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Team:Tufts/ribosponge
From 2014.igem.org
Problem
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Background
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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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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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
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).
The first of these, ECK120033737, is upstream and serves to prevent any read-through by a polymerase starting before the T7 when the sequence is inserted into a plasmid. Downstream, another pair of terminators, ECK120029600, and ECK120033736, serve to terminate transcription of the merRNA and prevent read-through by a polymerase moving along the reverse strand, and generating reverse-complementary RNA that would base pair with the merRNA and cancel out its effects, respectively. It should be noted that this last, reverse-oriented terminator may be superfluous in most cases, but was necessary for our application as we chose to place the fragment into LITMUS28i which has opposing T7 promoters. It is also likely that the merRNA is self-terminating due to the presence of a poly-U intrinsic terminator. Sites for PstI and BamHI were added to each end of this design in order to facilitate insertion into a plasmid. The resulting 686 base pair sequence was then synthesized by GenScript.
Our initial goal was to express this fragment in bacteria and spread it using M13 bacteriophage. As such, we chose LITMUS28i-I716104 as our vector. This plasmid contains the original NEB LITMUS28i phagemid backbone and a GTG-starting T7 polymerase which itself is under the control of a T7 promoter. Leaky transcription at this T7 promoter starts expression of the T7 RNA polymerase. The plasmid was generated by Dr. Monica Ortiz of the Endy group who shared it with us for use in our project.
Our synthetic DNA and the plasmid were digested with PstI and BamHI and ligated together to form the construct with which all of our biofilm experiments were run. The plasmid was then transformed into TSS competent E. coli JM109 and maintained via ampicillin selection. The plasmid was also transformed into E. coli ZK1056 provided to us by the Kolter group at Harvard Medical School. ZK1056 is a known biofilm-forming strain and its characteristics have been widely studied. In addition to these modifications, we also transformed the two strains with LITMUS28i-I716104.
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Results
Expectations
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Temperature Dependence
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Our Hypothesis
Applications
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Parts
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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
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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.
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