Coding
Art175

Part:BBa_K1659000

Designed by: Wei Chung Kong   Group: iGEM15_Oxford15   (2015-08-28)

Artilysin Art-175, an antibacterial fusion protein


This part contains the sequence for the antimicrobial protein Art-175.

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:


Secretion Tag Part Number
None BBa_K1659000
Flagellin 26-47 signal peptide sequence BBa_K1659001
DsbA 2-19 signal peptide sequence BBa_K1659002
YebF BBa_K1659003


Biology

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].


Usage

We aim to test this part for antibacterial activity by means of protein purification from host cell lysate after induction of gene expression. The antibacterial potency of this part will serve as a standard which we will compare our secretion-tagged modified Art-175 parts against.


Sequencing Trace Data

Characterization

To characterize this part, we moved the Art-175 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 Art-175 coding gene inducible by L-arabinose. This Art175[pBAD] plasmid is then cloned into E. coli MG1655.


Toxicity Testing


When Art-175 is produced and retained intracellularly, it should not be able to reach the peptidoglycan layer of its E. coli expression host and hence not be able to exert lysis activity due to obstruction by the inner membrane. As such, we set out to test this hypothesis by inducing the production of Art-175 using 0.2% L-arabinose and measuring cell density as a function of time.


Art175 growth

Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking. MG1655 pBAD/HisB is ''E. coli'' MG1655 having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.


The results above show that when E. coli MG1655 is not killed even under L-arabinose induction conditions that normally lead to gene overexpression in pBAD/HisB. We can infer from these results that either Art-175 is not being produced at all, or Art-175 is indeed being produced and that our hypothesis that it cannot kill our cells from intracellularly is correct. Given how the rest of the Art-175 parts family (BBa_K1659001, BBa_K1659002, and BBa_K1659003) are expressed and function as expected, it would be reasonable to think that in this part Art-175 is indeed being produced and is not lysing its expression host cells.

To better simulate the experimental conditions for protein purification, we subcultured MG1655 Art175[pBAD] with 1:20 dilution in antibiotic-supplemented LB media in a total volume of 20mL and let the culture grow at 37°C with shaking for 1.5 hours before inducing gene expression using 0.2% L-ara at 27°C for 4 hours. We measured the cell density before and after the 4-hour induction:


Bacteria type OD600 before induction with L-ara OD600 after induction with L-ara
MG1655 Art175[pBAD] 1.218 1.402
MG1655 pBAD/HisB 1.216 1.367


As seen in the cell density readings above, Art-175 does not induce cell lysis in its E. coli expression host at stationary phase even when its gene expression is induced by L-ara.

As such, we can conclude that this part works as expected in that its gene expression alone does not result in host-cell killing, and that it would henceforth be appropriate to pursue further downstream uses such as protein purification.




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.


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Categories
//collections/antimicrobial
Parameters
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