Part:BBa_K1659003

Artilysin Art-175 fused at N-terminal with porin-dependent YebF secretion signal protein

This part contains the sequence for the antimicrobial protein Art-175 with protein YebF 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_K1659003 is a composite of artilysin Art-175 (BBa_K1659000) with YebF, a protein reported to be naturally secreted into the extracellular medium by laboratory E. coli strains:

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. YebF

YebF is a 13kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory E. coli strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form [5]. Export from periplasm into the extracellular space takes places via the Omp pathway, whereby the electropositive dynamic region of YebF electrostatically helps load YebF onto the OmpF/OmpC porins at their electronegative periplasmic face, and after which the disordered N-terminal region of YebF gets threaded through the OmpF lumen [6]. YebF has been used successfully to mediate the secretion of recombinant proteins [7][8].

Usage

We fused YebF to the N-terminus of Art-175 to with the aim of facilitating the fusion protein's export from the host cell. 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 YebF-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 YebF-Art175 coding gene inducible by L-arabinose. This YebF-Art175[pBAD] plasmid is then cloned into E. coli MG1655.

Toxicity Testing

If the YebF protein works as expected in facilitating the secretion of the passenger Art175 moiety through the inner membrane, YebF-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 YebF-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 37°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.

Despite the fact that the 37°C incubation temperature in this experiment is more ideal for E. coli growth and cell division than protein production, a very significant extent of host cell killing is still observed. This implies that YebF is a more effective secretion tag than DsbA or Fla, giving rise to a higher concentration of Art-175 secretant causes greater host cell lysis.

In view of that, a host cell with a more robust peptidoglycan layer was required for investigation of YebF-Art175 expression. Dr Andreas Diepold, a postdoc based in our host lab cloned YebF-Art175[pBAD] into Y. enterocolitica IML421asd, a Biosafety Level 1 lab strain of Y. enterocolitica that is known to have a stronger peptidoglycan layer than lab strains of E. coli, the former requiring eight times more lysozyme to for peptidoglycan hydrolysis than the latter [9].

To investigate expression host cell lysis, IML421asd YebF-Art175[pBAD] and E. coli MG1655 YebF-Art175[pBAD] was subcultured with with 1:20 dilution in antibiotic-supplemented BHI media in a total volume of 20mL and the culture grown at 30°C with shaking for 1 hour before gene expression was induced using 0.2% L-ara at 28°C for 4 hours. The cell densities were measured before and after the 4-hour induction:

Bacteria type	OD600 before induction with L-ara	OD600 after induction with L-ara
MG1655 YebF-Art175[pBAD]	1.032	0.215
IML421asd YebF-Art175[pBAD]	0.917	0.682

Host cell lysis occurred to a smaller extent in IML421asd than MG1655 after 4 hours induction. Based on this data it is shown that IML421asd is a more appropriate expression host for this purpose than MG1655.

Using cell-free supernatant of IML421asd YebF-Art175[pBAD] to kill planktonic P. putida

A subculture of MG1655 YebF-Art175[pBAD] was prepared from its stationary culture, where 1mL of stationary culture was cleaned of its growth antibiotics and resuspended in fresh BHI media. The subculture was grown at 30°C for 1 hour before gene expression was induced using 0.2% L-ara at 28°C for 4 hours.

At the end of the 4 hours the supernatant of the subculture was isolated and incubated with freshly subcultured P. putida, and the cell density of the P. putida culture was tracked.

''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 to an even larger extent than was achieved with BBa_K1659002.

Using cell-free supernatant of IML421asd YebF-Art175[pBAD] to kill persister P. putida within biofilms

One of the major antibacterial resistance mechanisms which the biofilm state of growth confer to bacteria is their entering of metabolically-inactive "persister" state. Here we investigate whether or not the secretant of IML421asd YebF-Art175[pBAD] can kill persister P. putida.

P. putida biofilms pre-grown on 96-well plate for 3 days. Supernatants for cell-killing prepared using same procedure as above. Biofilms incubated with supernatants for 2 days at 27°C.

After incubation, number of persister cells remaining in biofilm measured using crystal violet staining, which stains whole bacterial cells but not lysed ones, and amount of crystal violet that adhered to biofilm-encased persister cells can be measured by dissolving biofilms using acetone and measuring optical density at 590 nm.

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.

Results above show that IML421asd YebF-Art175[pBAD] supernatant lyse persister cells to an appreciable degree.

We conclude that this part works as expected, with host cell lysis occuring to a large extent and the supernatant of gene expression subcultures being able to kill both planktonic and biofilm-encased P. putida.

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] Zhang, G., Brokx, S. & Weiner, J.H., 2006. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature biotechnology, 24(1), pp.100–104.

[6] Prehna, G. et al., 2012. A protein export pathway involving Escherichia coli porins. Structure, 20(7), pp.1154–1166.

[7] Fisher, A.C. et al., 2011. Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Applied and Environmental Microbiology, 77(3), pp.871–881.

[8] Hwang, I.Y. et al., 2014. Reprogramming microbes to be pathogen-Seeking killers. ACS Synthetic Biology, 3(4), pp.228–237.

[9] Diepold, A., Wiesand, U. & Cornelis, G.R., 2011. The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer. Molecular Microbiology, 82(2), pp.502–514.