A Gut Commensal Bacterium Promotes Mosquito Permissiveness to Arboviruses

Wu, Pa; Sun, Peng; Nie, Kaixiao; Zhu, Yibin; Shi, Mingyu; Xiao, Changguang; Liu, Han; Liu, Qiyong; Zhao, Tongyan; Chen, Xiaoguang; Zhou, Hongning; Wang, Penghua; Cheng, Gong

doi:10.1016/j.chom.2018.11.004

Cited by 181 publications

(136 citation statements)

References 54 publications

Supporting

Mentioning

132

Contrasting

Unclassified

Order By: Relevance

“…Symbiotic examples of these ride-sharing characteristics have been well-documented among arthropods and their microbiota. Specific instructive examples of beneficial interactions include two bacterial endosymbionts of the glassy-winged sharpshooter (Homalodisca coagulata) each of which synthesizes different essential metabolites for the host and possibly for each other (Wu et al, 2006) and a gut commensal bacterium of the Aedes aegypti mosquito that facilitates arboviral infection by altering the gut epithelial layer (Wu et al, 2019). An example of an antagonistic interaction in arthropods includes the inhibition of Plasmodium falciparum development within the midgut of Anopheles gambiae mosquitos resulting from generation of reactive oxygen species by an endogenous Enterobacter strain (Cirimotich et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

Stewart

Bloom

2020

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

The deer tick Ixodes scapularis transmits a variety of disease agents in the United States, spreading the bacteria that causes Lyme borreliosis, the protozoan agent of babesiosis, and viruses such as Powassan. However, a variety of other organisms have also evolved symbiotic relationships with this tick species, and it seems likely that some of these microbes have simultaneously coevolved mechanisms to impact each other and their tick host. The number of organisms identified as I. scapularis symbionts has increased seemingly exponentially with the advent of PCR and next generation sequencing technologies, but convincing arguments have proposed that some of these are of environmental origin, unadapted to surviving the physiological conditions of the tick or that they are artifacts of ultrasensitive detection methods. In this review, we examine the diversity of the known microbes occurring within the I. scapularis microbiome, the evidence for interactions between microbes, and discuss whether some organisms reported to be symbionts of I. scapularis are experimental artifacts.

show abstract

Section: Introductionmentioning

confidence: 99%

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

Stewart

Bloom

2020

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

show abstract

“…In recent studies that used qRT-PCR analysis to detect ZIKV RNA in mosquitoes, most used hydrolysis probes as fluorescent labels. More than half of the studies that used fluorescent probes to detect ZIKV RNA also used viral extraction kits and four did not describe how RNA extraction was performed [9,14,16,18,22,23,31,32,33,34,35,36,37,38,39,40,41,42,44,45,46,48,49].…”

Section: Discussionmentioning

confidence: 99%

“…The ZIKV 1086 / ZIKV 1162c primers and the 1107-FAM probe [15] can be considered the gold standard to ZIKV RNA detection in mosquito samples since it has been preferentially used in most recent studies [31,33,34,35,36,38,46,49,51]. The ZIKV 1086 and ZIKV 1162c have been successfully used to detect ZIKV RNA in cell lines, using SYBR Green as a dye instead the 1107-FAM [63], and could be a less expensive alternative to ZIKV detection in mosquitoes.…”

Section: Assay Sensitivity Reproducibility and Specificity Evaluationmentioning

confidence: 99%

“…In recent studies utilizing RT-qPCR to detect ZIKV RNA in mosquitoes, which mostly analyzed vector competence, it is possible to observe a methodological trend favoring the utilization of hydrolysis probes as a fluorescent label (83% of the papers in literature) and the use of viral RNA extraction kits to obtain the RNA templates (56% of the studies). However the number of studies could actually be higher since some of them do not specify the extraction RNA method used for the experiments [9,14,16,18,19,21,22,23,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51]. Although this approach has demonstrated to be relatively effective in detecting ZIKV RNA, since the isolation of viral RNA is prioritized, it does not permit the study of the gene expression in mosquito genes during viral infection using the same samples.…”

mentioning

confidence: 99%

See 1 more Smart Citation

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

Araujo

Feitosa-Suntheimer

Gold

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is qRT-PCR, yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito RNA. In this work we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing for confident detection. Methods: Our new oligo design to detect ZIKV RNA included in silico analysis of both viral and mosquito ( Ae. aegypti and Ae. albopictus )genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedes genomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes. Standard curves constructed with in vitro transcribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay. Results: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegypti immune response to virus infection. Conclusions: The experimental approach to detect ZIKV RNA in Aedes aegypti presented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

show abstract

“…In the vector biology field, gene knockout approaches can be used to interrogate the role of bacterial genes responsible for host-microbe interactions, while the ability to integrate genes into the bacterial symbiont genome has great potential for applied paratransgenic control strategies [10, 45–47]. Previously, manipulation of non-model symbionts that associate with insect vectors have has been accomplished by plasmid transformation [48–55] or stable transformation of the genome using transposons or integrative plasmids [56–63], but the use of CRISPR/Cas9 gene editing in insect gut symbionts has yet to be accomplished. For paratransgenic strategies, stable site-specific integration of transgenes into the symbiont genome is critical, and as such, the application of CRISPR/Cas9 gene editing technology to non-model bacteria that associate with insect vectors will stimulate research in this field.…”

Section: Introductionmentioning

confidence: 99%

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

Hegde

Nilyanimit

Kozlova

et al. 2018

Preprint

View full text Add to dashboard Cite

BackgroundSymbiotic 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 FindingsTo investigate the role of bacterial 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 an Enterobacter 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 adults 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 bacterial 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 these symbionts genome and demonstrated that these genes were functional in vitro and in vivo.Conclusions/SignificanceOur results shed insights onto 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.Author summaryMicrobiota profoundly affect their host but few studies have investigated the role of bacterial genetics in host-microbe interactions in mosquitoes. Here we applied the CRISPR/Cas9 gene editing system to knock out a membrane protein in Enterobacter, which is a dominant member of the mosquito microbiome. The mutant strain lacked the capacity to form biofilms, infected larvae and adults at lower titers, and had a reduced prevalence in adults. The lower prevalence in adults, but not larvae, likely reflects the difference in the modes of bacterial acquisition from the larval water of these two life stages. Importantly from an applied perspective, we also demonstrated that this editing technology can be harnessed for site-specific integration of genes into the bacterial chromosome. In proof-of-principle studies we integrated either a fluorescent protein or gene conferring antibiotic resistance into the bacterial genome and showed these transgenes were functional in mosquitoes. The specificity, flexibility, and simplicity of this editing approach in non-model bacteria will be useful for developing novel symbiotic control strategies to control arthropod-borne disease.

show abstract

A Gut Commensal Bacterium Promotes Mosquito Permissiveness to Arboviruses

Cited by 181 publications

References 54 publications

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

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

Contact Info

Product

Resources

About

A Gut Commensal Bacterium Promotes Mosquito Permissiveness to Arboviruses

Cited by 181 publications

References 54 publications

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection &amp; gene expression during ZIKV infection

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

Contact Info

Product

Resources

About

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection