Difference between revisions of "Part:BBa K1659001"

Latest revision as of 19:16, 18 September 2015

Artilysin Art-175 fused at N-terminal with flagellin 26-47 peptide segment

This part contains the sequence for the antimicrobial protein Art-175 with flagellar secretion signal peptide sequence fused to its N-terminus and a Hisx6 tag fused to its C-terminus.

Our Art-175 part collection comes in a family of four parts, one being Art-175 by itself and three others being Art-175 fused to different secretion signal sequences:

Biology

BBa_K1659001 is a composite of artilysin Art-175 (BBa_K1659000) with the 26-47 peptide segment of Salmonella typhimurium flagellin:

1. Art-175

Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolyins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Endolysins have a degree of specificity in terms of of the peptidoglycan chemotype which they can break down by means of the structural selectivity of their enzymatically-active domain (EAD) or cell wall binding domain (CBD).

Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydrolytic activity of endolysins from the outside. To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer can be covalently fused to endolysins to transport them past the outer membrane to reach the peptidoglycan layer.

Biers et al. fused the sheep myeloid antimicrobial peptide SMAP-29 to the N-terminus of the endolysin KZ144 to create Artilysin Art-175 [1]. Endolysin KZ144 has previously been shown to selectively exert cell wall lytic activity on the peptidoglycan chemotype A1γ (which Gram-negative bacteria such as P. aeruginosa, E. coli, and Salmonella typhimurium belong to), where the bacterial outer membranes have already been separately permeabilized, by targeting the fully N-acetylated glucosamine units present in that peptidoglycan chemotype [2]. On the other hand, SMAP-29 on its own exhibits broad antimicrobial activity by means of using its N-terminal ampiphathic α-helical region in conjunction with its C-terminal hydrophobic region to disrupt of the outer and inner membranes of bacteria. SMAP-29 on its own, however, is unsuitable for clinical applications because it is also hemolytic towards human erythrocytes [3][4].

Art-175, the product of their linkage, is able to use its SMAP-29 moiety to transport itself past the bacterial outer membrane and exert lytic activity on the peptidoglycan layer. It exhibits strong antibacterial activity against pathogenic P. aeruginosa strains PAO1 and PA14, being able to kill even persister cells effectively as it does not require active bacterial metabolism to exert its lytic activity. However, unlike SMAP-29 by itself, Art-175 cannot kill bacteria using SMAP-29's cell membrane disruption mechanism, and owing to the chemotype selectivity of KZ144 is hence ineffective against bacteria of other peptidoglycan chemotypes, such as S. aureus [1].

2. Flagellin 26-47 secretion signal

Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export Salmonella flagellin is its 26-47 amino acid residue segment [5].

Usage

We fused the Flagellin 26-47 signal peptide sequence to the N-terminus of Art-175 to with the aim of facilitating the fusion protein's export via the SRP pathway. A hexahistidine tag is also attached onto the C-terminus of the composite to allow for easy purification of the expressed protein via metal-affinity column chromatography.

In view of the fact that the N-terminus of the SMAP-29 is essential for its antibacterial activity, we wish to investigate whether and to what extent will the fusion of an N-terminal signal sequence onto the SMAP-29 moiety of Art-175 affect its antibacterial potency.

Since our project is on the topic of antimicrobial resistance, or more specifically biofilm-related ones, our aim is to use this part to create host organism strains that are capable of secreting Art-175, which is able to kill persister cells, in conjunction with biofilm-degrading enzymes such as DNase (BBa_K1659301) or Dispersin B (BBa_K1659211) to function as effective prophylaxis and/or treatment against undesirable biofilm-protected bacteria in both medical and industrial settings.

In terms of scaling up the production of Art-175, it would also be more desirable and efficient for the enzyme product to be available extracellularly as a secreted product rather than intracellularly, as the former would allow for a more streamlined harvesting process involving only the collection of the secretant-containing extracellular media as opposed to the need to process the host cells for batch lysis during each harvest.

Characterization

To characterize this part, we moved the Fla-Art175 coding sequence into the commercial expression vector pBAD/HisB by adding a BspHI restriction site to the 5' site of the coding sequence using PCR and performing digestion-ligation at BspHI(insert)-NcoI(plasmid) and PstI, making the expression of the Fla-Art175 coding gene inducible by L-arabinose. This Fla-Art175[pBAD] plasmid is then cloned into E. coli MG1655.

Toxicity Testing

If the flagellin 26-47 signal sequence works as expected in helping the secretion of the passenger Art175 moiety through the flagellar export system, Fla-Art175 should be able to escape into the extracellular medium thus allowing the Art175 moiety in the fusion protein to exert cell lytic activity on its expression host cell using its outer membrane-peptidoglycan disruption mechanism as detailed in the Biology section above.

As such, we set out to test this hypothesis by inducing the production of Fla-Art175 using 0.2% L-arabinose and measuring cell density as a function of time.

30°C incubation temperature failed to produce any evidence of host cell lysis.

At 27°C, induction of gene expression leads to decrease in cell density, suggesting that at this temperature Fla-Art175 is successfully produced and secreted, allowing it to exert lytic activity on the expression host.

We conclude that this part works as expected, with the 27°C induction data being especially useful as it allows contrast with BBa_K1659000 (Art-175, without secretion tag), in that when Art-175 is not secreted there is no cell lysis, whereas when Art-175 is secretion-tagged such as in this part there is cell lysis. This verifies the Art-175's mechanism for executing cell lysis as laid out in the original paper where it was first discussed.

References

[1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., … Lavigne, R. 2014. Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774–3784.

[2] Briers, Y. et al., 2007. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL. Molecular Microbiology, 65(5), pp.1334–1344.

[3] Skerlavaj, B. et al., 1999. SMAP-29: A potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Letters, 463(1-2), pp.58–62.

[4] Shin, S.Y. et al., 2001. Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide. Biochemical and biophysical research communications, 285(4), pp.1046–1051.

[5] Vondervizst, F., Sajó, R., Dobó, J., & Závodszky, P. (2012). The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella. In: Recombinant Gene Expression - Reviews and Protocols, Methods in Molecular Biology, 824, 131-143.

[6] Chilcott, G.S. & Hughes, K.T., 2000. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiology and molecular biology reviews : MMBR, 64(4), pp.694–708.