Antimicrobial peptide GL13K

GL13K: An antimicrobial peptide with therapeutic potential

Bacterial biofilms are a key obstacle in any medical device or implant application. Once established, bacterial biofilms are virtually impossible to eradicate under biological conditions. Long term use of antibiotics or biocides are problematic due to bacterial resistance and toxicity.

To address this need, we have used a biologically based approach to discover novel antimicrobial peptides (AMPs). Rather than screening vast peptide libraries, structural analysis was used to identify human proteins that could serve as a template for AMP design. This approach is a more direct and less time-consuming screening, and is predicted to present less toxicity and bacterial resistance due to the human origin of the new AMPs (no bacteria are known to be resistant to human AMPs).

The lead peptide emerging from this approach, GL13K was used to develop a novel antimicrobial coating that prevents biofilm formation, is bactericidal, can kill drug-resistant bacteria and exhibits low mammalian toxicity. The coating is very stable and resists degradation by mechanical, hydrolytic and/or enzymatic challenges.

We invite you to browse the information below to learn more about GL13K and the applied peptide coatings or contact the investigators directly for more information and collaborative/licensing opportunities.

The peptide

Therapeutic need

“Drug-resistant pathogens are a growing menace to all people, regardless of age, gender, or socioeconomic background. They endanger people in affluent, industrial societies like the United States, as well as in less-developed nations.” (Interagency-Task-Force-on-Antimicrobial-Resistance, 2011).

Antibiotic resistance is on the rise and mortality from bacterial infection and attendant sepsis is increasing. Many bacteria show a high incidence of resistance to available antibiotics and can develop antibiotic resistance during treatment with broad spectrum antibiotics. Increased resistance to common antibiotics has led to the reintroduction of polymyxin (colistin) as a last resort, although concerns over toxicity remain. To overcome antibiotic resistance, treatment with multiple antibiotics is recommended. Since few anti-bacterial agents are in development, there is a need to identify such agents either for use alone or in combination with existing antibiotics. Cationic antimicrobial peptides (AMP) have shown some promise as a new class of antibiotic, although several obstacles remain to be overcome.

Challenges facing AMPs

GL13KThe identification of AMPs led to extensive research with the hope of identifying novel antibiotics for the treatment of bacterial infections. However, despite promising in vitro results the use of AMPs in vivo has met with several challenges and no new AMP has reached clinical use to date. Some of the challenges associated with AMPs include host-toxicity; lack of activity under physiological conditions; post-translational modifications; and concerns over resistance to human host-defense proteins. We have introduced a new family of AMPs that includes peptide modifications to target specific antimicrobial activities and bacterial types and address several of the challenges faced by other AMPs.

Introduction of GL13K

In recent years, we have designed new antimicrobial peptides based on the sequence of the human salivary protein Parotid Secretory Protein (PSP; BPIFA2). A recent modification of these peptides produced the peptide GL13K, which has shown promise in preliminary in vitro and in vivo experiments. A further modification by introduction of D-amino acids has improved stability and folding to decrease the effective concentration and enhance activity in PBS without an apparent increase in toxicity. Thus, preliminary data for GL13K suggests that this peptide can overcome several of the challenges encountered with other cationic AMPs. The short (13 amino acids) linear structure and absence of post-translational modifications simplifies synthesis and lowers cost.

In addition to the positive profile described above, GL13K shares several of the potential advantages with other AMPs: The peptide is active against several bacterial species; rapid onset of killing; bactericidal activity; inactivates lipopolysaccharide and exhibits anti-inflammatory activity in vivo. Importantly, GL13K is active against drug-resistant bacteria. In fact, drug-resistant Pseudomonas aeruginosa showed increased susceptibility to GL13K. In general, resistance to AMPs has been slow to develop and the mechanism of action targeting bacterial cell membranes for entry into the cell allows for activity against metabolic inactive or dormant cells, an important advantage for treatment of biofilms. Indeed, GL13K has shown promising activity against both aerobic and anaerobic biofilms. Full eradication of P. aeruginosa biofilms can be achieved by a combination of GL13K and tobramycin.

