Project

Introduction

Mosquitoes killed around 725,000 human in a year and are listed as the top 1 cause of death. They are considered as the deadliest animal in the world. Mosquitoes carry mosquito-borne infectious diseases and transmit them by blood sucking from people to people.

A syringe is a medical tool for many purposes. It is used to collect blood from human body and inject drug or vaccine for disease control. However, it is difficult to be recycled and makes environment dangerous due to its needle. Biodegradable alternatives should be considered for environmentally friendly issue.

Poor-resource areas have problems controlling infectious diseases. There are lacks of healthcare volunteers, laboratory facility and even electrical power supply. Those make the situation more difficult when the epidemic occurs.

Genetically engineered (GE) mosquitoes have been designed to suppress populations and reduce mosquito-borne diseases. To solve the problems of pathogen-transmitting mosquitoes, biodegradable syringe and limited-resource countries, could GE mosquitoes be a smart approach in synthetic biology?

Pest Control

Pest control by fluorescence has been first demonstrated by Dr. Yu-Chan Chao in 1996 and published the research paper on Nature. The diamondback moth larvae were infected with recombinant baculoviruses carrying green fluorescence gene. His study increased the public awareness of benefits of the application of genetic engineering.

Xenosurveillance

Xenosurveillance is a novel biotechnology that utilizes blood-fed mosquitoes to conduct surveillance for human and livestock viral pathogens. It could be used to uncover infectious diseases that may soon cause epidemics.

To understand the stability of pathogens digested in midgut of mosquitoes, Dr. Yu Yang fed Anopheles stephensi mosquitoes with non-replicable dengue viruses. 4, 8, 16 and 24 hours post-meal, respectively, RNAs were extracted and subjected to qRT-PCR. The result showed the viral RNA decreased over time but remains detectable for 24 hours after blood feeding (Am J Trop Med Hyg., 2015).

Mosquito Signaling

Mosquito immune defense signaling involves well-studied and well-known Toll and Imd intracellular pathways to trigger antimicrobial peptide (AMP) production. Gram-positive bacteria induce Toll signaling, while Gram-negative bacteria induce Imd signaling. However, some promoters may be activated in a synergistic and cross-talk way. Even though Mosquito-borne viruses are controlled by immune signaling in mosquitoes in a currently unidentified pathway, AMP promoter activities were enhanced in mosquito cells in the presence of the viruses.

To investigate the regulations of AMP in Aedes aegypti cells, Dr. Rudian Zhang cultured Aag2 cells isolated from Aedes aegypti and fed the cells with bacteria such as E. coli, B. subtilis, etc. The RNAs of the cells were extracted 12 hours after bacterial challenge. The AMP gene expression levels were analyzed by qRT-PCR with specific AMP primers. The data represented many of AMP promoters can be activated by challenging with Gram-negative and Gram-positive bacteria. In addition, some are regulated synergistically by cross-talking Toll and Imd pathways such as GAM1, CecN and DefA (Front Cell Infect Microbiol., 2017).

Experimental Design

To genetically engineer mosquitoes as biodegradable syringes and applied in blood surveillance, we use synthetic biology technique to develop blood testing GE mosquitoes in three ways to detect universal mosquito-borne pathogens, human non-mosquito-borne HIV viruses and versatile human and livestock viruses, respectively.

First of all, to detect mosquito-borne pathogens, we created a GFP reporter system under the control of an AMP promoter (i.e., GAM1 promoter) through Toll signaling. The Toll forms dimer when sensing the pathogens followed by signaling to activate MyD88 and Rel1. The activated transcription factor Rel1 translocates to the nucleus and drives the AMP promoters to express antimicrobial peptides. We have tested this system with Gram-negative E. coli and Gram-positive B. subtilis.

Second, to detect non-mosquito-borne human viruses, we designed and developed a GFP reporter system for HIV with synthetic chimera receptor composed of extracellular human CD4 domain fused with transmembrane and intracellular drosophila Toll domains. Dimerization of CD4 constitutively activates Toll signaling and induces AMP (Drosomycin) expression. In the existence of HIV particles, the gp120 of HIV will attach CD4 and prevent the dimerization followed by stop the signaling. As a result, GFP expression will be decreased by time. The system has been tested in response to gp120.

For further extending the application to versatile blood-transmitted viruses, we designed and planned a viral glycoprotein display system on Toll. Viral specific antibody can catch the glycoprotein which will multimerize and activate the Toll signaling. In the existence of virus in the blood, the free form of viral glycoprotein can compete and block the binding sites of antibody receptor, resulting in inhibiting the signaling.

Reference

1.Nature (1996) Pest control by fluorescence. https://www.nature.com/articles/380396b0

2.Am J Trop Med Hyg. (2015) Feasibility of Using the Mosquito Blood Meal for Rapid and Efficient Human and Animal Virus Surveillance and Discovery. https://www.ncbi.nlm.nih.gov/pubmed/26416112

3.Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells. Rudian Zhang, et al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291090/

4.Cell. (1988) The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. https://www.ncbi.nlm.nih.gov/pubmed/2449285

5.N
at Rev Immunol. (2006) Toll-like receptors as molecular switches. https://www.ncbi.nlm.nih.gov/pubmed/16917510

6.J Immunol. (2011) The Drosophila Toll signaling pathway. https://www.ncbi.nlm.nih.gov/pubmed/21209287

7.Retrovirology. (2006) Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1524797/

8.J Immunol. (2006) Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC. https://www.ncbi.nlm.nih.gov/pubmed/16709847

9.J Immunol. (2006) Triggering of T cell activation via CD4 dimers. https://www.ncbi.nlm.nih.gov/pubmed/16622011

10.J Biol Chem. (2014) Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization. https://www.ncbi.nlm.nih.gov/pubmed/24550395

11.UniProtKB - P01730 (CD4_HUMAN) https://www.uniprot.org/uniprot/P01730

12.UniProtKB - P08953 (TOLL_DROME) https://www.uniprot.org/uniprot/P08953