species, isolated from A. aegypti, this study takes a genetics approach to understand the role of the ompA in biofilm formation and gut colonization.


Experimental setup: The study makes use of the no-SCAR (Scarless Cas9 Assisted Recombineering) method. This method combines the λ-Red system with a Cas9 system to enable gene editing without chromosomal markers for selection. This was used to create an ompA disruption mutant. The authors then compared the ompA mutant with the wild-type (WT) as well as a complement strain. Biofilm formation was assayed using crystal violet staining. In order to study effects on gut colonization, infection assays were carried out in mono-association to avoid the influence of any other microbes present in the gut. A. aegypti larvae and adults were assayed for microbial infection by homogenization and CFU counting. CRISPR was also used to insert the mCherry gene or a gentamicin resistance gene into the ompA sequence, effectively creating a mutant and also showing that the method could be used in Enterobacteria to make insertions. To visualize the colonization, midguts were dissected, fixed and stained.


Important Results: After observing biofilm-resembling structures in the gut of A. aegypti upon infection with an Enterobacter isolate, this study choose to examine the role of a bacterial gene, ompA, in biofilm formation and gut colonization. Crystal violet staining showed that the ompA mutant had a significant defect in biofilm formation as compared to the WT and complemented strain. In the infection assays, a reduction in bacterial counts was observed for the mutant in both larvae and adult mosquitos, indicating that the ompA gene is important for colonization in both life stages. The number of mosquitos infected, or the prevalence of the infection was much reduced for the ompA mutant in the adults, with only 45% adults infected as compared to WT (95%) and the complement strain (88%). No such difference was observed for the larval stage. This difference may be attributed to differences in colonization in different life stages. The functionality of the gene insertions of mCherry ( visualized as fluorescent bacteria on a plate as well as within the gut) and gentamicin resistance (ability to grow on gentamicin plates) shows that the CRISPR system can be used to create insertions in other non-model bacteria.


Interesting aspects of the study: This study shows a new application of CRISPR-Cas9 in enteric bacteria found in invertebrates. The ability to create functional gene insertions as well as deletions has great implications for our understanding of the host-microbe relation. The approach used in this study can be applied to gain more insights about the role of bacterial genetics in host colonization and survival within the host. Literature also suggests an influence of these gut bacteria on vectorial capacity, with some having inhibitory effects on viruses that use A. aegypti as a vector. Using this technique to create strains that reduce vectorial capacity could be a strategy to tackle the numerous illnesses caused by the vector A. aegypti. This approach is being used to tackle dengue by the use of Wolbachia-infected mosquitos, since Wolbachia interferes with pathogen transmission and also affects lifespan.

In summary, the findings of this preprint indicate an important role for ompA in gut colonization and biofilm formation and add to the pool of data correlating biofilms and gut colonization across various gut-associated microbes.


References/Further Reading:

  • Dickson, Laura B., et al. “Diverse laboratory colonies of Aedes aegypti harbor the same adult midgut bacterial microbiome.” Parasites & vectors1 (2018): 207.
  • Minard, Guillaume, Patrick Mavingui, and Claire Valiente Moro. “Diversity and function of bacterial microbiota in the mosquito holobiont.”Parasites & vectors1 (2013): 146.
  • Hoffmann, A. A., et al. “Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.” Nature7361 (2011): 454.
  • Hegde, Shivanand, Jason L. Rasgon, and Grant L. Hughes. “The microbiome modulates arbovirus transmission in mosquitoes.” Current opinion in virology15 (2015): 97-102.
  • Kramer, Laura D., and Alexander T. Ciota. “Dissecting vectorial capacity for mosquito-borne viruses.” Current opinion in virology15 (2015): 112-118.
  • Weiss, Brian, and Serap Aksoy. “Microbiome influences on insect host vector competence.” Trends in parasitology11 (2011): 514-522.
  • Reisch, Christopher R., and Kristala LJ Prather. “S carless C as9 A ssisted R ecombineering (no‐SCAR) in Escherichia coli, an Easy‐to‐Use System for Genome Editing.” Current protocols in molecular biology1 (2017): 31-8.


Tags: biofilms, gut colonization, mosquito, mosquito gut microbiota, ompa, vectorial capacity

Posted on: 21 September 2018


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