Team:Tufts/ribosponge

Problem

Biofilms present one of the largest problems facing healthcare in the United States today. The ability of bacterial pathogens to form biofilms is one of their most important virulence factors, as biofilm formation confers resistance to both antibiotics and natural host defense mechanisms. (1) These sessile bacterial growths, often of the genus Staphylococcus, form in the lungs of cystic fibrosis patients, in patients suffering from chronic infections, and on implant materials such as catheters. The CDC estimates that 65% of infections in developed nations are caused by persistent bacterial biofilms. (2) They are the leading cause of Healthcare-Associated Infections (HAI’s), of which there are two million instances and 100,000 resulting deaths each year in the United States alone. (3) The inability to treat biofilms with antibiotics and antimicrobials has lead the Tufts iGEM Team to seek novel methods for disrupting biofilm formation.
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Background

Not-so-magic bullets “Two years into the war the Army issued its first meager supplies of penicillin, instructing physicians to use the precious drug sparingly, in doses of about 5,000 units (less than a third of what would be considered a minimal penicillin dose for minor infections in 1993 ). In those early days before bacteria became resistant to antibiotics, such doses were capable of performing miracles, and the Army doctors were so impressed with the powers of penicillin that they collected the urine of patients who were on the drug and crystallized excreted penicillin for reuse on other GIs.” Laurie Garrett - The Coming Plague

Humanity is locked in a perpetual arms race against its pathogens. For the majority of our existence our species has relied upon its innate immune ability in order to survive disease. This has proven effective at a population level so far: no pathogen has driven us to extinction. However, in the last two centuries humanity has added new weapons to its disease fighting arsenal: antibiotics reduced bacterial infections from life-threatening to trivial matters. Antiviral drugs have rescued those infected with HIV from the brink of death. For a time, these drugs were so indisputably effective they were thought of as “magic bullets” and thrown at pathogens indiscriminately. Unfortunately, their efficacy has been diminishing. In a textbook example of selective pressure, the most susceptible bacterial pathogens were wiped out while those able to withstand antibiotics have thrived.
Antibiotics have several modes of action: beta lactams inhibit cell wall synthesis, aminoglycosides interfere with translation and lead to misfolded proteins, and fluoroquinones disrupt DNA gyrase and topoisomerase during cell replication. In response, bacteria have evolved the ability to degrade these molecules with enzymes such as the beta-lactamase, or to actively remove them from the intracellular space by means of efflux pumps. Such pathogens are deemed resistant to antibiotics as they can grow and persist in their presence unperturbed.

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Laying low “He who knows when he can fight and when he cannot will be victorious.” – Sun Tzu, The Art of War

Efflux pumps and hydrolytic enzymes are by no means the only evolutionary strategies which pathogenic organisms have in order to escape destruction. Many bacteria have the ability to reduce their metabolism to a minimum and enter a dormant stage in which little to no new cell wall construction, protein translation, or DNA replication occurs. (2) (4) (5) As a result, any cells that have in effect shut down are in practice impervious to the modes of action of modern antibiotics. Such cells do not need to be genetically resistant because their ability to become dormant allows them to tolerate the presence of antibiotics. (2) The strategy is effective not only against toxic chemicals, but also applicable during periods of overcrowding or low nutrient availability.
Cells entering a persistent biofilm undergo a transition from a more motile state to a sessile state, and use their extracellular projections to attach to one another and to their surroundings. In addition to clumping together, certain bacteria secrete a polymer which forms a protective matrix around the cells. In human wounds infected with P. aeruginosa this exopolymer matrix is effective at denying macrophages access and preventing them from digesting the dormant bacteria. Should antibiotic-susceptible members of the biofilm population be wiped out, the space enclosed by the exopolymer can be repopulated by any individual bacteria which carry resistance genes once therapy is discontinued. The persistence of these bacteria as a biofilm within the lungs of cystic fibrosis patients and in chronic wounds is well known. In addition, the CDC estimates that 65% of infections in developed nations like the United States are caused by persistent bacterial biofilms. (2)
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Signaling

The ability of bacteria to lie low is an indispensable evolutionary adaptation for P. aeruginosa, M. tuberculosis, and V. cholerae. In effect these bacteria have the ability to wait out and evade our immune systems or persist contrary to adverse environmental factors. Is there a unifying factor to these seemingly similar latent behaviors?
The chemical Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) was discovered in the late 1980s. (7) The compound is created when two molecules of guanosine triphosphate are joined in a reaction catalyzed by diguanylate cyclase (DGC). Originally found in Gluconacetobacter xylinus, natural and synthetic versions of the molecule were shown to activate the bacterium’s membrane-bound cellulose synthase. (8) In other bacteria it has been shown to promote the synthesis of adhesins and exopolymers necessary for biofilm formation. (5) (8) In order to test the effects of c-di-GMP research groups working with a range of bacterial species have upregulated the production of DGC enzymes responsible for the secondary messenger’s synthesis. (8) This modification caused the transgenic strains to become sessile and less virulent, implicating that the secondary messenger is the signal which informs the cells to switch to an inactive, sessile, and persistent state. (8) On the other hand, artificially stimulating the phosphodiesterase (PDE) enzymes which break down c-di-GMP resulted in motile populations unable to form a biofilm. (8) (See Figure)

Figure: Cyclic di-GMP, its synthesis and breakdown pathways, and its effects on bacterial phenotype. Taken from (9).

