1 CRISPR/Cas9-Mediated Gene Deletion of the Ompa Gene in an Enterobacter Gut

bioRxiv preprint doi: https://doi.org/10.1101/389957; this version posted August 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut 2 symbiont impairs biofilm formation and reduces gut colonization of Aedes 3 aegypti mosquitoes 4 5 Shivanand Hegde1, Pornjarim Nilyanimit2, Elena Kozlova1, Hema P. Narra1, Sanjeev K. 6 Sahni1, Grant L. Hughes3,4,# 7 8 1 Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA. 9 2 Center of Excellence in Clinical Virology, Chulalongkorn University, Bangkok, 10 Thailand. 11 3 Department of Pathology, Institute for Human Infections and Immunity, Center for 12 Tropical Diseases, Center for Biodefense and Emerging Infectious Disease. University 13 of Texas Medical Branch, Galveston, TX, USA. 14 4 Current address: Department of Vector Biology and Department of Parasitology, 15 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK. 16 #Corresponding author. Email: [email protected] 17 18 19 20 Running head: CRISPR/Cas9 engineering of a gut bacterium. 21 1 bioRxiv preprint doi: https://doi.org/10.1101/389957; this version posted August 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Abstract 23 Symbiotic bacteria are pervasive in mosquitoes and their presence can influence 24 development, reproduction, and immunity of their host. It is evident that environmental 25 and host genetic factors contribute in shaping the microbiome of mosquitoes, but we 26 have a poor understanding regarding how bacterial genetics affects colonization of the 27 mosquito gut. While CRISPR/Cas9 gene editing is a powerful tool to modify bacterial 28 genomes this approach has yet to be applied to insect symbionts. To demonstrate that 29 gene editing can be completed in non-model bacterial species isolated from insects and 30 to investigate the role of bacterial genes in gut colonization, we mutated the outer 31 membrane protein A (ompA) gene of an Enterobacter symbiont using the CRISPR/Cas9 32 system. The ∆ompA mutant had an impaired ability to form biofilms and poorly infected 33 Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adults, 34 the mutant had a significantly reduced infection prevalence compared to the wild type or 35 complement strains, while no differences in prevalence were seen in larvae, suggesting 36 bacterial genetic factors are important for adult gut colonization. Integration of genes 37 (antibiotic resistance and fluorescent markers) into the symbiont genome demonstrated 38 this technology can be exploited to develop novel symbiotic control strategies to 39 interfere with arboviral pathogens such Chikungunya, Zika and Yellow fever viruses 40 transmitted by Aedes mosquitoes. Our results shed insights onto the role of ompA gene 41 in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing 42 can be employed for genetic manipulation of non-model gut microbes. 43 44 2 bioRxiv preprint doi: https://doi.org/10.1101/389957; this version posted August 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 45 Importance 46 CRISPR/Cas9 gene editing approaches have revolutionized several biological fields, 47 however despite their applicability for altering bacterial genomes, few studies use this 48 technology in microbes that associated with eukaryotic hosts. Here we use this editing 49 approach to knockout a gene encoding a membrane protein in an Enterobacter isolated 50 from Aedes mosquitoes and show this gene is essential for biofilm formation and 51 promotes bacteria colonization of the gut. A reduced bacterial load of the mutant 52 compared to the wild type or complement strains, was seen in both larval and adult 53 mosquitoes, however this was most evident in adults, likely due differences in the mode 54 of acquisition of microbes at each life stage. Our work extends CRISPR/Cas9 genetic 55 manipulation into a new bacterial species, and in conjunction with other studies, 56 suggests that members within Enterobacteriaceae are amenable to genome 57 engineering by this approach. This study will facilitate the development of novel 58 microbial-based approaches to mitigate mosquito-borne disease. 59 60 Keywords: CRISPR/Cas9, Gut microbe, Gut colonization, Biofilm, 61 Paratransgenesis, Paratransgenic 62 3 bioRxiv preprint doi: https://doi.org/10.1101/389957; this version posted August 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 63 Introduction. 