Team:UCL/James/Description

Title

One

Text here.

Two

  • List.
  • List.
  • List.
  • List.
  • List.

Three

Text.

"Quote"

Joe Bloggs

Text.

Four.

Caption.

Five

Text.

Five (a)

Text.

Five (b)

Text.

Five (c)

Text.

Five (c) (i)

Text.

Delivery

A major part of our project involves investigating what is the best way to deliver our biofilm-degrading and antimicrobial enzymes to the site of infection in the urinary tract.

As we have mentioned above, patients with recurrent, complicated cases of UTI often get their infections from an already-inserted catheter which may have to be there and cannot be removed for a variety of other medical reasons. In view of that, we decided to conceptualize an initial delivery method which was centered on the catheter.

Our AlgiBeads design involves encapsulating our therapeutic, enzyme-secreting bacteria in sodium alginate beads. These beads are immobilized in a modified section of a catheter, from which the bacteria can secrete the therapeutic enzymes into the infected urinary tract. On our Design page, thorough consideration was given to the AlgiBeads delivery method, including issues of safety and practicality.

However, based on some preliminary data obtained for gene expression and diffusion rates, our computational models predicted that the equilibrium concentration of enzymes in solution based on the AlgiBeads delivery method would be too low when compared against the known concentrations required for biofilm degradation.

As such, we have had to instead consider an alternative delivery method - the introduction of our enzyme-releasing therapeutic engineered bacteria into the urinary microbiome, whereby the problem of low enzyme concentration in solution will be overcome by the close proximity between the therapeutic bacteria and the pathogenic bacteria. Another benefit of having therapeutic bacteria as part of the microbiome is of course that the treatment becomes preventive in nature, with the therapeutic bacteria now part of the bacterial community in the body constantly releasing pathogen-killing enzymes.

Of course, altering the microbiome comes with its own set of hazards, and we hope to mitigate it at least in part by doubling up the pathogen-killing mechanism as a population control mechanism for the engineered bacteria as well:

How our 3-part engineered microbe works:
1. Constant secretion of biofilm-degrading enzyme
2. Production and accumulation of antibacterial Art-175
3. Detection of pathogenic bacteria via quorum sensing
4. Permeabilization of inner membrane by T4 Holin
5. Access and lysis of host cell wall by Art-175
6. Release of Art-175 and lysis of target cell

Art-175 is normally prevented from reaching the cell wall of the expression host by the inner membrane. When a large amount of pathogenic bacteria is present, the quorum sensing signals trigger the production of T4 Holin, which permeabilizes the inner membrane, allowing Art-175 to reach the cell wall and degrade it. This causes lysis of the host cell and releases the accumulated Art-175 in a single high-concentration pulse, killing the pathogenic bacteria and achieving population control of the expression host at the same time.

Other safety aspects of this microbiome-modification design, including issues on immunogenicity, can be found here.

Results

Through our experimental work we were able to obtain preliminary evidence suggesting the validity of these points:

  • DsbA-DNase and DsbA-DspB can be secreted in a fully folded and functional state
  • Both DNase and DspB are able to degrade biofilms
  • Art-175 is able to exert cell lytic activity on planktonic E. coli and P. putida
  • Art-175 is able to kill a portion of biofilm-encased P. putida cells

The results and in-depth discussion of our experimental work can be found on the Experiments page.

Improving Part Function

Improving the function of another team’s part: BBa_K729004

Team UCL 2012 also had a part comprising Staphylococcal DNase with a DsbA tag upstream of it. We were interested in finding out:

  • Whether the DsbA 2-19 sequence is able to facilitate the export of this part of expression host organism E. coli MG1655
  • Whether the Staphylococcal nuclease can degrade E. coli biofilms (it was shown to degrade S. aureus biofilms in Mann et al, 2009)

Figure 14: SDS-PAGE of E. coli MG1655 BBa_K729004 [pBAD], 0% ara supernatant (A) and E. coli MG1655 BBa_K729004 [pBAD], 0.2% ara supernatant (B)

Figure 14 shows the successful DsbA-directed secretion of DNase across both cell membranes.

A is the supernatant of uninduced E. coli MG1655 BBa_K729004 [pBAD], whilst B is the supernatant of 0.2% induced E. coli MG1655 BBa_K729004 [pBAD]. The band is approximately 21 kDa, corresponding to the size of DsbA-DNase.

Figure 15: Expression host MG1655 BBa_K729004 [pBAD] biofilm growth assay

Figure 15 shows the effect of inducing the expression of BBa_K729004 [pBAD] on the ability of the host to form biofilms. The control (MG1655, pBAD/HisB, 0.2% ara) and MG1655, BBa_K729004[pBAD], 0% ara are both able to grow biofilms, as shown by the intensity of the crystal violet staining. When BBa_K729004[pBAD] is expressed, the intensity of the crystal violet staining is reduced, showing a diminished ability to grow biofilm. This data suggests that the secretion of DNase is able to inhibit biofilm formation.

Conclusion

Through our experimental work, we have successfully created and submitted 12 sequence-confirmed BioBrick parts, 7 of which we rigorously characterized for antibacterial and/or antibiofilm function. We validated that Art-175 and Microcin S are both potent antibacterials, the former of which is shown to be even capable of killing antibiotic-resistant biofilm-encased bacteria. On the antibiofilm side of things, we not only showed that the enzymes of interest, DNase and DspB, were successfully exported across both membrane layers of E. coli following our modification of them with secretion tags, but also proved that the enzymes are able to refold properly post-secretion such that they retain their enzymatic function.