Additional work has also provided further information about the mode of action of the c-di-GMP molecule itself. In silico computations predicted that the PilZ protein domain present in G. xylinus would bind the secondary messenger. As a result of this, PilZ domains have been identified in many bacterial proteins whose activity responds negatively to the presence of c-di-GMP. (7) One example, the YegR protein of free swimming E. coli, contains a PilZ site. Experiments show that the deletion of the phosphodiesterase enzyme responsible for c-di-GMP degradation in the E. coli strain leads to reduced swimming ability. In addition, subsequent deletion of the YegR protein (which binds and responds to the presence of c-di-GMP) restores motility to 80% of that exhibited by the wild type bacteria. (8)
Riboswitch aptamers (ligand-binding nucleic acid molecules) for c-di-GMP have been identified. (9) (10) These RNA aptamers can come before or after a transcribed gene. Examples of “on” and “off” regulatory c-di-GMP riboswitches have been observed in P. aeruginosa, M. tuberculosis, V. cholerae, Y. pestis, and other bacterial species which latent phases which are more tolerant of antibiotics and adverse conditions. (6) (8) (9) (10) It seems that this secondary messenger is the key molecule in persistent cells.

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The Plan

Killing the Messenger
“The most important secret in war consists of making one’s self the master of communications.” – Napoleon Bonaparte, Pensées

In summary, current data shows that if cyclic di-GMP is eliminated from the cells of a number of bacterial species, they cannot enter the persistent state. Theoretically, the pathogens can be prevented from shutting down and becoming tolerant to antibiotics or lack of nutrients. Experiments have already demonstrated this by overproducing the phosphodiesterases which break down the messenger or mutating the dyguanylate cyclases which produce it. (9)
Targeting c-di-GMP across all these species could be done by excising the enzymes which are responsible for synthesizing the molecule or adding more phosphodiesterases to break it down. Genetic editing that is targeted with this degree of specificity is difficult to accomplish in a clinical setting. Furthermore, the addition of new proteins would increase the metabolic burden associated with their translation.
A potential tool is presented by the gram-negative predator of gram-negatives, Bdellovibrio bacteriovorus. This organism also has two distinct life phases. A free-swimming attack phase allows it to roam and locate its prey. After finding a suitable host, B. bacteriovorus attaches to the unfortunate gram negative bacterium and digests it from within. A recent transcriptome analysis by Karunker and colleagues has shown that the most common RNA identified by sequencing was a non-coding segment which houses a c-di-GMP aptamer. (11) This RNA was only present during the motile attack phase. In addition, it accounted for just as many reads as all protein coding sequences combined and is about as common as structural nucleic acids such as rRNA. These results imply a different mode of action for the bacteriovorus riboswitch. The sequence acts more like a sponge which sequesters available c-di-GMP than a switch which responds to its presence. The discoverers have dubbed this 445nt transcript massively expressed regulatory RNA (merRNA). (11)
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Strategy and Methods

The merRNA from Bdellovibrio could be introduced into the genome of other bacterial species and placed under the control of an always-on constitutive promoter. Cyclic di-GMP RNA aptamers are known to bind 1000-fold more tightly to their messenger (KD ~ 1nM) than other regulatory proteins like PilZ (KD ~ 1µM). (10) Thus, these transcripts of the merRNA should in turn sequester the secondary messenger. As a result, the pathways responsible for the transition to a persistent state should not be activated. One advantage of this strategy is that the ribosponge is a non-coding RNA construct; the metabolic burden of transcription should be much lower than that demanded by the transcription and activity of a phosphodiesterase or genome editing enzyme.

We designed a strategy to express merRNA in E. coli and test its effects on biofilm growth using SnapGene molecular biology software. The merRNA sequence was obtained from Karunker et al (11) and identified in the Bdellovibrio bacteriovorus HD100 genome at position 1,073,741−1,074,185 with a length of 445 nucleotides.. Since the goal here was to physically sequester c-di-GMP in cells, we aimed to produce large quantities of the RNA and thus decided to place it under the control of the T7 promoter which is both constitutive and leads to very strong expression. In turn, this sequence is flanked by a set of three strong, naturally occurring terminators from a library characterized by Chen and colleagues (12).

