The role of S-layer proteins in adherence to IPEC-1 cells
The poor water-solubility of Lactobacillus S-layer proteins, resulting from the inherent self-assembly property of bacterial S-layers in vitro, sets limitations on what methods can be used to assess the adherence of S-layer proteins to a particular target. In order to avoid potential unspecific effects associated with protein precipitation in adhesion experiments, a protein presentation system, based on purified L. amylovorus cell wall fragments as S-layer protein carriers, was developed and used to study the role of the surface-located L. amylovorus Slp:s in adhering to IPEC-1 cells (see Figure 8B for an electron micrograph of purified CWF). This method is based on the inherent tendency of S-layer proteins to recrystallize in a native manner on CWF [42,43], which have been purified in such a way to remove all of the non-covalently attached components (including the endogenous S-layer proteins), but preserving the covalently attached polymeric components like teichoic acids and polysaccharides, thus ensuring the proper self-assembly of the recombinant Slp:s. However, purified cell wall fragments are of low density and have poor contrast, necessitating specific staining if one wishes to detect the CWF on epithelial cells. For Slp-coated CWF, an indirect immunofluorescence staining procedure with Slp-specific antibodies was used, but as we failed to obtain functional antibodies against purified cell wall fragments (data not shown), the detection of uncoated control CWF was based on their prior biotinylation and staining with labeled streptavidin after the adherence assay.
Figure 8  Adherence of S-layer protein-coated cell wall fragments to IPEC-1 cells. A) The IPEC-1 cell adherence of CWF, coated or uncoated by the indicated S-layer proteins, expressed by quantitative means. The mean number of adherent CWF was quantitated from 20 randomly selected fields of 3.5 x 104 μm2 and the results are presented as means and standard deviations from one representative experiment out of three; letters above the bars refer to Figures C-H below. B) An electron micrograph of purified CWF of L. amylovorus DSM 16698. Scale bar, 0.5 μm. C-H) Examples of the adherence of Slp-coated and uncoated L. amylovorus CWF to IPEC-1 cells as detected by fluorescence. The figures show the adherence of uncoated L. amylovorus DSM 16698 CWF (C) and the adherence of the following Slp/CWF complexes: DSM 16698 CWF/SlpA (D), DSM 16698 CWF/SlpB (E), DSM 16698 CWF/SlpC (F), DSM 20531T CWF/SlpA (G) and GRL 1117 CWF/SlpA (H). The rightmost figures display the corresponding fields viewed with phase contrast optics. The inset in (D) shows a magnified image of a cell wall fragment. Arrowheads in (G) indicate precipitated Slp. Scale bars, 10 μm.   Figure 8A shows the adherence of CWF, coated or uncoated by L. amylovorus cell surface-located Slp:s, to IPEC-1 cells. In Figures 8C-H, micrographs illustrating the results of the binding assay in (A) are shown. The adherence of all uncoated CWF was negligible, as exemplified by the adherence of the CWF of the strain DSM 16698 in Figure 8C. The major Slp:s of the L. amylovorus strains DSM 16698 (Figure 8D), GRL 1112 and GRL 1115 adhered poorly to IPEC-1 cells, although the intact cells of these strains were adhesive (Figure 2). The minor S-layer like protein SlpB of DSM 16698 exhibited some adhesiveness (Figure 8E), when compared to SlpA (Figure 8D) or SlpC (Figure 8 F) of the same strain. Surprisingly, the S-layer protein SlpA of the weakly adhering strain GRL 1117 (Figure 8H) and, to a lesser extent, the Slp:s of some of the other weakly adhering strains, e.g. DSM 20531T (Figure 8G) and GRL 1118 also displayed affinity for IPEC-1 cells.As detailed in Methods, special care was taken to minimize the formation of S-layer protein precipitates during the coating procedure of CWF. However, the presence of small Slp aggregates, as indicated by the small, dot-like, immunoreactive material among the coated cell walls, could not be completely avoided (see Figure 8G as an example). However, the quantification of the result by microscopic counting made it possible to ignore this undesirable signal, probably originating from unspecific and/or irrelevant binding.