Composite
Dsb-Art175

Part:BBa_K1659002

Designed by: Wei Chung Kong   Group: iGEM15_Oxford15   (2015-08-26)

Artilysin Art-175 fused at N-terminal with DsbA signal peptide


This part contains the sequence for the antimicrobial protein Art-175 with DsbA 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:


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

BBa_K1659002 is a composite of artilysin Art-175 (BBa_K1659000) with the 2-19 peptide segment of protein-folding factor DsbA:


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. DsbA 2-19 signal peptide sequence

DsbA is a thioredoxin fold-containing disulfide oxidoreductase protein found predominantly in Gram-negative bacteria, which functions as a protein-folding factor [5][6]. The 2-19 peptide sequence of DsbA is a signal sequence that can direct passenger proteins for co-translational export via the signal recognition particle (SRP) pathway [7][8]. It has recently been shown that the DsbA signal sequence is capable of mediating passenger protein secretion under a selection of different induction temperatures [9].


Usage

We fused the DsbA 2-19 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 DsbA-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 DsbA-Art175 coding gene inducible by L-arabinose. This DsbA-Art175[pBAD] plasmid is then cloned into E. coli RP437 ∆FliC.


Toxicity Testing

If the DsbA 2-19 signal sequence works as expected in helping the secretion of the passenger Art175 moiety through the Sec export system, DsbA-Art175 should be expected to exert cell lytic activity on its expression host cell using its peptidoglycan disruption mechanism (as detailed in the Biology section above) as it passes through the inner membrane and enters the periplasm, coming into contact with the peptidoglycan layer.

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


Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking. RP437 ∆FliC pBAD/HisB is ''E. coli'' RP437 ∆FliC having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.


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


flaArt175 growth 27

Expression host cell cultures were grown in a 96-well plate at 27°C with 200 rpm shaking. RP437 ∆FliC pBAD/HisB is ''E. coli'' RP437 ∆FliC having a blank pBAD/HisB plasmid transformed into it, and serves as the negative control in the experiment.


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


Using cell-free supernatant of E. coli RP437 ∆FliC DsbA-Art175[pBAD] to kill P. putida

In view of the toxicity test data implying the possibility of DsbA-Art175 being exported from its E. coli expression host, we decided to test the bacterilytic activity of the expression host's supernatant.

A subculture of RP437 ∆FliC DsbA-Art175[pBAD] was prepared from its stationary culture, where 1mL of stationary culture was cleaned of its growth antibiotics and resuspended in fresh LB media. The subculture was grown at 37°C for 1 hour before gene expression was induced using 0.2% L-ara at 30°C for 4 hours. The choice of 30°C induction temperature was made in view of the fact that induction of gene expression at 27°C is shown to result in death of host cells in the toxicity tests.

At the end of the 4 hours the supernatant of the subculture was isolated and incubated with P. putida pre-grown to mid-log phase, and the cell density of the P. putida culture was tracked.


flaArt175 growth 27

''P. putida'' incubated with supernatants in a 96-well plate at 27°C with 200 rpm shaking. The non-induced supernatant was prepared using the exact same steps as the induced supernatant, except Milli-Q water being added to the culture in place of L-ara.


The data shows that the supernatant of a L-ara-induced culture being able to kill P. putida.


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. Also, the fact that the supernatant of the host cell for this part is able to kill P. putida suggests that DsbA-Art175 is indeed exported successfully into the expression host's extracellular medium, and that the function of the Art-175 moiety is not affected by the process as well as the addition of an extra peptide sequence at its N-terminus.


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] Guddat, L.W., Bardwell, J.C. & Martin, J.L., 1998. Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization. Structure (London, England : 1993), 6(6), pp.757–767.

[6] Heras, B. et al., 2009. DSB proteins and bacterial pathogenicity. Nature reviews. Microbiology, 7(3), pp.215–225.

[7] Schierle, C.F. et al., 2003. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), pp.5706–5713.

[8] Steiner, D. et al., 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nature biotechnology, 24(7), pp.823–831.

[9] Božić, N. et al., 2013. The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting, recombinant α-amylase from Bacillus licheniformis ATCC 9945a. Process Biochemistry, 48(3), pp.438–442.


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