Applications

FE-SEM image of the morphology of disrupted S. gordonii bacteria wall cultured in the drip flow bioreactor on GL13K-coated surfaces. Ruptured bacteria showed protoplasts with localized disturbances of the bacterial membrane.

Titanium implants are used extensively in medicine and dentistry with a high success rate. However, depending on conditions, 20% of dental implants, 10% of orthopedic fixation devices, and up to 4 % of orthopedic joint replacements develop peri-implant infections over their lifetime. Bacteria can invade the inert implant surfaces where they establish biofilms that are largely out of reach for the host natural defenses and can infect the tissues surrounding the implant. Infection can occur at early and late stages after surgery. This increases the challenge for protecting the surface since continuous local and/or systemic prophylaxis is currently not clinically feasible. In fact, these bacterial biofilms are extremely difficult to eradicate with traditional antibiotics due to the low metabolism of cells in the biofilm. Dentistry has developed successful strategies to combat bacterial biofilms on tooth surfaces without the use of antibiotics. Studies in the highly accessible oral sites have shown that strategies that prevent biofilm formation allow host defenses to control the overall microbiome and maintain the health of surrounding tissues.

Using these principles, peri-implantitis can be prevented by inhibiting growth of biofilm and kill bacteria through contact with the implant surface over extended periods after surgery. To that purpose, we have developed a novel antimicrobial peptide coating, which unlike conventional antibiotics, is effective against cells that are not metabolically active.

The coating contains the peptide GL13K, which was developed from the human salivary protein Parotid Secretory Protein, and it is effective against planktonic or biofilm Gram positive and Gram negative bacteria with low host toxicity. The GL13K peptide is covalently coupled to titanium surfaces using silane chemistry.

The GL13K peptide coatings:

  • exhibit sustained antimicrobial activity against key pathogens of oral infections such as Porphyromonas gingivalis and Streptococcus mutans.
  • retain antimicrobial activity after sterilization by autoclaving and repeated cycles of exposure to biological fluids and bacterial challenge.
  • are cytocompatible with fibroblasts and osteoblasts.
  • are resistant to mechanical, hydrolytic, and enzymatic/proteolytic degradation
  • uniquely disrupt the integrity of the wall of gram positive bacteria such as Streptococcus gordonii.

Data

GL13K Molecular model of the GL13K peptide

GL13K GL13K kills P. aeruginosa within 5 min. GL13NH2 does not show bactericidal activity. Bacteria were incubated with each peptide for the time indicated and then plated on agar to determine colony formation.

GL13K Antimicrobial effect of GL13K-peptide coatings on S. gordonii cultured for 3d in the drip flow bioreactor. Live/dead cell fluorescence staining showed strong antimicrobial effect of the GL13K peptide coatings in comparison to all control surfaces. S. gordonii bacteria were killed on the GL13K coated surfaces.

Chen, Hirt, Li, Gorr, Aparicio, 2014. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms. PLOS ONE 9(11):e11579.

FE-SEM images of S. gordonii biofilms FE-SEM images of S. gordonii biofilms. General view (top row) and Close-up (bottom row) images of surfaces tested in the drip flow bioreactor. The biofilms did not form and grow on the GL13K coatings.

Chen, Hirt, Li, Gorr, Aparicio, 2014. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms. PLOS ONE 9(11):e11579.

FE-SEM image of the morphology of disrupted S. gordonii bacteria wall cultured in the drip flow bioreactor on GL13K-coated surfaces. Ruptured bacteria showed protoplasts with localized disturbances of the bacterial membrane. FE-SEM image of the morphology of disrupted S. gordonii bacteria wall cultured in the drip flow bioreactor on GL13K-coated surfaces. Ruptured bacteria showed protoplasts with localized disturbances of the bacterial membrane.

Chen, Hirt, Li, Gorr, Aparicio, 2014. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms. PLOS ONE 9(11):e11579.

