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.

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.