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.

↓Anopheles stephensi (primary vector of malaria)

↓Dengue virus serotype 2 (DENV-2)

↓in human red blood cell and serum (Key Biologics, LLC)

↓RNA extracted at 0, 4, 8, 16, 24 hours after blood feeding

↓DENV-2 copy by qRT-PCR

Results

In Anopheles stephensi, which does not allow DENV-2 to replicate, viral RNA titer will reduce gradually but remains detectable for at least

24 hours in the midgut.

Reference

1.Am J Trop Med Hyg. (2017) The Use of Xenosurveillance to Detect Human Bacteria, Parasites, and Viruses in Mosquito Bloodmeals.

2.PLoS Negl Trop Dis. (2015) Xenosurveillance: a novel mosquito-based approach for examining the human-pathogen landscape.

3.Am J Trop Med Hyg. (2015) Feasibility of Using the Mosquito Blood Meal for Rapid and Efficient Human and Animal Virus Surveillance and Discovery.

Living Syringe

•(In order) To ………., we conducted/performed/did……………..

•The result/data showed/presented/indicated…………..

•Taken our results and previous researches together, mosquitoes could be……………..

Experiment

↓Aedes aegypti

↓10% glucose water with or without heat-killed E. coli (109 cells/ml)

↓gDNA extracted at 0, 6, 18 hours after feeding

↓2 repeats for every group & one control of gDNA of E. coli

↓PCR with specific primer for A. aegypti or E. coli

Primers

•Aedes: (730 bp)
    -Act-2F (5′-ATGGTCGGYATGGGNCAGAAGGACTC-3′),
    -Act-2R (5′-TCGCACTTCATGATSGAGTTGTA-3′)

•E. coil: (970 bp)
    -K12IS-L (5’-CGCGATGGAAGATGCTCTGTA-3’)
    -K12-R (5’-ATCCTGCGCACCAATCAACAA-3’)

Result

Genomic DNAs of Aedes aegypti were confirmed by PCR with Aedes primers in all groups, indicating the integrity of the extracted DNA. Feeding heat-killed bacteria to the mosquitoes, genomic DNAs of E. coli can be detected in the group at 6hr post-feeding and slightly decreased at 18hr post-feeding, demonstrating the existence of non-dividing bacteria at least within overnight in the gut of a mosquito.

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

1.Appl Environ Microbiol. (1995) Rapid and accurate identification of Escherichia coli K-12 strains.

2.J Am Mosq Control Assoc. (2010) Universal primers for the amplification and sequence analysis of actin-1 from diverse mosquito species

3.PLoS One. (2010) Dengue virus inhibits immune responses in Aedes aegypti cells.

GFP System

To create a reporter system, we constructed a GFP expression vector. We amplified a constitutive promoter from Drosophila actin 5c gene and an eukaryotic poly A signal by PCR from pAc5.1 vector. The resulting DNA fragments were assembled with a BioBrick existing part of GFP to generate the reporter vector of Ac5- GFP-polyA / pSB1C3 (K2543004).

To test the reporter system, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA. GFP positive cells and intensity were analyzed 2 days after transfection.

EXPERIMENT

↓C6/36 cells (1.8 x 105 cells/well in a 96-well plate)
↓Liposome-mediated transfection and culture for 2 more days
↓Read fluorescence intensity at Ex/Em = 480/520 nm with a microplate reader
↓Observe GFP+ cells under a fluorescence microscope

RESULT

As data shown here, Ac5 is a strong and constitutive promoters which can drive GFP to high expression level in mosquito cells. And we can transfect more than 50% of GFP positive cell with liposome-mediated DNA delivery.

Mosquito Immune 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.

Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells.
Rudian Zhang, et al.

EXPERIMENT

↓Aag2 cell line from Aedes aegypti
↓E. coli or Bacillus at OD600 = 0.05 (≅ MOT = 10)
↓RNA extracted 12 hours after bacteria challenge
↓qRT-PCR with specific AMP primers

RESULT

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.

