Evaluation of Cell-penetrating-peptide (CPP) in Archaebacteria

Abstract

It is a well-known fact that earth contains a vast variety of microbiomes, in which the existence of those microorganisms effects the ecological state of the environment. For decades, humanity have studied these microorganisms and harvested their biological functions to aid the development of biotechnology. Although intensive research had been performed toward microorganisms such as bacteria, fungi, and algae, information related to archaea are still low. Due to their unique characteristic of being extremophiles, the tools needed to isolate and manipulate them are still lacking. This thesis proposes the utilization of cell-penetrating peptide (CPP), a short peptide capable of permeating through cell membrane into the cytoplasm, as a potential method for the isolation and manipulation of archaea cells such as introducing labeling molecules conjugated with quenchers and complementary nucleic acid into the target cells without harming the viability of the cell. Three different CPPs that exhibits different properties were used for the evaluation: the cationic and hydrophobic CPP ((KFF)3K), polycationic CPP (CPP A), and amphipathic CPP (CPP B). Using Haloarcula Japonica as the archaea model, the assay was conducted with both Haloarcula media and PBS as the medium to expose the cell in an environment where they could retain or removed of their S-layer, respectively. The result obtained from the evaluation showed a positive conformation of CPP permeation through the cell wall of the H. Japonica with (KFF)3K and CPP B, with CPP B showing more efficiency. This information shows that the S-layer of the H. Japonica served as a charge barrier that restrict positively charged CPPs. Phylogenetic analysis and surface glycoprotein sequence alignment of the H. Japonica revealed similarities of its cell wall structure with other members of the Haloarcula genus and other halophilic archaea species, which potentially means that CPP permeation using the information obtained from H. Japonica could be applied to a broader range of archaea species. Although currently the permeation efficiency of (KFF)3K and CPP B is still low, further optimization of the permeation condition will be conducted in future project to optimize the permeation efficiency.