Team:Pasteur Paris/Fighting
OUR SOLUTION
Our strategy is not to develop a system that kills directly Staphylococcus aureus, for example with an antimicrobial peptide or with a toxin/antitoxin system. In fact, S. aureus strains can develop resistance to this type of solution due to the selective pressure over time[1].
We chose to focus on preventing the formation of a biofilm by S. aureus, because it is the state in which pathogens develop resistance to both the immune system and to antibiotics[2], [3]. Communication within a bacterial community consists in the production and detection of signal molecules, in a system called quorum sensing. For instance, pathogens use quorum sensing to regulate and coordinate the biofilm’s architecture and the production of toxins and virulence factors[3]. This is why we focused on quorum sensing inhibitors as a promising antibiofilm strategy.
Quorum Sensing in Staphylococcus aureus
Quorum sensing (QS) is a mechanism of cell-cell communication. It works thanks to the regulation of gene expression that produces and releases chemical signal molecules called auto-inducers in response to fluctuations in cell-population density[4].
There are three major types of QS systems: the acylhomoserine lactone (AHL) QS system in Gram-negative bacteria, the autoinducing peptide (AIP) QS system in Gram-positive bacteria and the autoinducer-2 (AI-2) QS system in both Gram-negative and -positive bacteria[5].
Staphylococcus aureus is a gram-positive coccus, that uses AIP Quorum Sensing, allowing diverse arrays of physical activities, including regulation of colonization and virulence factors production[4].
In S. aureus, these mechanisms are controlled by the accessory gene regulator (agr) QS system that has a biphasic strategy that regulates one another to cause disease[6]. The agr system is a chromosomal locus that encodes two transcribed transcripts: RNAII and RNAIII (Figure 1).
The first system, RNAII, is a polycistronic transcript that originates from the promoter P2 and encodes for four proteins – agrA, agrB, agrC and agrD – that allow the sensing properties of S. aureus[4], [7]. First, a small peptide is encoded by agrD, and is transformed into an auto-induced peptide (AIP) by AgrB, a transmembrane protein. AIPs from all the bacteria accumulate in the extracellular environment. Another transmembrane protein, AgrC, binds the extracellular AIPs and in turn, phosphorylates AgrA (the intracellular regulator). When phosphorylated, AgrA has an increased affinity for promoters P2 and P3[4], [6]. Thus, there is an upregulation of all the agr system.
The promoter P3 allows the transcription of the RNAIII transcript, that is the regulatory molecule that upregulates the production of numerous secreted toxins[7].
When colonies of S. aureus multiply and grow, they secrete a protein called RAP. RAP accumulates outside the cells and when it reaches a threshold, it induces a histidine phosphorylation of its target molecule, TRAP. When TRAP is phosphorylated, it activates the agr system and the production of toxins.
Detection of S. aureus by our biofilm and inhibition of QS
We wanted our interface to produce a protein that would inhibit the development of S. aureus in the environment of the implant, but we didn’t want this protein to be secreted continuously and to accumulate outside the biofilm. We wanted our biofilm to start producing growth inhibiting molecules only in the presence of a pathogen.
We decided to use the Biobrick BBa_I746100 from iGEM Cambridge 2007 Team and to improve it by optimizing it for our chassis: E. coli BL21 strain.
We engineered the bacteria composing our biofilm by introducing the genes encoding for AgrC and AgrA proteins, the two proteins responsible for the detection of AIPs. They are encoded under the constitutive promotor BBa_J23107, from iGEM Berkeley 2006 Team
When AIPs are detected in the environment by the transmembrane protein AgrC, AgrA is phosphorylated and has an increased affinity for the promoter P2.