REFERENCE

1.Trends Parasitol. (2016) Mosquito Defense Strategies against Viral Infection.
2.Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells

AMP System

Aedes aegypti, a yellow fever mosquito, is a major arbovirus vector to transmit several diseases and spread mosquito-borne viruses such as dengue virus, Ziki virus, yellow fever viruses, etc. It can be genetically modified to control viral transmission. The cell line isolated from Aedes are widely used in the research. The full genome sequence and signaling pathways are defined in the literature. Therefore, it a good host for our study. In our project, we successfully cloned 3 AMP (GAM1, CecN, DefA) promoters from gDNA of Aedes aegypty and confirmed the sequencesby sequences by sequencing.

AMP promoters amplified by PCR

To create AMP reporter system, the AMP promoters of Aedes aegypti amplified from PCR were assembled with GFP-polyA /pSB1C3 (BBa_K2543003)

To test the function of the device, C6/36 cells were transfected with the vectors. And the mosquito cells were challenged with bacteria on 2 days after transfection.

EXPERIMENT

↓C6/36 cells were seeded at the density of 1.8 x 105 cell/well in a 96-well plate
↓Cells were transfected with the AMP-GFP-polyA vectors
↓E. coli was added on 2 days post-transfection at MOI=10
↓GFP positive cells and intensity were analyzed by a fluorescence microscope and a microplate reader at Ex/Em = 480/520 nm,respectively

GAM1-GFP-polyA / pSB1C3 challenged with E. coli

GAM1/CecN/DefA-GFP-polyA / pSB1C3 challenged with E. coli

RESULT

We successfully assembled three AMP promoters with GFP and poly A to pSB1C3 vector. The function of the devices were tested by challenging with E. coli. The intensities were 5.31-fold, 3.02-fold and 2.29-fold increase for E. coli-induced GAM1, CecN and DefA promoter activities, respectively. The GFP positive cells after induction were clearly observed under fluorescence microscope.

To test the AMP promoter in response of Gram-negative and Gram-positive bacteria, we challenged GAM1 promoter with E. coli and Bacillus subtilis, respectively.

EXPERIMENT

↓C6/36 cells were seeded at the density of 1.8 x 105 cell/well in a 96-well plate
↓Cells were transfected with the GAM1-GFP-polyA vector
↓E. coli or B. subtilis was added on 2 days post-transfection at MOI=10
↓GFP intensity was measured by a microplate reader at Ex/Em = 480/520 nm.

RESULT

The data represented in C6/36 cells showed that GAM1 promoter was not only activated by Gram-negative E. coli but also induced by Gram-positive B. subtilis. The result further indicated the AMP promoter activity may cross talk between Toll and Imd signaling pathways.

GAM1-GFP-polyA / pSB1C3 challenged with E. coli & B. subtilis

To apply GAM1 promoter as a biosensor, E. coli at various concentrations were added to the mosquito cells transfected with the GAM1-GFP-polyA / pSB1C3

EXPERIMENT

↓C6/36 cells were seeded at the density of 1.8 x 105 cell/well in a 96-well plate
↓Cells were transfected with GAM1-GFP-polyA or Ac5-GFP-polyA vectors
↓E. coli at MOI=2, 4, 8, 16, 32 were added on 2 days post-transfection
↓GFP intensity was measured by a microplate reader at Ex/Em = 480/520 nm.

RESULT

As figures shown below, the green fluorescence intensities driven by GAM1 promoter were increased dose-dependently in the presence of E. coli at MOIs from 2 to 32. The fluorescence expressed by Ac5 promoter was not influenced at the same condition. These results demonstrated GAM1-GFP reporter system can used in the mosquito cells as a biosensor in response of different concentrations of bacteria.

GAM1 promoter was dose-dependently induced by heat-killed E. coli

Ac5 promoter was not affected by heat-killed E. coli

REFERENCE

1.Aedes aegypti: From Wikipedia, the free encyclopedia
2.Science (2007) Genome sequence of Aedes aegypti, a major arbovirus vector
3.Crit Rev Eukaryot Gene Expr. (2017) Genetically Modified Aedes aegypti to Control Dengue: A Review.
4.Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells