Project

Mosquito as a natural syringe

Xenosurveillance

•Definition

•Application

•Paper1, 2, 3 showed that

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

Experiment

↓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

Result

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.

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

Then we go further to search whether there is someone who has the similar idea with us. We found that there are methods like phage display that can produce scFv (single chain variable fragment)_[12]. In our project, besides finding out the scFv combine to aflatoxin from a research published in 2012_[13], we will further more improve its function. In the traditional process, we have to bind gold nanoparticles to the antibody in order to observe the result on test strip. To replace this step, we put the sequence of Red Fluorescent Protein into DNA sequence when designing the fusion protein. What’s more the structure of scFv only contain the variable region of antibody, so the control line of the strip which have secondary antibody that bind to the constant region of primary antibody can’t work anymore.

To deal with this problem and facilitate the process of purification, we add a His-tag at the end of the fusion protein. After putting the three-dimensional structure of protein into consideration, we add a rigid linker between the RFP and scFv to avoid interference between the two proteins when folding.

What we do

To improve the way of making the test strip, we constructed the DNA sequence which is composed of scFv and Red Fluorescent Protein.There are two main advantages about this fusion protein. One is that it can reduce the cost to produce aflatoxin antibody compared with using hybridoma because our fusion protein can be expressed abundantly by E.coli. The other is that we don’t need to use gold nanoparticles to show the result. It could simplify the process of making the antibody tested on strip.

Reference

• 1. Bbosa, G.S., et al., Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis. Health, 2013. 5(10): p. 14.
• 2. Galvano, F., V. Galofaro, and G. Galvano, Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. Journal of Food protection, 1996. 59(10): p. 1079-1090.
• 3. Butler, W., Acute toxicity of aflatoxin B1 in rats. British journal of cancer, 1964. 18(4): p. 756.
• 4. Newberne, P.M., R. Russo, and G.N. Wogan, Acute toxicity of aflatoxin B1 in the dog. Pathologia veterinaria, 1966. 3(4): p. 331-340.
• 5. Williams, J.H., et al., Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. The American journal of clinical nutrition, 2004. 80(5): p. 1106-1122.
• 6. Creppy, E.E., Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology letters, 2002. 127(1): p. 19-28.
• 7. Adebo, O., et al., Review on microbial degradation of aflatoxins. Critical reviews in food science and nutrition, 2017. 57(15): p. 3208-3217.
• 8. Lapalikar, G.V., et al., F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the Actinomycetales. PLoS One, 2012. 7(2): p. e30114.
• 9. Taylor, M.C., et al., Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular microbiology, 2010. 78(3): p. 561-575.
• 10. Lavallie, E.R., et al., A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Nature biotechnology, 1993. 11(2): p. 187-193.
• 11. Chandler, J., T. Gurmin, and N. Robinson, The place of gold in rapid tests. Vol. 6. 2000. 37-49.
• 12. Hammers, C.M. and J.R. Stanley, Antibody phage display: technique and applications. The Journal of investigative dermatology, 2014. 134(2): p. e17.
• 13. Li, X., et al., Molecular characterization of monoclonal antibodies against aflatoxins: a possible explanation for the highest sensitivity. Analytical chemistry, 2012. 84(12): p. 5229-5235.