Display of heterologous proteins on the surface ofmicroorganisms, enabled by means of recombinant DNA technology,has become an increasingly used strategy in variousapplications in microbiology, biotechnology and vaccinology.This thesis presents two bacterial systems, designed forsurface display of chimeric proteins on the food-gradestaphylococcal speciesStaphylococcus xylosusandStaphylococcus carnosus, respectively.
To achieve expression and translocation of gene products,theS. xylosussystem uses the promoter and signal peptideofS.aureusprotein A (SpA), while theS. carnosussystem employes the promoter, signal peptideand a propeptide from aS. hyicuslipase gene construct. It was investigatedwhether the 207 amino acid propeptide region could be deletedor replaced, but it was found that, at least for inefficientlysecreted proteins, the presence of the propeptide wasbeneficial. Both systems utilize the anchoring regions of SpAto achieve cell surface anchoring of expressed heterologousproteins. Introduced into both expression systems is an albuminbinding protein (ABP) from streptococcal protein G, which isfound closest to the cell wall in surface anchored chimericproteins. This ABP region has been found to function as aspacer region to increase surface accessiblity, and has beenused for ABP-mediated affinity purification of chimeric surfaceproteins and as a reporter peptide in the monitoring of surfacedisplay of various target proteins.
For the purpose of investigating whether the staphylococcalsystems were suitable as vehicles for delivery of subunitvaccines, antigenic determinants of various origin, bacterial,viral and protozoan, have been expressed as surface displayedon the staphylococci. A malarial peptide M3, from thePlasmodium falciparumblood-stage antigen Pf155/RESA,was found to be successfully surface expressed onS. carnosuscells, as demonstrated by immunofluorescencestaining and immunogold electron microscopy. This was furtherassessed by an ABP-based colorimetric assay and byfluorescence-activated cell-sorting (FACS). In a comparativestudy, investigating the surface expression of differentfragments derived from the G glycoprotein of human respiratorysyncytial virus (RSV), it could be concluded that theS. carnosussystem was better than theS. xylosussystem, in its ability to translocateinefficiently secreted proteins and also in terms of the numberof surface displayed chimeric proteins. This was subsequentlycorroborated by a quantitative FACS study, taking advantage ofpurpose-designed calibration beads, indicating that theS. carnosussystem expresses approximately 104 chimericsurface proteins per bacterium, while theS. xylosuscells carry about 3 x 103. Furthermore, theVibrio choleraecholera toxin B subunit (CTB) was foundto be expressed in a functional form on both types of bacteria.As will be discussed, vaccination studies based on the systemsdescribed, have subsequently led to the eliciting of protectiveimmune responses in mice.
A functional scFv antibody fragment, reactive to human IgE,was successfully expressed on bothS. xylosusandS. carnosus, and the generated bacteria were capable ofbinding IgE. Such bacteria could potentially be evaluated aswhole-cell diagnostic devices. Furthermore, staphylococcalexpression of phage-selected "affibody" fragments, based on anengineeredS. aureusprotein A domain, have generated staphylococciwith specific binding ability to human IgE and IgA. Thecombination of phage and bacterial display would thus make itpossible to create bacteria with desired bindingspecificities.
The possibility of expressing metal-binding polyhistidylpeptides on the staphylococci was investigated and it wasindeed demonstrated that the generated recombinantstaphylococci had gained improved metal-binding ability. Theobtained results for surface display onS. xylosusandS. carnosus,suggest that the descibed strategies havethe potential to be applied in vaccine development, design ofnovel diagnostic devices and as tools for bioadsortion ofvarious metals of environmental concern.
Key words:affibody, albumin binding protein, bacterialsurfacedisplay, bioremediation, CTB, diagnostic, FACS, ,IgA-binding, IgE-binding, live bacterial vaccine deliveryvehicle, malaria, metal binding, quantification, RSV, scFvantibody fragment, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus,whole-cell affinity adsorbent.
© Patrik Samuelson, 1999
Stockholm: Bioteknologi , 1999. , 61 p.