bridge between the bacteria and the host. Several leptospiral adhesins have been identified and characterized as ECM-binding molecules [7–20]. The adhesion process is considered as a critical step in
promoting host infections. Therefore, it is a key event that deserves
to be investigated in leptospiral pathogenesis.
The process of cell adhesion is complex and includes receptorligand binding, changes in intracellular signaling pathways, and
modulation of cytoskeletal assembly [21]. Most of the methods
used measure the ability of a bacterial cell to interact with a specific
adhesion molecule or evaluate this interaction in the presence of
inhibitors [2]. The first interaction assay performed with leptospires used radiolabeled leptospiral suspensions and host cell monolayers [22]. Despite the fact that assays involving radioactivelabeled bacteria have been used successfully, hazards involved in
these manipulations have decreased their use. Moreover, assays that
involve host cells are considered laborious and expensive. The
development of recombinant DNA technology has made an
impressive impact in the number of recombinant proteins that can
be produced, and consequently, new methods to evaluate the adhesion process have been established. Here, we describe a method
based on a simple, fast, and sensitive colorimetric assay to investigate the initial interaction between recombinant leptospiral proteins and host components.
2 Materials
2.1 Extracellular
Matrix Components
All host macromolecules and the control proteins can be purchased
from Sigma-Aldrich, and solutions prepared and stored as
described by the manufacturer. Dilute all macromolecules to a
working concentration of 1 μg/well in PBS.
1. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4. Autoclave and
store at room temperature.
2. Tris buffer: 0.2 M Tris–HCl, pH 8.8.
3. Laminin: The solution of laminin from Engelbreth-HolmSwarm mouse sarcoma basement membrane is kept at
À20
C (see Note 1).
4. Collagen type 1: The solution of collagen type 1 of rat tail
tendon is stored at 2–8
C (see Note 2).
5. Collagen type 4: Collagen type 4 from Engelbreth-HolmSwarm mouse sarcoma basement membrane is reconstituted
in PBS containing 0.25% acetic acid at a concentration of
0.5–2.0 mg/mL, which is kept for several hours at 4
C with
occasional shaking (see Note 3).
172
Aline F. Teixeira and Ana L. T. O. Nascimento
promoting host infections. Therefore, it is a key event that deserves
to be investigated in leptospiral pathogenesis.
The process of cell adhesion is complex and includes receptorligand binding, changes in intracellular signaling pathways, and
modulation of cytoskeletal assembly [21]. Most of the methods
used measure the ability of a bacterial cell to interact with a specific
adhesion molecule or evaluate this interaction in the presence of
inhibitors [2]. The first interaction assay performed with leptospires used radiolabeled leptospiral suspensions and host cell monolayers [22]. Despite the fact that assays involving radioactivelabeled bacteria have been used successfully, hazards involved in
these manipulations have decreased their use. Moreover, assays that
involve host cells are considered laborious and expensive. The
development of recombinant DNA technology has made an
impressive impact in the number of recombinant proteins that can
be produced, and consequently, new methods to evaluate the adhesion process have been established. Here, we describe a method
based on a simple, fast, and sensitive colorimetric assay to investigate the initial interaction between recombinant leptospiral proteins and host components.
2 Materials
2.1 Extracellular
Matrix Components
All host macromolecules and the control proteins can be purchased
from Sigma-Aldrich, and solutions prepared and stored as
described by the manufacturer. Dilute all macromolecules to a
working concentration of 1 μg/well in PBS.
1. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4. Autoclave and
store at room temperature.
2. Tris buffer: 0.2 M Tris–HCl, pH 8.8.
3. Laminin: The solution of laminin from Engelbreth-HolmSwarm mouse sarcoma basement membrane is kept at
À20
C (see Note 1).
4. Collagen type 1: The solution of collagen type 1 of rat tail
tendon is stored at 2–8
C (see Note 2).
5. Collagen type 4: Collagen type 4 from Engelbreth-HolmSwarm mouse sarcoma basement membrane is reconstituted
in PBS containing 0.25% acetic acid at a concentration of
0.5–2.0 mg/mL, which is kept for several hours at 4
C with
occasional shaking (see Note 3).
172
Aline F. Teixeira and Ana L. T. O. Nascimento