Team:Pasteur Paris/Fighting

""

BACKGROUND

Biomedical implants, such as prostheses, catheters and several other implanted devices, can improve the patient’s quality of life. Nevertheless, because they are artificial they increase the risk of infection, handled from outside the body, and rarely have an antimicrobial property. In fact, device-associated infections account for 25.6% of all health-care-associated infections in the USA [1].

Orthopedic implant infections are caused by the development of pathogenic microorganisms on an inert material, allowing bacterial adhesion and biofilm formation on the implant. Biofilm-related infections frequently lead to chronic infections and require implant replacement. They are therefore responsible for a significant medical (on both the patient’s and surgeon's point of view) and economic burden [2] [3].

What are biofilms?

A biofilm is a community of microorganisms surrounded by an extracellular matrix produced by the microbes themselves. They can attach to various surfaces whether living or inert. Biofilm formation is a process that begins with the attachment of the microorganism, in our case bacteria, to a surface where they can grow under the control of specific biofilm genes expression. Bacteria living in a biofilm produce extracellular polymeric substances (EPS) composed mostly of water with polysaccharides, proteins, lipids and extracellular DNA [4] [5].

Stages of Biofilm formation

The process of biofilm formation applies to both Gram-negative and Gram-positive bacteria. It involves different stages, from adhesion of planktonic cells, microcolony formation, EPS production and biofilm maturation to dispersal stage, as shown in Figure 1.

Figure 1: Biofilm formation process. Adapted from E. Maunders et al., 2017[6]

How can biofilms form on an implanted device and elude immunity and antibiotic treatments?

A major issue in the fight against bacteria is their organization as biofilms. These bacterial aggregates strongly adhere to the biomaterial surfaces. In this bio-mechanical configuration, the implant-infecting biofilms can elude innate and adaptive host defenses as well as antibiotic therapies [7].

In fact, the biomaterial becomes encapsulated in layers of tissues which isolate the biofilm from the surrounding tissues and so shields it from the immune system. At the same time, the extracellular matrix also protects the microorganisms from immune cells [8].

Biofilms, by their architecture, give the bacteria that compose them an inherent resistance to antimicrobial agents by making it difficult for them to reach the bacteria [4]. Antibiotic treatments may lead to the removal of most of the bacterial population, but some of the bacteria frequently survive and can reconstitute the biofilm later when the antibiotic therapy is stopped [7]. The evasion of host defense and tolerance to antibiotics leads to the establishment of chronic infections.

Which microorganism is the most frequently responsible for prosthetic-joint infections?

Staphylococcus aureus, a Gram-positive coccus, is the most commonly isolated microorganism from orthopedic-associated infections, accounting for more than 40% of hip and knee prosthetic-joint infections shown by Table 1 [9]. Other species such as Staphylococcus epidermidis and Gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli are less frequent implant-infecting bacteria. The situation is alarming as implant-infecting S. aureus strains have high rates of antibiotic resistance against commonly used antibiotics (e.g. methicillin, gentamycin) [7].

Table 1: Distribution of organisms (the top 10 ranking organisms are identified to genus/species level) causing surgical site infection in patients undergoing knee and hip arthroplasty. [9]

What treatments are currently used?

In most cases, the treatment after a prosthetic-joint infection necessitates the withdrawal of the implant followed by several weeks of antibiotic treatment. Apart from the fact that it represents a heavy burden for the patient, the extended hospitalization length and reoperation represent a considerable excess of cost also for the health economy. A study led in France [9] estimates that the revision of infected prosthesis multiplies by a 3.6 factor the cost of the total procedure.

However, reoperation is almost always necessary, because as bacteria growing in biofilms are more resistant to antimicrobial therapies, dissolving biofilms using antibiotics is a complicated task. To fight this serious problem, new treatment strategies have already appeared, such as antimicrobial peptides (AMP), bacteriophages, matrix-degrading enzymes or inhibitors of Quorum-sensing [4]. An alternative approach to fight against prosthetic infections is to directly prevent pathogenic bacteria from forming a biofilm, thus blocking the pathogen development of resistance to the immune system and antibiotics.

OUR SOLUTION

Our strategy is to concentrate our efforts on fighting against S. aureus, since it is the leading cause of infections. S. aureus strains can develop resistance to antimicrobial peptides or toxin/antitoxin systems due to the selective pressure over time [10]. For this reason, we chose as an anti-biofilm strategy the disturbance of Quorum sensing. 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 [11] [12]. 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 [12]. This is why we focused on quorum sensing inhibitors as a promising antibiofilm strategy.

Quorum Sensing in S. 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[13].
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[14].
S. 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[13].

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[15]. 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[13], [16]. 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[16].
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.

Figure 1: The accessory gene regulator (agr) system in S. aureus. The P2 operon encodes the RNAII transcript, that corresponds to the signalling mechanism, and the P3 operon encodes the RNAIII transcript, that regulates the quorum response and virulence genes.

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 promoter 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)[16], [17].

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)[18].

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.

Figure 2: The detection proteins AgrC and AgrA are present in the engineered bacteria composing our biofilm, and thus, can detect the AIPs in the environment if S. aureus is present. AIPs will bind to AgrC, that will phosphorylate AgrA. This will induce the production and secretion of RIP, that inhibits S. aureus QS and thus, biofilm formation.

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

Visit the RESULTS to know more

REFERENCES

  • S. S. Magill et al., “Multistate Point-Prevalence Survey of Health Care–Associated Infections,” N. Engl. J. Med., vol. 370, no. 13, pp. 1198–1208, 2014.

  • T. Lamagni, S. Elgohari, and P. Harrington, “Trends in surgical site infections following orthopaedic surgery,” Curr. Opin. Infect. Dis., vol. 28, no. 2, pp. 125–132, 2015.

  • M. Sciences, “Annals of Microbiology and Research,” no. May, 2018.

  • L. Drago and M. Toscano, Biofilm formation and the biological response. Elsevier Ltd., 2016.

  • C. N. Melton and G. G. Anderson, Biofilms and Disease: A Persistent Threat, 4th ed. Elsevier Inc., 2018.
  • E. Maunders and M. Welch, “Matrix exopolysaccharides; the sticky side of biofilm formation,” FEMS Microbiol. Lett., vol. 364, no. 13, pp. 1–10, 2017.

  • C. R. Arciola, D. Campoccia, and L. Montanaro, “Implant infections: Adhesion, biofilm formation and immune evasion,” Nat. Rev. Microbiol., vol. 16, no. 7, pp. 397–409, 2018.

  • M. Ribeiro, F. J. Monteiro, and M. P. Ferraz, “Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions,” Biomatter, vol. 2, no. 4, pp. 176–194, 2012.

  • T. Lamagni, “Epidemiology and burden of prosthetic joint infections,” J. Antimicrob. Chemother., vol. 69, no. SUPPL1, pp. 5–10, 2014.

  • 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.