This year, in our project, we constructed a new expression vector working effectively in the fungus and submitted our parts and transforred into Metarhizium anisopliae 128 to increase the adhesion, penetration and immune avoidance capacity of the modified Metarhizium anisopliae. Therefore, we performed experiment on three aspects and the experimental characterizations of our parts are shown as follow.

Adhesion：HsbA

In this part, a strong promoter, PgpdA allows the HsbA protein to be expressed without induction.

The HsbA( BBa_K2788000 ) from Beauveria bassiana encodes a kind of membrane surface hydrophobic protein which helps our spores adhere to the wax on the cockroach body surface. Moreover, with the overexpression of HsbA, our spores can more effectively adhere to the cockroach. Then it will follow as spores’ germination, germinal tube, appressorium and the next penetrating process.

This part was insert into the expression vector by restriction sites EcoRI and PstI (Fig.1), and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.

Fig.1 Construction of expression vector HsbA-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: BBa_K2040101 and BBa_K2040102 , and HsbA comes from the Beauveria bassiana ARSEF 2860. The PgpdA-HsbA-TtrpC part is connected to the pBC plasmid through the BioBrick site.

We transformed the expression vectors into Metarhizium anisopliae 128 by the method of Xiaoling Wang, and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis.

The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernate were electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.(Fig.3)

Fig.4 One of the wild-type Metarhizium anisopliae 128 groups: P1 is the picture before treated and P2 is the picture after treated. The spores quantity is 40 in P1 and 102 in P2. And there are only two left that have not been washed away and moved. The adherence rate is 2/40=5.0%

It’s obvious that it happens great change（circle in red）in the position and number of spores in the observing area of wild-type Metarhizium anisopliae 128 experimental groups.

Fig.5 One of the Metarhizium anisopliae HsbA transformant groups: P3 is the picture before treated and P4 is the picture after treated. The spores quantity is 42 in P3 and 52 in P4. And there are 41 spores left that have not been washed away and moved. The adherence rate is 41/42=97.6%

It’s obvious that it almost happens no change except the place (circled in red) in the position and number of spores in the observing area of Metarhizium anisopliae HsbA transformant experimental groups.

Finally, the following chart (Fig.6) can be obtained by statistical data of four areas in all experimental groups.

Fig.6 M.a128 is the wild-type Metarhizium anisopliae 128 groups, and the adherence rate in average is 26.7%. HsbA is the Metarhizium anisopliae 128 HsbA transformant groups, and the adherence rate in average is 97.7%.

In conclusion, this result well confirmed that Metarhizium anisopliae HsbA transformant certainly enhanced the capacity of adhesion.

Penetration: Bbchit

In order to make Metarhizium anisopliae penetrate the corpus callosum more efficiently, we transferred Bbchit( BBa_K2788001 ), a chitinase gene from Beauveria bassiana ARSEF 2860, which encodes a chitinase that can be secreted extracellularly. The chitin of the body surface decomposes, thereby destroying the wall of the corpus callosum, so that the genus Metarhizium enters the sputum.(Fig.7)

Fig.7 Construction of expression vector Bbchit-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: BBa_K2040101 and BBa_K2040102, and Bbchit comes from the Beauveria bassiana ARSEF 2860. The PgpdA-Bbchit-TtrpC part is connected to the pBC plasmid through the BioBrick site.

We have a part encoding Chitinase, the Bbchit express and function intracellularly. We constructed a shuttle vector to transform this part and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis.(Fig.8)

The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining.(Fig.9)

To determine the activity of chitinase, we improved it according to the DNS colorimetric method of Kan Zhuo, Xiaozhen Shi. First, the standard curve was drawn with different concentration gradients of glucose solution, and 0.5 ml of the wild-type and transformed type 1, 3, 5, 7, 9 and 12 days of culture solution were respectively taken for enzyme activity test: the crude enzyme obtained after filtering the culture solution was used. The solution was mixed with 0.5 ml of 1% chitin colloid, reacted at 37 ° C for 60 min, and then added to a 0.5 ml DNS boiling water bath for 10 min. The absorbance of the obtained product was measured and the enzyme activity was calculated. There were three groups of wild-type and transformed type. Parallel, three parallel experiments were performed in each group, and the final data were averaged.