GL13K membrane disruption mechanism Schematic of the GL13K membrane disruption mechanism, (left) planar supported bilayer, (center) GL13K interacts with lipid head groups and (right) after reaching a threshold concentration, GL13K causes membrane destabilization by removing parts of it forming peptide lipid micelles or stable supra molecular structures.

Balhara, V., R. Schmidt, S.U. Gorr, and C. Dewolf. 2013. Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K. Biochimica et Biophysica Acta. 1828:2193-2203

Scanning electron microscopy of 24-h P. aeruginosa biofilm formed on the pegs of a Calgary device. Scanning electron microscopy of 24-h P. aeruginosa biofilm formed on the pegs of a Calgary device. (A) Biofilm on a representative control peg not exposed to antimicrobials. (B) Magnification (×20) of the area indicated by a white box in panel A. (C and D) Images of pegs treated with 100 μg/ml (70 μM) GL13K for 4 h at low (C) and high (D) magnifications.

Hirt and Gorr, 2013. Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 57:4903-4910

Antibacterial activity in vivo. Antibacterial activity in vivo. Romaine lettuce leaves were injected with 10 μl P. aeruginosa (108 cells/ml) in the presence or absence of 200 μg/ml of antibacterial peptides. (A) representative experiment showing the absence of infection at mock infected sites (No PAO1), infection through the midrib after injection of bacteria (Pa) or localized infection around the injection sites when bacteria were co-injected with GL-13-NH2 (Pa + GL13).

Gorr, S.U., J.B. Sotsky, A.P. Shelar, and D.R. Demuth. 2008. Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal/permeability increasing-like protein family. Peptides. 29:2118-2127

SEM images of the morphology of disrupted S. gordonii bacteria wallSEM images of the morphology of disrupted S. gordonii bacteria wall cultured in a drip flow bioreactor on GL13K-coated surfaces. GL13K-peptide coatings ruptured the cell wall of multiple bacteria (A). Two types of broken bacteria were identified. Bacteria with empty shell-like cell wall structures (solid arrows in A). Close up of these emptied broken walls can be observed in B, D, E. Bacteria that showed the protoplast (dashed arrows in A). Close up of bacteria exposing the protoplasts can be observed in C, D, F, and G. Arrows in C and E point to cells with broken walls at the polar and septum areas, respectively. Arrows in F and G point to protoplasts with localized disturbances of the bacterial membrane.

Chen, Hirt, Li, Gorr, Aparicio, 2014. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms. PLOS ONE 9(11):e11579.

GL13K-peptide coatings are resistant to shear stresses during surgical implantation. GL13K-peptide coatings are resistant to shear stresses during surgical implantation. Fluorescence-labeled GL13K peptide coatings on a dental implant surfaces A) before implantation in PU foamed blocks that simulate Type-2 bone (0.32 g/cm3, Pacific Research Laboratories, Inc., USA); and B) after implantation and removal from the GL13K-coated implants following standard surgical procedures. C) experimental set-up and implant screwed to the most coronal part of the implant (insert), after implantation and before extraction.

 

Funding

Research reported on this web site was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers R01DE017989, R01DE12205, and R90DE023058; the Office of the Vice-president for Research at the University of Minnesota through the Grant-in-Aid of Research, Artistry, and Scholarship Program; and the 3M Foundation through a 3M Non-Tenured Faculty Award. Parts of this work were carried out in the University of Minnesota I.T. Characterization Facility, which receives partial support from NSF through the MRSEC program.

Publications

Characterization of the GL13K peptide family

Hirt, H., and S.U. Gorr. 2013. Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 57:4903-4910.

Balhara, V., R. Schmidt, S.U. Gorr, and C. Dewolf. 2013. Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K. Biochimica et Biophysica Acta. 1828:2193-2203.

Abdolhosseini, M., S.R. Nandula, J. Song, H. Hirt, and S.U. Gorr. 2012. Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity. Peptides. 35:231-238.

Abdolhosseini, M., J.B. Sotsky, A.P. Shelar, P.B. Joyce, and S.U. Gorr. 2012. Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain. Molecular and Cellular Biochemistry. 359:1-8.

