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Determining the toxicity of Cry19Aa against Ag55(anopheles gambiae) cell lineDr Adang’s Lab

Research done from 1/11/16 to 5/1/16 Supervison under Ruchir Mishra P.H.D student

Mihir Panchal

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Determining the toxicity of Cry19Aa against Ag55(anopheles gambiae) cell linesSummary:

The overall purpose of this laboratory research conducted from 1/11/16 to

5/1/16 in Dr. Adang’s molecular lab was to understand how to better control

mosquito populations in regions susceptible to diseases in which mosquitoes are a

vector. The use of Bacillus thuringiensis Cry19Aa toxin was central to our research.

Working with a doctoral student (Ruchir Mishra), our research project focused on

the affects of the BT Cry19Aa toxin on the cell membrane proteins of different

species of mosquitoes (Culex, and Anopheles), Cry19Aa is also especially effective

against Culex strains resistant to Cry4Aa, Cry4Ba, Cry11Aa toxins, investigating this

resistance and its effectively to mosquito cells was key to our research.

In order to conduct our research we used Ag55 cell lines. Ag55 cell lines have

been generally considered relatively easy to study Cry interaction and cellular

responses to those interactions. Thus we used Ag55 cells to carry out assays to

figure out the toxicity of Cry19Aa to Ag55 cell and larval cell lines. Preliminary

results showed that trypsin present in activated Cry19Aa was cytotoxic to Ag55 cells

and these results can give insights to other more efficient methods for mosquito

control.

Introduction:

Mosquito control has been central to human civilization for over millennia,

many civilizations have tried to control mosquitoes and the various disease they are

vectors for such as malaria, Chickungunya, Dengue, West Nile virus and various

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other emerging diseases such as the Zika Virus. The most common form of mosquito

control is sanitation and insecticides (Teixeira, 2012).

The ability of diseases vectored by mosquitoes to disrupt life and increase

morbidity, especially in the developing world, where Malaria kills nearly 660,000

people every year (Nadjm,2012) led us to research more effective toxins against

different types of mosquitoes. We decided to use Bacillus thuringiensis, a toxin of the

bacterial origin that targets the larval stages of certain mosquitoes. A key advantage

to using B. thuringiensis toxins is they affect few non target species, which makes

them much more environmentally friendly (Roh,2009). Bt(Bacillus thuringiensis)

toxins incorporate various different Cry and Cyt toxins, the crystal aggregation

which these toxins form contains at least four major toxic compounds. Cry and Cyt

toxins both are pore-forming toxins that lyse midgut epithelial cells by inserting into

a target cell membrane and forming pores.

Our research focused on Cry19Aa, a toxin found in the pBtoxis plasmid which

is isolated from B.thuringiensis subsp. Jegathesan . Our research targeted Cry19Aa

because it is known to be toxic to Anopheles and Culex but not to Aedes and it is also

toxic to Culex strains resistant to Cry4Aa, Cry4ba, and Cry11Aa, showing no cross-

resistance. The lack of resistance suggests a mechanism of action distinct from other

Bti toxins and Cry11Ba. This lack of resistance makes studying and investigating

Cry19Aa and its affectivity compared to other toxins of importance for developing

better ways of controlling mosquito-vectored diseases.

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Understanding the efficiency and rate at which the Cry19Aa protein kills the

Ag55 cells, a cell line of Anopheles gambiae, can lead to further research to

genetically develop even more effective toxins against mosquito cells.

Experimental Methods:

In order to perform our experiment, we first grew a cell culture model conducive

for investigating Cry toxin action. The cell cultures we used and found effective were

the Ag55 cell lines. Ag55 cell lines were previously effective for investigating

malarial plasmodium research. After growing 1x10^6 Ag55 cell lines, we prepared

activated Cry19Aa using trypsin. Additional details and order of the experiment is

provided below:

Culturing Cry19Aa toxin:

A single colony was seed cultured in 5ml LB supplemented with 10ul erythromycin

(10mg/ml in ethanol filtered and stored at -20 degrees Celsius) to make a final

concentration of 20ug/ml, shake at 250-350rpm at 30 degrees Celsius overnight.

First we prepared a medium to grow the toxin, we added 2g/L Peptone, 5g/L Yeast

extract, 12.54g/L K2HPO4(Dibasic), 2.31g/L KH2PO4 (Monobasic), we also filter

sterilized and added 5 ml 20% glucose + 1.23% MgSO4 , 1 ml 7.5% Cacl -2H2O

,1 ml 0.15 MnSO4- 7H2O and 1ml 0.014% FeSO4- 7H2O to the Erlenmeyer flask.

and then add 2ml Erythromycin stock (10mg/ml), Shake at 250-300 rpm at 30

degrees Celsius. After one day, we added 1L of sodium phosphate solution

[(Na2HPO4 8g/L (Na2HPO4-12H2O 20.18g) + NaH2PO4 5g/L (NaH2PO4-H2O 5.75g)]

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into the growing medium. After this, we cultured the toxin for another two days

until complete sporulation. Then we centrifuged the solution having sporulated

Cry19Aa at 5000rpm for 20 minutes. Then we discarded the supernatant. Next, we

purified the Cry19Aa.