We calculate activity based on the standard curve formula: U=（A540+0.03279）/2.202 (Fig.10), a summary of the data at different times is made into a line chart as follows.We can see that after 12 days the transformed type’s enzyme activity is still growing and the wild-type is falling.(Fig. 11)

Fig.10 Glucose standard curve

Fig.11 changes in chitinase activity over time.

In order to verify the function of Bbchit from a macro level, we improved Kan Zhuo's chitin transparent circle method for verification. We stained the czapek solid medium without chitin colloids in red with 0.1% Congo red dye solution and then cultured wild-type and transformed Metarhizium. Compared with the size of the colony, the size of the transparent circle was compared to obtain the transformed type. The size of the transparent circle is the diameter of the chitin transparent ring (R2) and colonies. The ratio of the diameter (R1), expressed as R2/R1.The conclusion that the chitinase activity of Metarhizium anisopliae is enhanced.

Fig.12 A: wild-type Metarhizium anisopliae 128 produced transparent zone on Czapek chitin - induced medium; R1: 8mm, R2: 9mm; R2/R1=9/8 B: Metarhizium anisopliae HsbA transformant produced transparent zone on Czapek chitin - induced medium; R1’: 8mm, R2’: 12mm; R2’/R1’=12/8=3/2

Therefore, these results well confirmed that the chitinase activity of Metarhizium anisopliae Bbchit transformant is about 1.3 times that of wild-type Metarhizium anisopliae 128. Our modified fungus certainly enhanced the capacity of penetration.

Immune avoidance: MCL1

During evolution, insects have developed a very strong immune system against entomopathogenic fungi^[1] In hemolymph, hemocytes, and plasma play important roles in eliminating the fungi that are present in the hemolymph,^[2] But arm race between parasitic fungi and their insect hosts never stops. The fungus has mechanisms to overcome the immune systems of insects.,for example, the lower level of β-1,3-glucan in the cell surface does not stimulate the host immune response^[3]. We extract a gene named MCL1 from Metarhizium robertsiiARSEF 23, which encodes collagen-like protein to combine and block β-1,3,-glucan.^[3]This gene works like putting an “invisible cloak ”on the fungus so that immune avoidance can happen.

In this part, PgpdA is a strong promoter that allows MCL1 to be expressed without induction. of hemolymph.

Fig.13 Construction of expression vector MCL1-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: BBa_K2040101 and BBa_K2040102, and MCL1 come from the Metarhizium robertsii ARSEF 23. The PgpdA-Bbchit-TtrpC part is connected to the pBC plasmid through the BioBrick site.

We transferred the expression vector MCL1-pBC by CaCl ₂ -PEG induction method, then screen transformant by G418 resistance genes and colony PCR.PCR product was identified by agarose gel electrophoresis (Fig.14)

Fig.14 0.8%Agarose Gel Electrophoresis of colony PCR product of the positive clones. The PCR product showed two signal bands at 335 bp and 1817bp respectively, which correspond to the length of M.a primer PCR product and MCL1 primer PCR product. Lane 1: M.a primer PCR product; Lane 2: MCL1 primer PCR product; Lane M: DL marker.

The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total RNA by using RNAiso Plus(TAKARA), reverse transcription by using TAKARA PrimeScript™ RT reagent Kit, then perform quantitative PCR. (Fig.15)

Fig.15 quantitative PCR analysis demonstrating WT M.anasopliae and genetically enhanced M.anasopiae mRNA levels, which was increased significantly in transformant. (p <0.05)

We gain the total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernate was electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.

Fig.16 SDS-PAGE analysis of total protein of wild-type Metarhizium anisopliae 128 and genetically enhanced Metarhizium anisopliae 128. Lane 1: Marker Ladder；Lane 2：Metarhizium anisopliae 128；Lane 3: recombinant strain Metarhizium anisopliae 128.