Gorr, S.U. 2012. Antimicrobial peptides in periodontal innate defense. Frontiers of Oral Biology. 15:84-98.

Gorr, S.U., and M. Abdolhosseini. 2011. Antimicrobial peptides and periodontal disease. Journal of Clinical Periodontology. 38 Suppl 11:126-141.

Gorr, S.U., M. Abdolhosseini, A. Shelar, and J. Sotsky. 2011. Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein. Biochemical Society Transactions. 39:1028-1032.

Gorr, S.U. 2009. Antimicrobial peptides of the oral cavity. Periodontology 2000. 51:152-180.

Gorr, S.U., J.B. Sotsky, A.P. Shelar, and D.R. Demuth. 2008. Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal/permeability increasing-like protein family. Peptides. 29:2118-2127.

Geetha, C., S.G. Venkatesh, L. Bingle, C.D. Bingle, and S.U. Gorr. 2005. Design and validation of anti-inflammatory peptides from human parotid secretory protein. J Dent Res. 84:149-153.

Bingle, C.D., and S.U. Gorr. 2004. Host defense in oral and airway epithelia: chromosome 20 contributes a new protein family. Int. J. Biochem. Cell Biol. 36:2144-2152.

Geetha, C., S.G. Venkatesh, B.H. Fasciotto Dunn, and S.U. Gorr. 2003. Expression and anti-bacterial activity of human parotid secretory protein (PSP). Biochemical Society Transactions. 31:815-818.

Biofunctional peptide coatings

X. Chen, H. Hirt, Y. Li, S.U. Gorr, C. Aparicio. 2014. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms. PLOS One. Accepted.

E. Salvagni, G.Y. Berguig, E. Engel, J.C. Rodriguez-Cabello, G. Coullerez, M. Textor, F.J. Gil, J.A. Planell, C. Aparicio. 2014. A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces. Colloids and Surfaces B: Biointerfaces 114: 225-233.

Y. Li, X. Chen, A. Ribeiro, E. Jensen, K.H. Holmberg. J.C. Rodriguez-Cabello, C. Aparicio. 2014. Hybrid nanotopographic surfaces obtained by biomimetic mineralization of statherin-inspired elastin-like recombinamers. Advanced Healthcare Materials 3:1638-1647.

K.V. Holmberg, M. Abdolhosseini, X. Chen, Y. Li, S. U. Gorr, C. Aparicio. 2013. Bio-inspired Antimicrobial Peptide Coating for Dental Implants. Acta Biomaterialia 9(9): 8224-31.

X. Chen, P. Sevilla, C. Aparicio. 2013. Surface biofunctionalization by covalent co-immobilization of oligopeptides. Colloids and Surfaces B: Biointerfaces. 107:189-97.

X. Chen, Y. Li, C. Aparicio. 2013. Biofunctional Coatings for Dental Implants. In Thin Films and Coatings in Biology. Biological and Medical Physics -Biomedical Engineering Series; ed. S. Nazarpour Springer-Verlag. ISBN 978-94-007-2592-8. pp 105-143.

Patents

US 8569449 B2
Synthetic peptides and peptide mimetics 
Inventors: Sven-Ulrik Gorr
Assignee: University of Louisville Research Foundation, Inc.
Summary of invention :The present inventors have designed several peptides based on the sequences of human Parotid Secretory Protein (PSP, also called SPLUNC2 or C20ORF70).

WO 2011036326 A3
Novel recombinant proteinic polymers and method for bioactivating surfaces with said polymers
Inventors: Conrado Aparicio and 9 more
Assignee: Universitat Politècnica de Catalunya, Universidad de Valladolid
Summary of the invention: The invention relates to proteinic polymers or mixtures thereof comprising domains that can promote the nucleation/growth and/or adhesion of calcium phosphates of biological interest, and domains that can promote cellular adhesion by means of peptides of the proteins of the extracellular matrix. The invention also relates to methods for activating materials for osseous implants using said proteinic polymers and to the implants that can be obtained by said methods.

