Novel surface display system for proteins on non-genetically modified gram-positive bacteria

Abstract

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

FIG. 1.

Schematic diagram of the organization of the general display vector pPA3. Abbreviations: P_NisA, inducible nisA promoter; usp45_ss, usp45 signal sequence; c-myc, region encoding an epitope for immunodetection; MCS, multiple cloning site. Cross-hatched boxes indicate the LysM repeat sequences.

FIG. 2.

Fluorescence microscopic (A and B), scanning electron microscopic (C and D), and transmission electron microscopic (E and F) images of L. lactis cells and L. lactis GEM particles. (A, C, and E) Untreated cells; (B, D, and F) GEM particles. Typical examples are shown. The samples in panels A and B contained bound PA3. The bar in panel D represents 1 μm. (G) Coomassie brilliant blue-stained SDS-PAA gel of total protein extracts of 5 × 10⁸ L. lactis cells (lane 1) and an equal number of L. lactis GEM particles (lane 2). The sizes of the marker proteins (lane M) (in kDa) are indicated on the left.

FIG. 3.

Colorimetric assay for detection of surface-displayed PA6 concatemers on lactococcal GEM particles. Different amounts of PA6 concatemers were added to 2.5 × 10⁹ GEM particles. After binding, particles were collected by centrifugation and washed twice with PBS buffer. Surface-displayed PA6 concatemers were detected with horseradish peroxidase-conjugated anti-c-myc antibody (Roche). GEM particles were washed three times with PBS and developed with 1 ml of the horseradish peroxidase substrate 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid (ABTS) (Fluka)-H₂O₂. Absorbance at 405 nm was determined after a suitable time. ▪, PA6; ▴, 2PA6; •, 3PA6; ▾, 4PA6; ⧫, PA3.

FIG. 4.

Effect of different acid pretreatments on the binding capacity of L. lactis particles. α-PA was added to an equal number of GEM particles. Bound α-PA was determined with the chromogenic substrate amylose azure. Bar A, no treatment; bar B, 10% TCA; bar C, 0.2% TCA; bar D, 5.6 M HAc; bar E, 0.01 M HCl; bar F, 0.72 M lactic acid; bar G, 0.56 M formic acid. The means for three determinations (<15% difference) are shown.

FIG. 5.

Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase activities on GEM particles carrying α-PA and β-PA at different ratios. Sample A, incubation with 1 volume of cell-free culture supernatant containing β-PA; sample B, incubation with 1 volume of cell-free culture supernatant containing α-PA; sample C, incubation with 1 volume of cell-free culture supernatant containing α-PA and 1/3 volume of β-PA; sample D, incubation with 1 volume of cell-free culture supernatant containing α-PA and 2/3 volume of β-PA; sample E, incubation with 1 volume of cell-free culture supernatant containing α-PA and 1 volume of β-PA. The mean value for three determinations (<15% difference) was normalized to the activities for samples A and B, which were defined as 100%. Solid bars, nitrocefin as the substrate (specific for β-lactamase activity); gray bars, amylose azure as the substrate (specific for α-amylase activity).

FIG. 6.

IgG-specific serum antibody response to the CSP[2xB] epitopes. Groups of 10 mice were immunized nasally or subcutaneously with GEM particles (CSP[2xB]-PA). The sera were obtained as described in Materials and Methods.