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Results

Expectations

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Temperature Dependence

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Our Hypothesis

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Applications

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Parts

BBa_K1427000

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BBa_K1427001

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Notebook

  Lab Notebook - June
  Lab Notebook - July
  Lab Notebook - August
  Lab Notebook - September
  Lab Notebook - October

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Works Cited

1. Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice. Rong Wang ... Shu Y. Queck, Michael Otto. January 4, 2011 J Clin Invest. 2011;121(1):238-248. doi:10.1172/JCI42520.
http://www.jci.org/articles/view/42520
2. Persister cells, dormancy and infectious disease. Lewis, Kim. January 2007, Nature, pp. 48-56.
3. NIH Scientists Identify Mechanism Responsible for Spreading Biofilm Infections Finding Could Lead to Treatment to Prevent Infection Associated with Catheters and Medical Implants. Monday, Dec. 6, 2010. National Institute of Allergy and Infectious Diseases (NIAID)
http://www.niaid.nih.gov/news/newsreleases/2010/Pages/biofilmMechanism.aspx
4. BACTERIAL BIOFILMS: FROM THE NATURAL ENVIRONMENT TO INFECTIOUS DISEASES. Hall-Stoodley, Luanne, Costerton, J. William and Stoodley, Paul. February 2004, Nature Reviews | Microbiology, pp. 95-108.
5. Signals, Regulatory Networks, and Materials That Build and Break Bacterial Biofilms. Karatan, Ece and Watnick, Paula. 2, 2009, Microbiology and Molecular Biology Reviews, Vol. 73, pp. 310-347.
6. Phytoplankton-linked viable non-culturable Vibrio cholerae O1 (VNC) from rivers in Tucuman, Argentina. Seeligmann, Claudia, et al., et al. 4, 2008, Journal of Plankton Research, Vol. 30, pp. 367-377.
7. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Tischler, Anna and Camilli, Andrew. 3, 2004, Molecular Microbiology, Vol. 53, pp. 857-869.
8. Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis. Tamayo, Rita, Pratt, Jason and Camilli, Andrew. 2007, Annual Review of Microbiology, pp. 131-148.
9. Principles of c-di-GMP signalling in bacteria. Hengge, Regina. April 2009, Nature Reviews | Microbiology, pp. 263-273.
10. Riboswitches in Eubacteria Sense the Second Messenger Cyclic Di-GMP. Sudarsan, N, et al., et al. July 18, 2008, Science, pp. 411-413.
11. A Global Transcriptional Switch between the Attack and Growth Forms of Bdellovibrio bacteriovorus. Karunker, Iris, et al., et al. April 16, 2013, PLOS|ONE.
12. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Chen, et al., June 02, 2013, Nature Methods.

Works Referenced

12. Todar, Kenneth. Mycobacterium tuberculosis and Tuberculosis. Todar's Online Textbook of Bacteriology. [Online] [Cited: December 16, 2013.] http://textbookofbacteriology.net/tuberculosis_2.html.
13. Tuberculosis: What We Don't Know Can, and Does, Hurt Us. Russel, David, Barry, Clifton and Flynn, JoAnne. May 14, 2010, Science, pp. 852-856.
14. Mycobacterium tuberculosis: success through dormancy. Gengenbacher, Martin and Kaufmann, Stefan. 2012, Federation of European Microbiological Societies, pp. 514-532.
15. Structural and mechanistic determinants of c-di-GMP signalling. Schirmer, Tilman and Jenal, Urs. 2009, Nature Reviews, pp. 724-735.
16. Cyclic di-GMP mediates Mycobacterium tuberculosis dormancy and pathogenicity. Hong, Yuzhi, et al., et al. 2013, Tuberculosis, pp. 625-634.
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19. Mycobacteriophage and their application to disease control. McNerney, R. and Traore, H. 2005, Journal of Applied Microbiology, pp. 223-233.
20. Ortiz, Monica. Enabling population-level programming via DNA messaging. Northeastern University. [Online] [Cited: December 20, 2013.]
http://madrid.ccs.neu.edu/biocom2/wp-content/uploads/2013/01/Monica_Ortiz.pdf.
21. Engineered cell-cell communication via DNA messaging. Ortiz, Monica E and Endy, Drew. 2012, Journal of Biological Engineering.
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http://parts.igem.org/Part:BBa_B0015. 27. iGEM07_Berkeley_UC. T7rnap (T7 RNA Polymerase) - GTG Start Codon. Registry of Standard Biological Parts. [Online] 06 09, 2007. [Cited: December 21, 2013.]
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