Healios: Working with Staph

Introduction - Helios

Approximately 30% of the human population is colonized with Staphylococcus aureus making it a human pathogen and a commensal bacteria species. It is the main cause of medical-related infections including MRSA (methicillin-resistant S. aureus)[1]. Research has shown an increase in the emergence of multiple antibiotic-resistant strains of S. aureus resulting in approximately 100,000 deaths in 2019 [9]. The prevalence of these infections combined with a lack of new antibiotic synthesis has called for new and unique treatment options.

Bacteriophage therapy has been identified as a means of antimicrobial treatment using naturally lytic bacteriophages. Bacteriophages, also known as phages, are viruses that only infect bacterial cells. Phages can be encoded with protein sequences that will bind to their outer coating causing the phage to display specific proteins on its surface.[9] This process allows phages to bind to specific targets, in our case S. aureus.

Photodynamic light therapy (PDT) has been proven to combat drug-resistant pathogens, including S. aureus [7] effectively. For PDT to work, a photosensitizing compound has to be concentrated in the cells so that when specific wavelengths of light are applied, only the cells containing the compound are killed. Highly reactive oxygen species are the catalyst for cell death by releasing energy and causing the cells to rupture. Current research has shown that PDT has a positive impact on the immune response, improving bacterial cell death rates [7]. Despite their effectiveness, these PDT compounds can damage healthy cells via ROS or exposure to heat. To combat this, the pathogenic cells need to be specifically targeted by binding the photosensitizing agents to the phage cells prior to contact with patients.

Our project Healios aims to combine multiple treatment methods to safely and effectively eradicate S. aureus while mitigating damage to healthy cells. This will include photodynamic light therapy with an engineered M13 bacteriophage capable of specifically binding to S. aureus. Bacteriophages carrying fused peptides Gp3 or Gp8 for binding against various S.aureus surface structures will be chemically conjugated to a photosensitizing dye known as Rose Bengal. Wavelength-specific photodynamic light will then be used to activate the dye after the phages have bound to S. aureus. This will generate reactive oxygen species and lyse the pathogen while leaving healthy cells unharmed. Healios consists of two genetic elements - one helper phage and one plasmid for producing the fusion proteins - to generate a modular system that allows for rapid change of gene expression and phage targeting via GoldenGate Cloning. This process allows for any desired peptide to easily be fused, not just those used to encode for S. aureus binding. This will not only enable the optimization of phage production against S. aureus but also allow for rapid adaptation by other researchers in future projects.

Figure 1. Helios two plasmid systems for the production of Gp3 and Gp8 and the helper phage to allow for the binding of S. aureus. (This image was created in BioRender and inspired by Petrosino et al ).

When Bacteria Go Rogue - Staphylococcus aureus

Our team chose S. aureus as the main focus due to its increasing prominence and potential to cause various infections, from minor skin issues to major bloodstream infections. S. aureus can produce toxins and develop resistance to multiple antibiotics, including a prominent variant known as MRSA (methicillin-resistant S. aureus). This bacteria is the leading cause of infections in healthcare settings and spreads rapidly through communities. Its unique virulence factors make it a significant concern for immunocompromised individuals, those with preexisting conditions, the elderly, and young children. As traditional treatments become less effective, researchers are seeking alternative treatments to combat this growing public health threat. Ultimately, S. aureus poses significant treatment, prevention, and public health challenges.

M13 - The Model Bacteriophage

The current treatment for S. aureus involves a series of broad-spectrum antibiotics such as vancomycin and daptomycin. However, S. aureus has proven to evolve and develop multiple antibiotic resistance tools. Thus our project aims to engineer a bacteriophage that can be used as an alternative to traditional treatment methods. Bacteriophages are viruses that only infect bacterial cells and are, therefore, not harmful to humans. M13 is a filamentous bacteriophage found in Escherichia coli that acts as a harmless and highly effective delivery platform as it can undergo significant genetic modification. This bacteriophage is commonly used in phage display where most peptides are presented on proteins pIII and pVIII [9]. M13 phages can be engineered to display peptides or nanobodies that bind to specific pathogenic bacteria and can be conjugated with a variety of photosensitizers. The ample use of the M13 bacteriophage in previous research allows us to understand molecular biology better, aiding in greater ease of genetic modification.[7]

Our genetic engineering and chemical conjugation strategy is based on the work of Petrosino et al.[7]. M13 phages will be modified to specifically bind to S. aureus by altering the existing surface proteins Gp3 and Gp8 which are responsible for binding their bacterial targets. The genetic devices that will be created are as follows: 1) a plasmid encoding either structural protein GP8 or GP3 fused with an S.aureus binding peptide, controlled by easily exchangeable promoter and ribosome binding sites, and 2) a helper phage that produces all other structural proteins. By removing from the helper phage the packaging sequence responsible for guiding the phage genome into the particle and adding it to the plasmid, we will increase the number of phage particles created and available for subsequent conjugation to the photosensitizing dye.

S. aureus Retargeting of M13

S. aureus surface proteins were identified and selected through in-depth literature reviews and expert consultation. Three peptides with high S. aureus binding affinities were selected including SSYGGS [10], RVRSAPSSS[2], and GIGKFLHSAGKFGKAFVGEIMKS[9]. Since M13 binds naturally to E. coli the surface binding proteins must be altered to retarget S. aureus. The peptides correlated to E. coli binding will be translationally fused to either the Gp3 or Gp8 proteins of the M13 phage.

