title: Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine.
abstract: Tuberculosis (TB) is a disease caused byMycobacterium tuberculosisorMycobacterium bovisand still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in bothEscherichia coliandLactococcus lactisand displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenicMycobacterium smegmatiswas engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered intoE. coli-Mycobacteriumshuttle plasmids and expressed intransImmobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens.
  
  
  
  E-MBB, and anM. smegmatisvector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated andMycobacterium bovisBCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens.IMPORTANCETuberculosis (TB) is a disease caused byMycobacterium tuberculosisorMycobacterium bovisand still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.|AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Lee
authors.affiliation: AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Parlane
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.|PolyBatics, Palmerston North, New Zealand.
authors.forename: Bernd H A
authors.initials: BHA
authors.lastname: Rehm
authors.affiliation: AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand.
authors.forename: Bryce M
authors.initials: BM
authors.lastname: Buddle
authors.affiliation: AgResearch, Hopkirk Research Institute, Palmerston North, New Zealand axel.heiser@agresearch.co.nz.
authors.forename: Axel
authors.identifier: http://orcid.org/0000-0003-0389-6132
authors.initials: A
authors.lastname: Heiser
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism; D000217:Acyltransferases / Q000276:immunology / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001704:Biopolymers / Q000378:metabolism*; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D006885:Hydroxybutyrates / Q000378:metabolism; D007111:Immunity, Cellular; D051379:Mice; D008810:Mice, Inbred C57BL; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020102:Mycobacterium smegmatis; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011091:Polyesters / Q000008:administration & dosage / Q000378:metabolism*; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins; D014376:Tuberculosis / Q000517:prevention & control; D032581:Tuberculosis Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014611:Vaccination; D000094122:Polyhydroxybutyrates
chemical_list: D000102:Acetyl-CoA C-Acyltransferase; D000217:Acyltransferases; D000429:Alcohol Oxidoreductases; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001704:Biopolymers; D006885:Hydroxybutyrates; D011091:Polyesters; D054813:Polyhydroxyalkanoates; D011993:Recombinant Fusion Proteins; D032581:Tuberculosis Vaccines; C093276:ESAT-6 protein, Mycobacterium tuberculosis; C072681:Mycobacterium tuberculosis antigens; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C023744:acetoacetyl-CoA reductase; C112580:antigen 85A, Mycobacterium tuberculosis; C077225:poly(3-hydroxyalkanoic acid) synthase
keywords: Mycobacterium smegmatis; polyhydroxyalkanoate synthesis; tuberculosis; tuberculosis vaccines