Transcript araC
Teaching self-destructing Salmonella new tricks to fight cancer Wei Kong and Roy Curtiss III Arizona State University, Tempe, Arizona, USA Cancer is one of the leading causes of death in the world An estimated 289,000 Americans are expected to die from lung and other three most common cancers (colon, breast and pancreatic) in 2014 http://www.lung.org Tumor hypoxia- a major hazard of cancer therapy http://www.mc.pref.osaka.jp Development of novel therapies targeting hypoxic cancer cells Bacteria have been investigated as anticancer agents: • • • • • • • • Salmonella Escherichia Clostridium Bifidobacterium Caulobacter Listeria Proteus Streptococcus, etc. Live, attenuated or genetically-engineered Vector Bacteria Immunotherapeutic agents Toxin Spore Salmonella as anti-cancer therapy Motile S. Typhimurium exists as facultative anaerobes, allowing them to survive in both oxygenated and hypoxic conditions. S. Typhimurium strains have been shown to preferentially accumulate in tumours. Importantly, S. Typhimurium was shown not only to colonize large established tumors but also exhibit the property to invade and affect metastases. Self-destructing Salmonella - Programmed regulated lysis system For antigen delivery: For DNA vaccine delivery: Programmed regulated lysis system for antigen delivery In vivo vitrowithout with arabinose arabinose RASV Chromosome Host cell araC PBAD murA araC PBAD C2 araC PBAD lacI Plasmid araC PBAD murA asd P22PR Ptrc Ag The gut epithelium SCV Release Ag by lysis Endosome escaping RASV multiply and antigen synthesis nucleus DAP-less and Muramic acid-less Death in Host Strain with Regulated Lysis System Vector The genotype of 11283: 11283 LB Agar LB Agar + 0.2% Arabinose + 50g/ml DAP 11283 (pYA3681) asdA::araC PBAD c2 PmurA::araC PBAD murA (wza-wcaM) relA::araC PBAD lacI TT pmi Prfc::TT araC PBAD rfc pagP::Ptrc lpxE pYA3681 araC PBAD regulated murA+ asdA+ LB Agar LB Agar + 0.2% Arabinose Recombinant host-vector strain displaying arabinose-dependent growth and regulated cell lysis in the absence of arabinose Self-destructing Salmonella for DNA vaccine delivery- Vector pYA4272 (pYA3650-EGFP) DTS (II) pYA4685 (pYA4545-EGFP) 5’-ggggactttcc ggggactttcc tccccacgcg ggggactttcc gccacgggc ggggactttcc ggggactttcc INT-407 Vero The synthesis of EGFP from pYA4545 harboring EGFP gene in INT-407 and Vero cell line Self-destructing Salmonella for DNA vaccine delivery- Strain Genotype of the ideal RASV strain: asdA::TT araC PBAD c2 PmurA::TT araC PBAD murA relA (wza-wcaM) pmi pagP::Ptrc lpxE fljB fliC endA sifA PhilA::Ptrc lacO hilA tlpA sseL RASV harboring DNA vaccine vector pYA4545 PhilA :: Ptrc lacOhilA (Hyper-invasive) Host Cell pYA4545 ∆sifA Transcription factors NLS SCV ∆tlpA ∆sseL Apoptosis/ pyroptosis ∆asdA ∆PmurA Salmonella cell lysis Importin family members RA N nucleus Programmed Salmonella regulated lysis system delivering antigens or DNA vaccine induced mucosal and systemic antibody and T-cell responses protective immunity against various diseases Teaching self-destructing Salmonella new tricks to fight cancer Exhibit diminished toxicity of lipid A to lessen inflammatory responses. Rapid release of vacuolar Salmonella from the endosome to increase the efficacy of delivery and expression of a DNA vaccine. Allow in vivo maximized Salmonella localization in tumors. Modulate Salmonella swimming behavior for enhanced tumor targeting and tumor homing A. Bacterium swimming test on soft agar plate 4.3 cm 3.0 cm 11371 (Ptar::Ptrc lacOtar) 3761 Chemo attractants: 100 μM Aspartate B. Graph of swimming test result * • • • ΔPtar::PtrcΔlacO tar ΔPtsr::Ptrc ΔlacOtsr Δtrg *The data of graph were collected from 4 independent experiments, ρ < 0.05 Swimming Distance (cm) Genetically modification of chemoreceptor genes 3761 11371 The ratio of Salmonella colonization in tumor vs. spleen Reduced normal tissue fitness and enhance tumor fitness by genetically modifying RASV strains Deletion of purA gene: • eliminate the production of adenosine monophosphate (AMP) 500000 450000 400000 350000 300000 Day 1 250000 Day 3 200000 Day 5 150000 Day 7 100000 50000 Deletion of htrA gene: • Eliminate a bifunctional stress protein HtrA that is required by many bacterial pathogens to successfully cause infection 0 tumor specific fitness of indicated strains was determined in BALB/c CT-26 subcutaneous tumor bearing mice post IT injection Anti-tumor efficacy assay of anti-cancer Salmonella strains by intratumoral injection using whole body bioluminescence imaging breast cancer subcutaneous mouse model Day 0 Day 4 Day 8 11517 BSG Χ11517 (pYA4545) χ11517 (pYA4545 + FasL) PmurA::TT araC PBAD murA asdA::TT araC PBAD c2 (araC PBAD)::P22 PR araBAD (wza-wcaM) pmi relA::araC PBAD lacI TT pagP::Plpp lpxE endA PhilA::PtrclacOhilA Ptar::Ptrc lacO tar Ptsr::Ptrc lacO tsr trg Acknowledgements Project of programmed regulated lysis system: Dr. Roy Curtiss III Dr. Shamaila Ashraf Dr. Keith Ameiss Dr. Xiangmin Zhang Dr. Maria Dolores Juarez-Rodriguez Project of RASV anti-cancer therapeutic vaccine: Dr. Roy Curtiss III Mr. Matthew Brovold Mr. Jeffrey Tully Mr. Lee Benson Ms. Chelsea Warren National Institutes of Health (NIH/NIAID) National Institutes of Health (NIH/NCI) U. S. Department of Agriculture (USDA)