Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus Species

Abstract

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.

FIG 1

SDS-PAGE (A) and Western blotting (B) of lysates of L. lactis cells expressing DARPins in fusion with the Usp45 secretion signal and the LysM-containing cA domain, stained with Coomassie brilliant blue. I_07, DARPin I_07-cA fusion; I_19, DARPin I_19-cA fusion; Cont., control containing empty plasmid pNZ8148. DARPin-cA fusion proteins are indicated with arrows.

FIG 2

Flow cytometry (A), whole-cell ELISA (B), and fluorescence microscopy (C) results for L. lactis cells expressing DARPins I_07 and I_19, detected with FITC-conjugated human IgG. Cont., control containing empty plasmid pNZ8148. MFI, mean fluorescence intensity; A₄₅₀, absorbance at 450 nm. Error bars denote standard deviations. *, significant difference (t test; P < 0.05) in comparison to control. (C) (Left) Fluorescence images. (Right) Bright-field images. Bars = 20 μm.

FIG 3

Flow cytometric (A) and whole-cell ELISA (B) analyses of Lactobacillus sp. cells after incubation with DARPin I_07-cA fusion-containing growth medium and detection with FITC-conjugated human IgG (gray bars). MFI and A₄₅₀ values were normalized to those for the control samples (white bars). (C) Quantification of LTA on the surfaces of Lactobacillus spp. by whole-cell ELISA using an LTA-specific monoclonal antibody. ACID, Lb. acidophilus ATCC 4356; BULG, Lb. delbrueckii subsp. bulgaricus ATCC 1184; CASE, Lb. casei ATCC 393; GASA, Lb. gasseri ATCC 33323; GASK, Lb. gasseri K7; PARA, Lb. paracasei ATCC 25302; PLAN, Lb. plantarum ATCC 8014; REUT, Lb. reuteri ATCC 55730; RHAM, Lb. rhamnosus ATCC 53103; SALI, Lb. salivarius ATCC 11741. *, significant increase in IgG binding (t test; P < 0.05) in comparison to control.

FIG 4

Flow cytometric analysis of LysM-mediated nonrecombinant surface display on L. lactis after treatment of bacteria with different concentrations of the antibiotics erythromycin (A) and chloramphenicol (B). Binding of the DARPin I_07-cA fusion to the bacterial surface was detected with FITC-conjugated human IgG (gray bars). MFI values were normalized to those for the control samples (white bars), in which bacteria were incubated with immunoglobulins without prior binding of the DARPin I_07-cA fusion to the bacterial surface. Error bars denote standard deviations. *, significant difference (t test; P < 0.05) between the pair of samples indicated.