Team:CCU Taiwan/Proof Of Concept
Team:CCU Taiwan/Proof Of Concept
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Overview
In our design, thrombin cleaves the thrombin site of CBD-AMPs to release D2A21, DPK-060 and TAT-DPK-060 from CBD. The AMPs D2A21 and DPK-060 are responsible for cleaning the extracellular MRSA infection, while TAT-DPK-060 is responsible for cleaning the latent MRSA in macrophages.
To prove this concept, we need to perform three major experiments:
1. Confirming that the thrombin will release D2A21, DPK-060 and TAT-DPK-060 from CBD protein.
2. The released AMPs, D2A21 and DPK-060, are able to kill extracellular bacteria.
3. The released TAT-DPK-060 could penetrate into macrophages and kill latent bacteria.
Achievement
Due to the COVID-19 pandemic outbreak in Taiwan in May, we can not finish all the related experiments and prove the whole concept. But we still do our best and wait for the reopening of labs after the pandemic.
For the 1 st major experiment, we need to confirm that the thrombin will release D2A21, DPK-060 and TAT-DPK-060 from CBD protein.
▲ Figure 1: The release mechanism of antimicrobial agents.
In this part, we successfully constructed the vectors expressing CBD-D2A21, CBD-DPK-060, and CBD-TAT-DPK-060. We also induced the protein expression of CBD-D2A21, CBD-DPK-060, and CBD-TAT-DPK-060. Next, we will perform the thrombin cleavage experiments and examine whether the D2A21, DPK-060 and TAT-DPK-060 would be released by thrombin cleavage.
For experiment results supporting the achievement, please check details in Part C in the Result section.
For the 2 nd major experiment, we need to confirm that the released D2A21 and DPK-060 are able to kill extracellular bacteria.
▲ Figure 2: The mechanism of AMP-Cell disruption.
In this part, the thrombin-released D2A21 and DPK-060 from the 1st major experiment is necessary. However, these AMPs are not available yet. Instead, we ordered the commercial D2A21 and DPK-060 peptides to perform the MIC assay. In the MIC analysis using E. coli , we found that the MIC of DPK-060 and D2A21 is 7.752 µM and 2.972 µM (Table 1). In the MIC analysis using S. aureus , we found that the MIC of DPK-060 and D2A21 is 9.304 µM and 6.759 µM (Table 1).
For more detailed experiment results supporting the achievement, please check details in Part E in the Result section.
Besides, we think that the biosafety of D2A21 and DPK-060 should be taken into consideration. Therefore, we perform hemolysis and cytotoxicity assay to examine whether D2A21 and DPK-060 will harm red blood cells and macrophages. Please check details in Part F in the Result section.
▲ Table 1: The MIC of D2A21 and DPK-060 against
E. coli
and
S. aureus
. MIC
90
is defined as the minimal concentration
required to inhibit the growth of 90% of microorganisms.
For the 3 th major experiment, we need confirming that the released TAT-DPK-060 could penetrate into macrophages and kill latent bacteria.
▲ Figure 3: The mechanism of CPP.
In this part, we first perform the cell penetrating experiment, in which we showed that the TAT can bring cargo (i.e. eGFP) into macrophage (Figure 4). To further confirm that TAT can bring DPK-060 into macrophage, we have designed a His-tag between TAT and DPK-060. We will incubate the cells with TAT-His- DPK-060 and detect their phagolysosomal location by ICC co-staining using antibodies against His-tag and LysoTracker, which is a marker of tracking late endosomal and lysosomal vesicles. To examine whether the TAT-His- DPK-060 has antimicrobial activity, we will stain bacteria with the live/dead BacLight kit, which can distinguish between viable S. aureus (green) and dead S. aureus (red) with the TAT-His-DPK-060 [1] .
▲ Figure 4: The ICC staining result of actin and eGFP expression in differentiated U937
cells. The differentiated U937 cells incubated with the medium containing eGFP or TAT-eGFP
were fixed and ICC-stained with actin (red) and eGFP (green). The nuclei (blue) was counterstaining
by DAPI. Scale bar was shown.
