Thrombin-releasable AMPs or TAT-AMPs
We apply AMPs or TAT-AMPs in our dressing to kill both extracellular and intracellular MRSA on the wounds, but constitutively activated AMPs may induce unexpected immune responses. In addition, the AMPs or TAT-AMPs need to be stably anchored on the collagen layer. Therefore, we tried to design AMPs with collagen anchors that can be released only when necessary.
For the collagen anchors, we cloned the collagen binding domain (CBD) from the FN1 gene (see Design for details). Inspired by the 2019 iGEM team Linkoping_Sweden, we designed a linker containing thrombin cleavage site to link AMP/TAT-AMPs and CBD. When the dressing is on the wound, the secreted thrombin will cut the thrombin cleavage site to release the AMP.
Learn from lectures and re-design
Notably, the cleavage of the canonical thrombin cleavage site will leave two residues at the N-terminal of released AMPs, impacting the activity of AMPs or TAT . Petrassi et al. showed that the P1’ and P2’ positions of the thrombin cleavage site could endure the amino acid substitution to some extent without seriously hampering the cleavage ability . Therefore, it is possible to merge the N-terminal of AMP to the P1’ and P2’ positions of the thrombin cleavage site and generate thrombin-released AMPs without extra residues. Therefore, we substituted the amino acids at P1’ and P2’ positions of the thrombin cleavage site with the first two amino acids at the N-terminal of AMPs.
▲ Figure 1: The preferred amino acids at P1’ and P2’ positions of the thrombin cleavage site from the reference .
To prove our design, we constructed four vectors expressing fusion proteins to mimic the thrombin cleavage site linked AMPs (Table 1). To easily monitor the result of thrombin cleavage, we replaced the AMP with eGFP and saved the first two amino acids from the N-terminal of AMPs. The expressed fusion proteins are subjected into the thrombin cleavage experiment and the result is demonstrated by SDS-PAGE analysis.
▲ Table 1: Fusion proteins for thrombin activity test.
Test- hold by virus outbreak
After constructing the vector expressing the CBD-AMP mimic proteins, we start to express the CBD-AMP mimic proteins, and prepare for thrombin cleavage experiment. Unfortunately, the COVID-19 pandemic has come back to Taiwan, and we need to follow the rules and to slow down the experiment.
Although we could not finish the thrombin cleavage experiment under COVID-19 pandemic now, we have prepared to restart the experiment as soon as possible. We believe that, through the experimental design, we can clearly show the ability of our AMPs released by thrombin.
The CTSS-releasable AMPs
MRSA can remain latent in human cells, such as macrophage, to escape from antibodies, antibiotics or AMPs . To tackle the problem of MRSA latency, we designed a TAT linked AMP (TAT-AMP) to deliver the AMP into the macrophage.
The TAT-AMP will enter into macrophages through macropinosomes and fuse with phagolysosomes. In phagolysosomes, endopeptidases cathepsin D, F, O, L, and S are responsible for degrading the foreign objects . The survey of preference cleavage sequence of the cathepsin family members indicated that DPK-060 and D2A21 would not be attacked by cathepsins in the lysosome (See Table 3 in
Design). This result also suggested that cathepsins are good candidates to release the AMPs from TAT in phagolysosomes. Since cathepsin S (CTSS) is specifically expressed in antigen presenting cells (e.g., macrophages), we selected DPK-060 as intracellular AMPs and added a CTSS cleavage site to release DPK-060 in lysosomes in macrophage.
▲ Figure 2: Biobrick for intracellular bacteria.
Build & Test
Because it is too expensive to get commercial CTSS enzymes, we decided to perform molecular docking to examine the preference CTSS cleavage site. Click Model for more details.
The modeling results indicated that the CTSS cleavage sites could efficiently dock onto CTSS, suggesting successful cleavage. We may extend this result to model different combinations of amino acids around the CTSS cleavage site, and predict the CTSS cleavage sequence in silico. Furthermore, we can apply the same process to other cathepsin family members whose cleavage sequence remains unclear.
▲ Figure 3: The tripeptide (VGG shown in orange; LGG shown in purple) docked to cathepsin S.