In conclusion, we achieved our aim of creating bacterial "living therapeutics" - strains of bacteria genetically engineered to secrete functional antibiofilm and antimicrobial proteins towards the treatment of UTIs.

Future

To develop our project beyond a proof-of-concept design, we would adopt a more suitable chassis, such as Lactococcus lactis. L. lactis has been widely used as a expression host for the production of proteins in both the medical and food industries. Being a Gram-positive species of bacteria, it is less likely to be killed by the same mechanisms as major Gram-negative pathogens such as E. coli and P. aeruginosa (e.g. Art-175's peptidoglycan lysis ability is specific for Gram-negative bacteria). On top of that, being Gram-positive means that it will not pose the problems of endotoxicity brought about by the outer membranes of Gram-negative bacteria. Using E. coli as our host was purely a starting point, in view of its ease-of-use as well as availability of pre-existing resources.

In addition to secreting antibiofilm/antimicrobial proteins, a comprehensive treatment for UTIs would be a bacteria engineered to also sense and move towards biofilms. We conducted extensive literature review on this in the early stages of the project but, due to the time restraints of a summer project, could not put our ideas into practice. With further work, we would incorporate both a sensing and chemotaxis mechanism into our design.

Nurses, doctors and professors all raised to us the issue of targeting the multiple bacterial and fungal species that are involved in UTIs, highlighting the fact that the problem extends further than E. coli and P. aeruginosa. We have explored how we would approach this in the Practices page.

Beyond the scientific issues of implementation, thinking seriously about the questions of ethics and public acceptance is also crucial for the further development of synbio-based medical therapies especially in view of the fact that it is currently illegal to even bring genetically-modified organisms outside of the laboratory environment. We have explored this theme also in the Practices page.

References

  1. Global Report on Surveillance of Antimicrobial Resistance: 2014. WHO.
  2. Johnson, J.R., 2004. Laboratory diagnosis of urinary tract infections in adult patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 39(6), p.873; author reply 873–874.
  3. Zalewska-Piatek, B. et al., 2013. Biochemical characteristic of biofilm of uropathogenic Escherichia coli Dr+ strains. Microbiological Research, 168, pp.367–378.
  4. Sievert, D.M. et al., 2013. antibiotic-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America, 34(1), pp.1–14. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23221186.
  5. Fux, C. a. et al., 2005. Survival strategies of infectious biofilms. Trends in Microbiology, 13(1), pp.34–40.
  6. Flemming, H.-C. & Wingender, J., 2010. The biofilm matrix. Nature reviews. Microbiology, 8(9), pp.623–633. Available at: http://dx.doi.org/10.1038/nrmicro2415.
  7. Høiby, N. et al., 2010. Antibiotic resistance of bacterial biofilms. International Journal of antibiotic Agents, 35(4), pp.322–332.
  8. Briers, Y. et al., 2014. Art-175 is a highly efficient antibiotic against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. antibiotic Agents and Chemotherapy, 58(7), pp.3774–3784.
  9. Gunzer, F., 2013. Bacterially-formed microcin S, a new antibiotic peptide, effective against pathogenic microorganisms, e.g. enterohaemorrhagic Escherichia coli (EHEC), European Patent EP2557163A1.
  10. Antibiotics - Side effects. Avaolable from: http://www.nhs.uk/Conditions/Antibiotics-penicillins/Pages/Side-effects.aspx [5/06/2015]
  11. C. M. Kunin, “Urinary tract infections in females,” Clinical Infectious Diseases, vol. 18, no. 1, pp. 1–12, 1994.
  12. J. W. Warren, “Catheter-associated urinary tract infections,” Infectious Disease Clinics of North America, vol. 11, no. 3, pp. 609–622, 1997
  13. A. P. Lenz, K. S. Williamson, B. Pitts, P. S. Stewart, and M. J. Franklin, “Localized gene expression in Pseudomonas aeruginosa biofilms,” Applied and Environmental Microbiology, vol. 74, no. 14, pp. 4463–4471, 2008.
  14. L. Zhang and T. Mah, “Involvement of a novel efflux system in biofilm-specific resistance to antibiotics,” Journal of Bacteriology, vol. 190, no. 13, pp. 4447–4452, 2008.
  15. J. Klebensberger, A. Birkenmaier, R. Geffers, S. Kjelleberg, and B. Philipp, “SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa,” Environmental Microbiology, vol. 11, no. 12, pp. 3073–3086, 2009
  16. U. Römling and C. Balsalobre, “Biofilm infections, their resilience to therapy and innovative treatment strategies,” Journal of Internal Medicine, vol. 272, no. 6, pp. 541–561, 2012
  17. Hale, S.P., Poole, L.B. & Gerlt, J. a, 1993. Mechanism of the reaction catalyzed by staphylococcal nuclease: identification of the rate-determining step. Biochemistry, 32(29), pp.7479–7487
  18. Vondervizst, F., Sajó, R., Dobó, J., & Závodszky, P. (2012). The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella. In: Recombinant Gene Expression - Reviews and Protocols, Methods in Molecular Biology, 824, 131-143.
  19. 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.
  20. Heras, B. et al., 2009. DSB proteins and bacterial pathogenicity. Nature reviews. Microbiology, 7(3), pp.215–225.
  21. 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.
  22. 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.
  23. 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.
  24. 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.
  25. 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.
  26. Hwang, I.Y. et al., 2014. Reprogramming microbes to be pathogen-Seeking killers. ACS Synthetic Biology, 3(4), pp.228–237.
  27. Dramatic rise seen in antibiotic use. Available from: http://www.nature.com/news/dramatic-rise-seen-in-antibiotic-use-1.18383?WT.mc_id=TWT_NatureNews [17/09/2015]