Article Figures & Data
Figures
- Fig. 1.
Predicted amino acid sequence of the mature CbsA protein of L. crispatus JCM 5810. Below the CbsA sequence are shown conserved regions in eight lactobacillus S-layer sequences identified by the MEME program for protein motif detection. The other S-layer proteins were SlpA (Genbank accession number X89375 ) (5), SlpH1 (X91199 ) (9), CbsB (AF079365 ), M247 (AJ007839 ), SlpB (X89376 ; 6), SlpnA (AF253043 ) (Martinez and Pouwels, unpublished), and SlpnB (AF253044 ) (Martinez and Pouwels, unpublished). The amino acid substitutions between CbsA and Slpa or SlpnB, as well as the deletion sites, are indicated in boldface type above the CbsA sequence. The amino acid positions where the switches in the hybrid CbsA-SlpA and CbsA-SlpnB molecules were constructed are also shown.
- Fig. 2.
SDS-PAGE analysis of selected S-layer proteins. Lanes: a, CbsA extracted from L. crispatus JCM 5810 cells; b, His-CbsA from recombinant E. coli; c, the hybrid S-layer protein CbsA1–287/SlpA290–413; d, His-CbsA D130N; e, His-CbsA Δ91–96; f, His-CbsA1–287; g, His-CbsA42–287; h, His-CbsB. The migration distances of molecular mass marker peptides are indicated on the left in kilodaltons.
- Fig. 3.
Transmission electron microscopy of the CbsA preparations. The S-layer proteins are His-CbsA from recombinantE. coli (A), the polypeptide His-CbsA1–250 (B), the polypeptide His-CbsA1–287 (C), and the CbsA protein with the substitution KSDV257TANN (D). Bar, 100 nm.
- Fig. 4.
Binding of His-tagged S-layer proteins to extracellular matrix proteins immobilized on plastic was determined by ELISA technology with monoclonal anti-His-tag antibodies and secondary antibodies. (A) Binding in increasing concentrations of the His-CbsA1–287 polypeptide to type I collagen (●) and BSA (▾). (B) Binding of the polypeptide (1.2 mM) to wells coated with increasing concentrations of type I collagen (●) or BSA (▾). (C) Binding of S-layer proteins to type IV and I collagens, laminin, fibronectin, and BSA immobilized on plastic. A405nm, absorbance at 405 nm.
- Fig. 5.
Adherence of FITC-tagged cells of L. crispatus JCM 5810 to frozen section of chicken colon detected by fluorescence microscopy. (A) FITC-tagged bacteria adherent on the colon tissue. (B) The same microscopic field for staining with the TRITC-labeled monoclonal antibody against chicken type III collagen. (C) The same field by light microscopy. (D) Adhesion of FITC-labeled JCM 5810 cells after removal of the S-layer by guanidine hydrochloride extraction. (E) The same microscopic field for anti-type III collagen staining. The bacteria were tested at 108cells/ml. Arrows indicate connective-tissue regions rich in type III collagen, arrowheads indicate basolateral regions of epithelial cells, and e indicates epithelial cells. Bar, 100 μm. Note the adherence of JCM 5810 cells to type III collagen-rich areas and to basolateral aspects of the epithelial cells as well as the poor adhesion by JCM 5810 cells lacking the S-layer.
- Fig. 6.
Aggregation of L. crispatuscells in the presence of S-layer proteins. Bacteria (2 × 109) were incubated for 1 h with the His-CbsA proteins (10 μg) in PBS, stained with methylene blue, and then examined by light microscopy. (A) JCM 5810 cells without added His-CbsA protein. (B to G) Cells in the presence of His-CbsA (B), His-CbsA1–287 (C), His-CbsA288–410 (D), His-CbsA1–250 (E), His-CbsA1–212 (F), and His-CbsA42–287 (G). Bar, 10 μm.
Tables
- Table 1.
Binding of 125I-labeled type I and IV collagen to the S-layer proteins and formation of a crystalline layer by the proteins
Test protein Binding (%)aof Formation of crystalline layerb 125I-collagen type I 125I-collagen type IV Mean Range Mean Range Native or His-tag fusion S-layer proteinc CbsA 120 81–145 122 66–162 + His-CbsA 100 100 + SlpA <1 0–1 <1 0–1 + His-SlpA <1 0–1 <1 0–1 + His-CbsB <1 0–1 <1 0–1 + His-SlpnB 5 4–6 2 0–4 + Hybrid His-tag fusion S-layer protein His-CbsA1–212/SlpA208–413 <1 0–1 1 0–2 − His-SlpA1–207/CbsA213–410 <1 0–1 <1 0–1 − His-CbsA1–287/SlpA290–413 61 22–111 72 40–92 + His-SlpA1–289/CbsA288–410 <1 0–1 <1 0–1 + His-CbsA1–28/SlpnB20–409 <1 0–1 <1 0–1 + His-SlpnB1–19/CbsA29–410 105 87–153 97 74–117 + His-CbsA1–81/SlpnB73–409 <1 0 <1 0–1 − His-SlpnB1–72/CbsA82–410 78 27–131 61 44–86 + His-CbsA1–194/SlpnB187–409 <1 0–1 <1 0–1 + His-SlpnB1–186/CbsA195–410 <1 0–1 <1 0–1 + His-CbsA1–212/SlpnB205–409 <1 0–1 <1 0–1 + His-SlpnB1–204/CbsA213–410 <1 0–1 <1 0–1 + His-CbsA1–250/SlpnB250–409 <1 0–1 <1 0–1 − His-SlpnB1–249/CbsA251–410 <1 0–1 <1 0–1 − His-CbsA1–287/SlpnB287–409 54 39–96 73 47–98 + His-SlpnB1–286/CbsA288–410 2 0–3 4 4–5 + Polypeptide His-CbsA1–212 2 0–2 <1 0–1 − His-CbsA1–250 1 0–2 1 1–2 − His-CbsA1–287 77 65–83 73 27–77 + His-CbsA42–287 4 2–5 <1 0–1 − His-CbsA288–410 2 2–3 <1 0–1 − Mutated CbsA protein His-CbsA Δ22–26 2 0–2 8 5–15 + His-CbsA Δ91–96 22 20–24 27 19–33 + His-CbsA NNN14INL 88 50–131 74 32–100 + His-CbsA F19S 83 53–109 80 52–109 + His-CbsA D130N 56 40–68 64 39–78 + His-CbsA N226A 33 24–44 34 22–40 + His-CbsA KSDV257TANN <1 0–1 <1 0–1 + His-CbsA K257T 91 80–102 92 83–99 + His-CbsA S258A 10 6–13 9 4–13 + His-CbsA D259N 77 63–87 87 80–98 + His-CbsA V260N <1 0–1 1 0–1 + His-CbsA TA264SK 52 44–64 49 36–68 + His-CbsA P268A 51 34–66 44 36–54 + Control BSA <1 0 ↵a Results are mean values from at least six independent assays; the range gives the lowest and the highest value obtained in the assays. The binding value shown by His-CbsA was assigned as 100% in each individual test.
↵b Formation of the S-layer was determined by transmission electron microscopy of the purified proteins negatively stained with phosphotungstate acid.
↵c “Native” denotes to the S-layer extracted from Lactobacillus cells. His-tag fusion proteins were isolated from E. coli cells.
