Food Safety Interventions: Adhesin-Specific Nanoparticles for Removal of Foodborne Pathogens

Transcription

1 Food Safety Interventions: Adhesin-Specific Nanoparticles for Removal of Foodborne Pathogens Jeremy Tzeng Biological Sciences Clemson University IFT International Nanotechnology Conference, August 1, 2007

2 Research Interests Development of Preventive/Therapeutic Approaches that Minimize the Emergence of Drug Resistant Microorganisms Problems associated with the use of antibiotics Emerging multiple antibiotic resistant bacteria due to mutations/recombination Undesirable side effects Increased costs for the development of new antibiotics

3 Research Approaches Development of TiO 2 Self-Disinfecting Surface Development of a Narrow-Spectrum Bacteriocin for Anti-Acne Acne Applications Evaluation of Nutraceuticals for their Anti-bacterial and Anti-tumor tumor Properties Evaluation of Nutraceuticals for Anti-bacterial Efflux and Anti-tumor tumor p-glycoprotein p Applications Development of Bacterial Adhesin-Specific Nanoparticles

4 Bacterial Adhesins Copyright: Sigma-Aldrich

5 Host-Pathogen Interactions Earliest events in bacterial infections Required for extracellular colonization or internalization Colonization mediated by bacterial adhesins Adhesins recognize and bind to specific receptor moieties of host cells Receptor binding activates new genes important in the pathogenic process Blocking of interactions -> > effective prevention

6 Bacterial Adhesins Hair-like appendages called pili or fimbriae that extend out from the bacterial surface Or, directly associated with bacterial surface nonpilus adhesins. e.g. intimin of E. coli O157:H7

7 Examples of Bacterial Adhesins and their Receptors Sharon, N., and I. Ofek, Glycoconj. J. 17, (2000) Organism Adhesin Receptor E. coli Type 1 pili (FimH) D-mannose Intimin Tir Campylobacter jejuni CadF Fibronectin

8 E. coli ORN178 (FimH +, GFP)

9 E. coli ORN208 (FimH -, GFP)

10 Objectives Development of carbohydrate biofunctionalized nanoparticles (bio-np) that have affinity for pathogen adhesins Evaluation of these synthesized bio-np (mimicking host cell surface receptors) for their binding to pathogens Development of alternative non-antibiotic treatment for bacterial infections

11 Approaches Multivalent nanoparticles specific to adhesins of targeted pathogens Aggregation of pathogens mediated by nanoparticles prevents binding to host cells

12 Nanoparticle-Bacterial Bindings Nanoparticles Bacterial cell High NP Concentration: Bacterial Isolation Intermediate NP Concentration: Bacterial Agglutination Low NP Concentration: Bacterial Tagging

13 Nanoparticle Chemical Structure: Mannose Functionalization Mannose as Biofunctional group Polystyrene core Polyethylene glycol tether

14 Nanoparticle Design Strategy Functionalized PEG side chains extending from hydrophobic polymer backbone chain. Diagram illustrates the self assembly into the nanoparticles followed by photochemical curing.

15 TEM/SEM Images of Nanoparticles 200 nm Journal of Biomedical Nanotechnology Vol.1, 61 67, 2005

16 E. coli -NP Interaction a b TEM images showing the agglutination of E. coli ORN178 mediated by D- mannose-tethered nanoparticles (a,b) Lower magnification and (c,d) higher magnification (e) E. coli ORN178 only (similarly with bare nanoparticles) (f) E. coli ORN208 with the same D-mannose-tethered polymeric nanoparticles. c e d f Journal of Nanoscience and Nanotechnology, Vol.5, , 2005

17 Aggregation Mediated by NPs ORN178 with Man-NPs ORN178 with Man-NPs ORN208 with Man-NPs ORN178 with Gal-NPs Journal of Biomedical Nanotechnology, Vol.1, 1 6, 2005

18 Nanoparticles Mediated CFU Reduction of E. coli % CFU reduction E.coli ORN178 with NP µg NP/million bacterial cells

19 Journal of Biomedical Nanotechnology, Vol.2, 1 10, 2006 Acute Nanoparticle Exposure Sensitivity Studies A. Dorsal C. Conjunctival sac E. Aerosolization and inhalation chamber B. Dermal D. Ocular site after 48 hr F. Histological slide of lung tissue

20 Advantages Low cost Polystyrene core Simple carbohydrate biofunctional groups Easy transportation and storage condition Neither antibiotic nor antibody approach Less prone to the effects of mutations vs. an antibiotic approach Mutation in adhesin lost of ability to attach to host cells through the same mechanism

21 Campylobacter jejuni Nanoparticles jejuni-specific A B ASM 2007

22 Single-Walled Nanotube

23 D-galactose Nanotubes

24 Binding of SWNT to Targeted E. coli O157:H7 Strain C7927

25 Cell Aggregations Mediated by Galactose Biofunctionalized SWNT ASM 105 th National Meeting, 2005

26 Applications Purging of enteropathogens from intestinal tracks of commercial poults to allow for establishment of beneficial micro flora Purging of enteropathogens from intestinal tracks of commercial poultries prior to their process Displacing technology Non antibiotic approach for the prevention and treatment of infectious diseases Adhesin-specific specific drug delivery Diagnostic applications

27 Microarray for the Screening of Receptor Molecules

28 Adhesin in Biosensor Applications Current target capturing molecules used in biosensor applications: DNA or RNA sequences Proteins or antibodies Highly specific High cost Short shelf life Requires special instrument Availabilities of specific antibodies Antibodies showing cross reactions

29 Adhesin in Biosensor Applications Fabrication of microchip surface with receptor molecules specific for targeted microorganisms. Binding of microorganisms to microchip surface could cause changes in conductivities, resonance frequencies, or weight real-time biosensor for food safety applications

30 Intellectual Property US patent application entitled Adhesin- Specific Nanoparticles and Process for Using Same filed in 2003.

31 Funding: USDA SC-LIFE/HHMI Acknowledgements Collaborators: Clemson University R. Latour, Dept. of Bioengineering F.J. Stutzenberger, Dept. of Biological Sciences Y.-P. Sun, Dept. of Chemistry George Luo, Dept. of Biological Sciences North Carolina State University Jesse Grimes, Dept. of Poultry Science University of Tennessee Jun Lin, Dept. of Animal Science