Part:BBa_K1659211

Dispersin B fused with DsbA, with mutation at nucleotide 636 (T to C)

This part contains the sequence for the biofilm-degrading enzyme, Dispersin B (BBa_K1659200), with a conservative T -> C point mutation at nucleotide number 636, fused with an export signal at its N-terminus.

Biology

BBa_K1659211 is a composite of point-mutated Dispersin B (BBa_K1659210) with the 2-19 peptide segment of protein-folding factor DsbA:

1. Dispersin B

Dispersin B is an enzyme produced by Aggregatibacter actinomycetemcomitans, a species of bacteria found in the human oral cavity that grows almost exclusively in the form of biofilms. A. actinomycetemcomitans uses Dispersin B as a means of spreading its colonies by degrading a portion of its mature biofilm and releasing cells that were previously adherent, allowing them to propagate through liquid medium a form new biofilms on other surfaces. Kaplan et al identified the gene that coded for Dispersin B and characterized the protein using E. coli as the expression host [1].

Structural analysis of Dispersin B showed that the enzyme only works specifically against the β-1,6-glycosidic linkages found in poly-N-acetylglucosamine (PGA), which is a polysaccharide structural element found in the biofilms of E. coli, S. aureus, and S. epidermidis, but not in P. aeruginosa [2][3][4].

In the active site, Glu184 serves as the acid/base catalytic residue while Asp183’s negative charge stabilizes the cationic intermediate which has a less favourable ring conformation, promoting the forward direction in the anchimeric-assisted double SN2-style mechanism. Selectivity for 1,6-NAG polymers over 1,4-NAG is because of the anchimeric-assisted mechanism (neighbouring group-participation within the same molecule) that requires the close proximity between the -OR and -NHCOCH3 functional groups to work.


Image source: Manuel et al, 2007 [3]

Dispersin B is currently patented and licensed to Kane Biotech which is developing a wound care spray based on it [5][6]. When used in conjunction with triclosan as a surface prophylactic agent, Dispersin B effectively inhibited the formation of biofilms on both the internal and external surfaces of urinary catheters [7].

2. DsbA 2-19 signal sequence

DsbA is a thioredoxin fold-containing disulfide oxidoreductase protein found predominantly in Gram-negative bacteria, which functions as a protein-folding factor [8][9]. 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 [10][11]. It has recently been shown that the DsbA signal sequence is capable of mediating passenger protein secretion under a selection of different induction temperatures [12].

Usage

We fused the DsbA 2-19 signal peptide sequence to the N-terminus of Dispersin B 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 terms of scaling up the recombinant enzyme prooduction, it would 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.

As far as enzyme function is concerned, we are interested in the antibiofilm activity of Dispersin B against the biofilms formed by antibiotic-resistant strains of E. coli found in urinary tract infections. However, in the interest of lab usage safety, for our wet lab work we will only test the antibiofilm potency of DspB against Biosafety Level 1 laboratory strains of E. coli. Ultimately, we aim to use antibiofilm enzymes such as Dispersin B in conjunction with antibacterial enzymes such as Art-175 as an alternative treatment option to antibiotics in biofilm-related bacterial infections.

Characterization

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

Purification of Secreted Protein

MG1655 DsbA-DspB[pBAD] subcultured 1:20 in LB media (total volume 500mL), grown at 37°C for 1 hour then induced with 0.2% arabinose at 30°C for 4 hours. Supernatant purified using nickel-affinity chromatography using the following set of buffers:

- Resuspension buffer: 20 mM Tris (pH 8.0), 500 mM NaCl. - Wash buffer: 20 mM Tris (pH 8.0), 500 mM NaCl containing 5 mM imidazole. - Elution buffer: 20 mM Tris (pH 8.0), 500 mM NaCl) containing 100 mM imidazole.

10uL of eluate was mixed with SDS and run through a PAGE gel:

Lane D is where the stained eluate was loaded (DsbA-DspB is a 45kDa protein); Ladder used was 2-Color SDS Marker

This shows DsbA signal sequence successfully facilitates the export of DspB from MG1655.

Inhibition of Host Cell Biofilm Formation

Stationary cultures of and MG1655 DsbA-DspB[pBAD] subcultured 1:100 in fresh LB media and inoculated into 96-well plate incubated at room temperature for 3 days with or without 0.2% arabinose as gene expression inducer accordingly. Planktonic cells removed through very gentle rinsing with Milli-Q water, and adherent biofilms stained using 0.1% crystal violet solution. Stained biofilms dissolved in 80-20 ethanol-acetone and optical density at 590 nm measured (the higher the OD, the more stained biofilm).

Analysis was done alongside BBa_K1659301

Data shows that expression of DsbA-DspB inhibits host cell biofilm formation.

Co-culturing of Host Cell with normal biofilm-forming E. coli to inhibit normal E. coli from forming biofilms

Similar subculturing method as above; MG1655 DsbA-DspB[pBAD] co-cultured in 96-well plate with normal biofilm-forming E. coli in 8:2 ratio and left on bench for 5 days. If MG1655 DsbA-DspB[pBAD] were actually only inhibiting its own biofilm formation intracellularly instead of via the effect of a secretant, it would not be able to inhibit the biofilm formation of a normal biofilm-forming E. coli present in the same culture.

The data show that MG1655 DsbA-DspB[pBAD] inhibit the normal biofilm-forming E. coli from forming biofilms to a certain extent.

We conclude that this part works as we have conclusively proven:

- Secretion of hexahistidine-tagged DsbA-DspB using nickel-affinity chromatography

- That secreted DsbA-DspB inhibits biofilm formation of expression host

- That secreted DsbA-DspB inhibits biofilm formation of normal biofilm-forming E. coli present in co-culture

References

[1] Kaplan, J.B. et al., 2003. Detachment of Actinobacillus actinomycetemcomitans Biofilm Cells by an Endogenous beta-Hexosaminidase Activity. Journal of Bacteriology, 185(16), pp.4693–4698.

[2] Ramasubbu, N. et al., 2005. Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans. Journal of Molecular Biology, 349, pp.475–486.

[3] Manuel, S.G. a et al., 2007. Role of active-site residues of dispersin B, a biofilm-releasing beta-hexosaminidase from a periodontal pathogen, in substrate hydrolysis. FEBS Journal, 274(22), pp.5987–5999.

[4] Wang, X., Iii, J.F.P. & Romeo, T., 2004. The pgaABCD Locus of Escherichia coli Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation. Journal of Bacteriology, 186(9), pp.2724–2734.

[5] University Of Medicine And Dentistry Of New Jersey, (2011). Dispersin B polynucleotides and methods of producing recombinant DspB polypeptides. US7989604 B2.

[6] Kane Biotech, 2011. Access online at: http://www.kanebiotech.com/dispersinb.html

[7] Darouiche, R.O. et al., 2009. Antimicrobial and antibiofilm efficacy of triclosan and DispersinB combination. Journal of Antimicrobial Chemotherapy, 64(May), pp.88–93.

[8] 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.

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

[10] 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.

[11] 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.

[12] 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.