Investigators

Sven Ulrik-Gorr

Sven-Ulrik Gorr, PhD

sugorr@umn.edu

  • University of Copenhagen, Denmark, Cand. Scient (MSc), 1977-1984 (Biochemistry)
  • University of Basel, Switzerland, Post-grad, 1984-1985 (Pharmacology)
  • University of Copenhagen, Denmark, PhD, 1987-1990 (Cell Biology)
  • University of Louisville, Kentucky, Res. Assoc., 1985-1988 (Cell Biology)

After research training in Denmark, Switzerland and the U.S., I joined the faculty of the University of Louisville and in 2009 moved to the University of Minnesota. I have over 20 years experience as an independent investigator with additional roles as a research administrator at the National Institutes of Health and the University of Minnesota. Our research is focused on the function of salivary glands and salivary proteins and the design of antimicrobial peptides based on salivary protein structures. Thus we have studied the salivary secretory protein Parotid Secretory Protein (BPIFA2) for 10+ years. In 2001, we were the first to report the cDNA sequence of the human protein and found that the protein appeared to be related to host-defense and lipid-binding proteins. Based on this proposed similarity we designed and tested potential antimicrobial peptides. The lead peptide, GL13K, is active against drug-resistant bacteria, planktonic and biofilm bacteria, Gram negative and Gram positive bacteria and appears to act by removing lipid micelles from the cell membrane. We have built on these observations with active collaborations to further characterize these novel peptides and their innovative use as antibacterial coatings for implant surfaces. 

Conrado Aparicio

Conrado Aparicio, Ph.D.

apari003@umn.edu

  • Technical University of Catalonia, Spain, MSEng, 1997 (Mechanical Engineering)
  • Technical University of Catalonia, Spain, PhD, 2005 (Biomaterials)
  • Northwestern University, Chicago, Illinois, Post-doc, 2006-2007 (Nanobiomaterials)

I am an investigator with a research focus on dental biomaterials, their design, synthesis, as well as the characterization and analysis of their physical, chemical, mechanical, and biological properties.

I am a materials engineer with 15 years experience in the field of biomaterials for dental and orthopaedic applications. I initially worked on developing new surface treatments to improve osseointegration of dental implants that resulted in a patent that was licensed to a company that produces dental implants. Our work received the European Award in Basic Research in Dentistry.

Later, I expanded my research interests to applications of peptides and recombinant polymers in tissue engineering and regenerative medicine as a result of my work at the Institute for BioNanotechnology in Medicine at Northwestern University.

I arrived to the University of Minnesota in 2008 and started a research program on biological and hybrid coatings for dental and orthopaedic applications. We have established different methodologies to multi-functionalize metallic surfaces anchoring bioactive peptides or recombinamers in collaboration with local and International research groups. Indeed, our successful collaboration with Sven-Ulrik Gorr, Ph.D. at the University of Minnesota School of Dentistry has resulted in the development of GL13K-peptide coatings that have shown sustained resistance to degradation with broad antimicrobial activity and promising applications for dental implants, dental abutments, orthodontic appliances, total joint implants, intramedular pins, orthopaedic external fixators, osseointegrated prosthesis, etc.

My background and record of scholarly publications reflects a multi- and interdisciplinary approach to biomaterials and biomechanics science and engineering, including several biomolecular, in vitro cellular, and in vivo studies. 

Co-Investigators

Gorr Lab:

  • Massa Abdolhosseini, post-doctoral fellow
  • Chitta Geetha, post-doctoral fellow
  • Helmut Hirt, post-doctoral fellow
  • S. Rao Nandula, Research Associate
  • Anu Shelar, M.S. student
  • Jonathan Song, DDS student
  • Julie Sotsky, research assistant
  • In memoriam: S.G. Venkatesh, post-doctoral fellow

Aparicio Lab: 

  • Xi Chen, PhD student
  • Kyle Holmberg (now Kyle H. Vining), DDS
  • Yuping Li, post-doctoral fellow
Collaborators