64 Mosquitoes harbor a community of microbes within their guts. In general, the gut- 65 associated microbiome of mosquitoes tends to have low species richness but can differ 66 greatly between individuals and habitats1-8. Importantly, these microbes can modulate 67 many host phenotypes, several of which can influence vectorial capacity9-11. As such, it 68 is imperative that we understand how the microbiome is acquired and maintained within 69 mosquito vectors. While environmental factors unquestionably influences mosquito 70 microbiome composition and abundance2-4,8, and studies are elucidating the role of 71 microbial interactions5,7,12,13 and host genetic factors14-18 in shaping the microbiome, we 72 have a poor understanding regarding bacterial factors that influence colonization of the 73 mosquito gut. 74 75 In other invertebrates, several bacterial genes have been implicated in gut colonization. 76 For example, a genome wide screen exploiting transposon-sequencing found a suite of 77 genes from the bacterium Snodgrasselia involved in colonization of the honey bee gut19. 78 These bacterial genes were classified into the broad categories of extracellular 79 interactions, metabolism and stress response19. Knock out of a purine biosynthesis 80 gene in Burkholderia impaired biofilm formation and reduced bacterial colonization rates 81 in a bean bug. Biofilm formation was also shown to play a role in virulence of 82 pathogenic Pseudomonas in artificial infections of Drosophila, with strains that lacked 83 the capacity to form biofilms being more virulence to the host, while a hyperbiofilm strain 84 was less virulent than the WT strain20. In other blood feeding invertebrates, bacterial 85 genetics also appears critical for host colonization. Knockout of the type II secretion 4 bioRxiv preprint doi: https://doi.org/10.1101/389957; this version posted August 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 86 system in Aeromonas veronii reduced infection in Hirudo verbena leeches21. In Tsetse 87 flies, the outer-membrane protein A (ompA) gene of Sodalis glossinidius is essential for 88 symbiotic interactions22. Sodalis mutants lacking the ompA gene poorly colonized the fly 89 gut compared to the wild type (WT) Sodalis22 and the mutant strain also had a reduced 90 capacity to form biofilms23. Heterologous expression of the ompA gene from pathogenic 91 Escherichia coli in Sodalis mutants induced mortality in the fly implicating this gene as a 92 virulence factor in pathogenic bacteria22. Taken together, these studies suggest that 93 bacterial genetic factors are critical for host colonization of invertebrates and that biofilm 94 formation facilitates symbiotic associations in insects. 95 96 In mosquitoes, few studies have investigated how bacterial genetics affect gut 97 colonization. However, evidence from experimental evolution studies suggests bacterial 98 genetics plays a critical role. In two separate studies, Enterobacter was selected for 99 increased persistence in the gut of Anopheles gambiae mosquitoes, the major malaria 100 vector in sub-Saharan Africa, by repeatedly infecting mosquitoes with strains that 101 persisted in the gut for longer periods of time24,25. Transcriptomics comparisons of 102 effective and ineffective colonizers in liquid media identified 41 genes that were 103 differentially expressed between these two strains25, further implicating the importance 104 of bacterial genetics in mosquito infection, however the role of these genes in 105 colonization of the mosquito gut has not been resolved. In a separate study, in vitro 106 screening of a transposon mutant library of Enterobacter identified a waaL gene mutant 107 that was insensitive to oxidative stress26. The waaL gene encodes an O antigen ligase 108 which is needed for attachment of the O antigen to lipopolysaccharide and the mutant 5 bioRxiv preprint doi: https://doi.org/10.1101/389957; this version posted August 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 109 was found to have lower rates of colonization of the midguts of Anopheles 110 mosquitoes26. 111 112 Gene knockouts approaches in bacteria provide compelling evidence of the role of 113 bacterial genes in host-microbe interactions21-23,26-28. In general, most studies use 114 transposon mutagenesis for gene knockout, which requires screening of

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