Difference between revisions of "Part:BBa K4607008"

The biobrick consists of the polycationic nonapeptide (PCNP) which is capable to destabilize the lipopolysaccharide binds on Gram-negative baterium. The principle about the PCNP is related to the ionic interactions between the phosphate groups, divalent cations, and hyrdophobic lipids' stacking, where the PCNP acts as a destabilizing. This part has been fused with endolysins in order to increase their capability to lyse Gram-negative bacterium, demonstrating that the addition of the PCNP allows the endolysin introduction into the bacteria cell membrane. It part have been evaluated in Escherichia coli. The peptide has a lenght of nine amino acids [1].

Sequence and Features

Usage and Biology

As a brief contextualization, bovine mastitis is the result of the infection of the bovine mammary glands caused by pathogenic microorganisms, mainly gram-positive and negative bacteria. This disease reduces milk quality production to a great extent and produces painful damage to the bovine. The main treatment for mastitis is the use of diverse antibiotics, therefore the overuse and misuse of them have caused a real problem in the development of multidrug-resistant pathogens [2]. Our team has conducted an extensive investigation to find an alternative treatment for bovine mastitis without risking the environment.

The principle behind our proposal is the use of fused proteins based on efficient bacteriophage endolysins. The function of a bacteriophage is to infect bacteria in order to kill them. Once the bacteria are infected and the virions are mature, they release holins, which are enzymes that create pores in the inner cell membrane. Endolysins now have access to the cell wall, so they can degrade it. Endolysins have lytic activity for the purpose of setting free the phage progeny to continue infecting other cells [3]. Endolysins are composed of two main domains: the N-terminal, which represents the catalytic domain, and the C-terminal, which is a cell wall binding domain, which interacts by binding itself to the bacterium's cell wall, activating the catalytic region, and causing cell wall lysis. However, the average endolysin lifetime is 20 minutes [4] [3].

The main purpose of the PCNP is to counteract the endolysins' disadvantages to penetrate the Gram-negative cell membrane. The PCNP have the

albumin binding domain (ABD) is to counteract the problems related to the brief in vivo time life of the endolysins. These domains have the capacity to increase the lifetime of antibodies, proteins, and enzymes through the incorporation of their sequences into the fusion protein. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part consists of an affinity-maturated variant of the streptococcal protein G which has been used for LysK expression, with results of up to 34 hours in increasing the lifetime of the protein in mice [1]. The best results have been achieved with the following conformation: CHAP domain-ABD-SH3 domain [5]. The domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9. The average ABD-endolysin lifetime is about 30 hours [1].

References

[1] Briers, Y., Walmagh, M., Van Puyenbroeck, V., Cornelissen, A., Cenens, W., Aertsen, A., Oliveira, H., Azeredo, J., Verween, G., Pirnay, J.-P., Miller, S., Volckaert, G., & Lavigne, R. (2014). Engineered endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens. MBio, 5(4), e01379-01314. https://doi.org/10.1128/mBio.01379-14 ‌