In order to verify the ability of immune-avoidance of M.anasopliae,we inject hyphae homogenate into cockroaches as experimental groups and normal saline as control groups, then extract hemolymph in 0.5h, 1h , 2h, 4h, 8h, 12h and 24h. Meanwhile extract hemolymph from cockroaches that were not injected as group 0h. Count the nodules formed of hemocytes(Criteria: more than 10 hemocytes assemble closely)(Fig.17) and observe hemolymph smear under phase contrast microscope(Fig.18) <4>

Fig.17 Change of nodules formed of hemocytes in cockroach hemolymph after injecting hyphae homogenate. At the time point of 0.5h,1h and 8h, the nodules caused by WT M.anasopliae is significantly higher than transformant which means immune-avoidance occurs in genetically enhanced M.anasopliae.(p <0.05)

Fig.18

Fig.18. Immune response of cockroaches’hemocytes to Metarhizium anisopliae spores under phase contrast microscope.A.B.C. hemolymph without injection. The number of hemocytes is relatively large and the shape is normal. Almost no nodule formation; D,G,J,M,P,S,V:control,almost no nodules formed; E:0.5h of WT injection,blood cells aggregated to form nodules,which means immune response on hypha;F: 0.5h of transformant injection,almost no nodule formed;H:1h of WT injection,more nodules was formed;I :1h of transformant injection, nodules started to form but was less than WT group;K:2h of WT injection , nodules were becoming larger;L:2h of transformant injection, nodules were smaller than WT group;N:4h of WT injection, nodules were less and smaller than 2h;O:4h of transformant injection, almost no nodules formed;Q:8h of WT injection, nodules were Most disintegrating.R:8h of transformant injection, Metarhizium anisopliae was growing in the shape of yeast;T:12h of WT injection, the number of hemocytes and nodules decreased; U:12h of transformant injection, blood cells were destroyed and cell debris can be seen;W:24h of WT injection, blood cells’ structure were destroyed, a large amount of cell debris cen be seen; X:24h of transformant injection, Metarhizium aerated in large numbers and few blood cells left. Wild-type M.anasoplise triggers more intense immune response than MCL1 transformant, while hemocytes degraded more in transformant groups.

Suicide switch

In order to confirm the limited concentration of Tryptophan, we did a macro experiment. We put Metarhizium in an L-Tryptophan concentration gradient Petrie dishes from 0.05% to 0.14%. with solid and liquid czapek. After six days of cultivation, it can be seen in the

following results:

We can see the ten photos that, in the solid czapek, the Metarhizium can stay alive at the L-Trp concentration of 0.09% or higher while it could not grow well or die at a lower concentration. We can also see that Metarhizium could not survive without L-Tryptophan.

We put the Metarhizium in 100mL liquid czapek with different concentration L-Tryptophan, after 6 days of cultivation, here is the result:

Chart 1. Dry Weight of Metarhizium in different concentration of L-Tryptophan

Fig.18 Dry Weight of Metarhizium in different concentration of L-Tryptophan

We can see the chart and graph that, in the liquid czapek, the Dry Weight of Metarhizium stay in a stable level from 0% of [Trp] to 0.08% of [Trp], because it didn’t grow at this low concentration. At the L-Trp concentration of 0.09% or higher, its dry weight grow as the concentration of L-Tryptophan grow.

From our experiment we can see that, the limited Tryptophan concentration for Metarhizium to survive is 0.9% of Tryptophan.

Reference

[1] Gottar M, Gobert V, Matskevich AA, Reichhart JM, Wang CS, Butt TM, et al. Dual detection of fungal infections in Drosophila through recognition of microbial structures and sensing of virulence factors. Cell. 2007; 127(7): 1425–1437.

[2] Glupov VV. Mechanisms of resistance of insects, in: Glupov V. V. (ed.) Insect Pathogens: structural and functional aspects, Moscow, Kruglyi god, 2001, pp 475–557

[3] Wang C, St Leger RJ,A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses.Proc Natl Acad Sci U S A. 2006 Apr 25; 103(17):6647-52.