Part:BBa_K1659210

Dispersin B with mutation at nucleotide 582 (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 582. This mutation-modified Dispersin B also comes with a DsbA 2-19 signal sequence upstream of it, in BBa_K1659211.

Biology

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].

Usage

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.

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.