Purification of Cry19Aa:

Resuspended the pellet in 80ml of CWI(0.1M NaCl, 2%Triton X-100, 20mM Bis-Tris

pH 6.5) and sonicated it for 3 miniutes twice. Then Centrifuged and re-suspended

the pellet in 80ml of CWI. Then we repeated the above step with CWI twice and with

ultra pure H2O once. Then once again repeated the above step with CWII (1M Nacl)

twice and with ultra pure water once. After that we re-suspended the pellet in 5ml

of ultra pure H2O.

Separation of spores and crystals:

To further separate spores from crystals, we used a discontinues NaBr gradient. We

prepared a 30-60% NaBr gradient, after the gradient formed in the tubes we

overlayed the pellet on the gradient. Then we ultra centrifuged the gradient at

20,000 rpm for 1 hours and 15 minutes. This caused a layer of crystals to float up to

the top; we then extracted the crystals and washed them twice with ultra pure

water.

Solubilization and trypsin activation of Cry19Aa crystals:

To solubilize the cry19aa crystals, we solubilized the crystals in carbonate

buffer(Na2CO3/NaHCO3,buffer, pH 11.5) and 10mM DTT. We solubilized the crystals

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at 37OC overnight. After solubilization, we added trypsin in 1:10(weight by weight

ratio).

Anion exchange chromatography was performed using Buffer A= 50 mM Carbonate

buffer + 100mM NaCl(pH 11.50) and Buffer B= 50mM Carbonate buffer + 1 M

Nacl(pH 11.50) and this separated the activated cry19Aa from other protein

degrading toxin proteins. Then we carried out a concentration course and time

course assay.

To carry out the Cry19Aa concentration course and time course quantitate assay on

Ag55 cells to measure toxicity:

We seeded the 1x10^6 cells in the 7 wells of the 12 wells in the cell culture

plate.

The cells were treated with different concentrations of trypsin activated

Cry19Aa.

Live and dead cells were counted after 16 hours of incubation for

concentration course assay @ 2,4,6,8,24 hours for time course assay with

trypsin activated Cry19Aa using Trypan blue and Hemocytometer(an

instrument for visual counting of number of cells). Cells were counted from 4

corners. Each corner square having 16 small squares.

We then calculated the percentage of cells viable. (% cells viable/total

number of cells)x100.

We then repeated the experiment 3 and 2 times for time course and

concentration course respectively.

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This part of the experiment helped us measure the toxicity and affectivity of

Cry19Aa to Ag55 cells.

Results:

The size of the activated Cry19Aa toxin was approximately 42 kDa. This is

confirmed below in the gel-electrophoresis. Lane 1 is the marker while lane 2 is the

solubilized Cry19Aa. While lane 3 is trypsin activated Cry19Aa.

The approximate band length of trypsin activated Cry19Aa was 42 kDa. This was

obtained using SDS PAGE electrophoresis. The results of the toxicity of trypsin

activated Cry19Aa on the Ag55 cell lines is shown below. We obtained these results

using an inverted phase contrast microscope.

Lane 1 2 3

97.4 66.2

45

31

kDA

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The results show rapid toxicity to Ag55 cell lines by 8 minutes. Using these pictures,

we were able to carry out a concentration and time course assay. The results of the

time and concentration course are also shown below.

Viability of cells was checked at 16 hrs. after incubation with activated Cry19Aa using trypan blue.

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Trypan Blue Ag55 cell viability time course assay. Blue: Control (0.8 nM Carbonate buffer pH 9.6). Red: 0.8 nM Cry19Aa. Readings were taken at 2 hrs , 4 hrs , 6 hrs , 8 hrs & 24 hrs.

Discussion:

Our results were consistent in showing that the trypsin activated Cry19Aa

toxin was indeed cytotoxic to the Anopheles gambiae cell lines. The time course and

concentration course assay showed that the percentage of viable cells with

increasing Cry19Aa concentration decreased significantly with larger Cry19Aa

concentrations. The time course assay also showed that after 24 hours of exposure

to Cry19Aa, the cell viability decreased to zero. These results show that Cry19Aa is

an efficient toxin that could be used to effectively control major mosquito vectored

diseases around the world by eliminating mosquitoes rapidly from the area. Further

research on the Cry19Aa toxin and its mechanism on cell membrane proteins of the

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Ag 55 cell lines could give insights into developing or discovering even more

efficient toxins for mosquito control.

References

Nadjm, B. (2012). Malaria: An update for physicians. PubMed. Retrieved April 27,

2016, from http://www.ncbi.nlm.nih.gov/pubmed/22632637

Roh et al.2009. Mutagenic analysis of putative domain II and surface residues in

mosquitocidal Bacillus thuringiensis Cry19Aa toxin. FEMS Microbiol

Lett. 295(2):156-63.

Teixeira Correa et al. 2012.Cytotoxicity Analysis of three Bacillus thuringiensis

subsp. Israelensis δ-Endotoxins towards Insect and Mammalian Cells. PLoS ONE

7(9): e46121

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