In our engineered bacteria, P2 encodes for a protein called RNAIII Inhibiting Peptide (RIP). RIP is a small peptide (seven amino-acids) that has been proven to inhibit biofilm formation of S. aureus. Indeed, RIP competes with RAP by phosphorylating TRAP, which leads to an inhibition of TRAP phosphorylation. This leads to the inhibition of RNAIII transcription and thus to the attenuation of virulence factor production, inhibition of cell-cell communication and decreased adhesion capacities (Figure 2)[7], [8].
Because we needed our peptide to be secreted by our biofilm, we targeted RIP to the periplasm of E. coli by using the Sec-dependent Type II secretion system. Our peptide is fused to an amino-terminal signal sequence, that is recognized by the chaperone SecB, then addressed to SecA and translocated across the inner membrane through the SecYEG complex. One advantage of this system is that the signal sequence gets cleaved during translocation through SecYEG. Several different signal sequences have been characterized (all formed of 18 to 30 amino acids)[9].
Following the advice of Dr. Jean-Michel Betton, Research Director in Structural Microbiology at the Institut Pasteur, we chose to try two different signal sequences to export our peptides, which he knew were efficient: MalE and DsbA. Usually, a secretion machinery called “secreton” enables the release of proteins extracellularly, but this is not very well characterized yet, and it is a complex machinery composed of more than 10 proteins, so we did not plan to exploit it. However, since RIP is only a 7 amino-acid protein, a leaky release from the periplasm to the medium should be sufficient to obtain RIP in the medium.
To sum up, in the presence of S. aureus in the environment of our biofilm, our engineered bacteria secretes a small peptide, RIP, that competes with RAP, and inhibits the formation of a pathogenic biofilm. Unlike common antibiotics, RIP inhibits cell-to-cell communication rather than killing pathogenic bacteria. Thanks to such an approach, we avoid the development of antibiotic resistance by pathogens and we force pathogens to remain in their planktonic stage, in which they stay susceptible to the host immune system.
RESULTS
Achievements:
- Successfully cloned a part coding for RIP in pBR322 and in pSB1C3, creating a new composite part
- Successfully cultivate S. aureus biofilms with different supernatants
Next steps:
- Clone the sensor device with inducible RIP production upon S. aureus detection
- Improve the characterization of RIP effect on biofilm formation
REFERENCES
- D. Lebeaux and J. Ghigo, “associées aux biofilms Quelles perspectives thérapeutiques,” 2012.
- M. R. Shakibaie, “Bacterial Biofilm and its Clinical Implications,” Ann. Microbiol. Res., vol. 2, no. 1, pp. 45–50, 2018.
- K. I. Wolska, A. M. Grudniak, Z. Rudnicka, and K. Markowska, “Genetic control of bacterial biofilms,” J. Appl. Genet., vol. 57, no. 2, pp. 225–238, 2016.
- J. M. Yarwood and P. M. Schlievert, “Quorum sensing in Staphylococcus infections,” J. Clin. Invest., vol. 112, no. 11, pp. 1620–1625, 2003.
- G. Brackman and T. Coenye, “Quorum Sensing Inhibitors as Anti-Biofilm Agents,” Curr. Pharm. Des., vol. 21, no. 1, pp. 5–11, 2014.
- C. M. Waters and B. L. Bassler, “QUORUM SENSING: Cell-to-Cell Communication in Bacteria,” Annu. Rev. Cell Dev. Biol., vol. 21, no. 1, pp. 319–346, 2005.
- Y. Gov, A. Bitler, G. Dell’Acqua, J. V. Torres, and N. Balaban, “RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: Structure and function analysis,” Peptides, vol. 22, no. 10, pp. 1609–1620, 2001.
- A. Giacometti et al., “RNA III Inhibiting peptide inhibits in vivo biofilm formation by drug-resistant Staphylococcus aureus,” Antimicrob. Agents Chemother., vol. 47, no. 6, pp. 1979–1983, 2003.
- R. Freudl, “Signal peptides for recombinant protein secretion in bacterial expression systems,” Microb. Cell Fact., vol. 17, no. 1, pp. 1–10, 2018.