Figure 2. Retargeting of the M13 bacteriophage to S. aureus after chemical conjugation of the photosensitizing compound Rose Bengal. (This image was created in BioRender and inspired by Petrosino et al ).

Photodynamic Light Therapy - Photosensitizers Engage

Photodynamic therapy (PDT) in recent years has shown promising effects for treating various infections. When PDT is coupled with the photosensitizing dye Rose Bengal and exposed to specific wavelengths of light reactive oxygen species are generated.[4] When the photosensitizer is activated two types of photooxidative reactions occur simultaneously.[8] During type I the excited photosensitizer (PS) reacts with a cellular substrate and gives up an electron or hydrogen atom creating radicals. These radicals include peroxides, hydroxyl groups, and superoxides which initiate cytotoxic reactions. Type II involves the transfer of energy from the excited PS directly to oxygen.[6] This type produces excited singlet oxygen, the most important ROS in PDT-induced apoptosis. Rose bengal, commonly used as a stain, exhibits one of the highest elevated quantum yields of singlet oxygen making it an optimal candidate for this experiment.[4] Rose Bengal dye was selected as our photosensitizing agent as it has previously been approved for ocular diagnostics and is widely available. Additionally, it is capable of generating a triplet excited state resulting in the effective production of ROS[7]. Rose Bengal was bound to the major capsid protein Gp8 of the M13 phage, which allowed for the specific photosensitizing of infected S. aureus.

Hardware

The complete Healios system will involve the clinical application of the S. aureus targeting M13 phages. We envision that patients with confirmed S. aureus infections can go into their local urgent care clinics where the physicians can disinfect their wound using the Healios system. The critical component will be an LED light source that can activate the Rose bengal dye bioconjugated to the M13 phages. Rose Bengal dyes are best activated by green or blue LEDs. We will develop a prototype LED light lamp mounted on a telescopic stand. By doing so we would be able to treat patients of various body types but would also have a device that can be easily adjusted depending on the location of the infection. The hardware will then work in tandem with the modified phage to ensure the lysis of S. aureus.

Figure 3. Healios Lamp final design.

Future Directions

Moving forward, we hope our flexible bacterial retargeting system can be adapted by other iGEM teams or research labs to treat other bacterial infections with the eventual goal of having this system made available in hospital settings. We would like to make this available in more accessible locations like local urgent care clinics. To accomplish this, we would like to seek partnerships with biotechnology and pharmaceutical companies to perfect our methods and improve our design for more effective applications.

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References

  1. C. Tong, S. Y., Davis, J. S., Eichenberger, E., & Holland, T. L. (2015). Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management. Clinical Microbiology Reviews, 28(3), 603-661. https://doi.org/10.1128/CMR.00134-14
  2. De Plano, L. M., Carnazza, S., Messina, G. M., Rizzo, M. G., Marletta, G., & Guglielmino, S. P. (2017). Specific and selective probes for Staphylococcus aureus from phage-displayed random peptide libraries. Colloids and Surfaces B: Biointerfaces, 157, 473-480. https://doi.org/10.1016/j.colsurfb.2017.05.081
  3. Fischer, Beat B, et al. “Photosensitizers Neutral Red (Type I) and Rose Bengal (Type II) Cause Light-Dependent Toxicity in Chlamydomonas Reinhardtii and Induce the Gpxh Gene via Increased Singlet Oxygen Formation.” Environmental Science & Technology, vol. 38, no. 23, 1 Dec. 2004, pp. 6307–13, https://doi.org/10.1021/es049673y. Accessed 1 May 2023.
  4. Kurosu, M., Mitachi, K., Yang, J., Pershing, E. V., Horowitz, B. D., Wachter, E. A., Lacey, J. W., Ji, Y., & Rodrigues, D. J. (2022, January 5). Antibacterial activity of pharmaceutical-grade Rose Bengal: An application of a synthetic dye in antibacterial therapies. Molecules (Basel, Switzerland).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8746496/
  5. Mayo Clinic Staff. (n.d.). Staph infections. https://www.mayoclinic.org/diseases-conditions/staph-infections/symptoms-causes/syc-20356221
  6. Panzarini, Elisa & Inguscio, Valentina & Dini, Luciana. (2011). Overview of Cell Death Mechanisms Induced by Rose Bengal Acetate-Photodynamic Therapy. International Journal of Photoenergy. 2011. 10.1155/2011/713726.
  7. Petrosino, A., Saporetti, R., Starinieri, F., Sarti, E., Ulfo, L., Boselli, L., Cantelli, A., Morini, A., Zadran, S. K., Zuccheri, G., Pasquini, Z., Di Giosia, M., Prodi, L., Pompa, P. P., Costantini, P. E., Calvaresi, M., & Danielli, A. (2023). A modular phage vector platform for targeted photodynamic therapy of Gram-negative bacterial pathogens. iScience, 26(10), 108032. https://doi.org/10.1016/j.isci.2023.108032
  8. Sabbahi, S., Ben Ayed, L. & Jemli, M. Staphylococcus aureus photodynamic inactivation mechanisms by rose bengal: use of antioxidants and spectroscopic study. Appl Water Sci 8, 56 (2018). https://doi.org/10.1007/s13201-018-0693-y Dow