1. Inoculate culture at 1 Â 10
6 bacteria/mL to fresh EMJH
medium and incubate them during 7–10 days (see Note 5).
2. Centrifuge the culture in 250 mL or 500 mL bottles at
6500 Â g for 25–30 min (see Note 6).
3. Resuspend the pellets in pre-warmed 37
C endotoxin-free
DPBS and pool them in a 50 mL plastic tube (see Note 7).
4. Centrifuge again to obtain a pellet that should be around
1–5 Â 10
12 bacteria.
5. Carefully discard the supernatant and store the pellet at À80
C
or use right away for LPS extraction.
3.2 Fast Extraction
for O Antigen Analysis
The fast extraction protocol relies on the incubation of the leptospires with proteinase K to digest proteins and release the lipopolysaccharides from the bacterial membranes. This method is much
faster than the classical hot/water extraction and requires less
bacterial material, but does not allow the study of the immunological properties of the LPS because it is not separated from the rest of
the bacterial cell wall components. Described by Murray et al. [1]
for LPS mutant analyses, this technique has been widely used and
modified. The protocol described hereafter is based on the original
protocol modified by Eshghi and collaborators [7].
1. Resuspend the small bacterial pellet in endotoxin-free DPBS
containing 0.1% SDS at the concentration of 10
9 bacteria/mL.
2. Sonicate the bacteria in ultra-sound bath for 45 s.
3. Incubate the lysate with proteinase K at a final concentration of
30 μg/mL overnight at room temperature.
4. Mix the lysate with Laemmli buffer, denature the sample at
99
C for 10 min, and perform the silver staining analysis
(described in Subheading 3.4.2) using the amount
corresponding to 10
7 bacteria per well on the gel.
3.3 Classical
Purification for
Immunological Studies
3.3.1 Hot Water/Phenol
Extraction
Like other bacterial LPS, leptospiral LPS can be purified by the
classical hot water/phenol extraction described in the literature by
Westphal et al. [6]. Nevertheless, LPS are usually extracted from the
aqueous phase, whereas the leptospiral LPS are essentially obtained
in the phenol phase because of their structural peculiarities (Fig. 2).
1. Resuspend the bacterial pellet in hot endotoxin-free water
(temperature above 68
C) (see Note 8). Add the same volume
of pre-heated 90% phenol (68
C) to the solution (see Note 9).
For 5 Â 10
12 bacterial pellet, add 20 mL of water and 20 mL of
90% phenol.
2. Perform the extraction in a 50 mL endotoxin-free plastic tube
by incubating the bacteria with the water/phenol mixture for
Purification of LPS from Leptospira
57
6 bacteria/mL to fresh EMJH
medium and incubate them during 7–10 days (see Note 5).
2. Centrifuge the culture in 250 mL or 500 mL bottles at
6500 Â g for 25–30 min (see Note 6).
3. Resuspend the pellets in pre-warmed 37
C endotoxin-free
DPBS and pool them in a 50 mL plastic tube (see Note 7).
4. Centrifuge again to obtain a pellet that should be around
1–5 Â 10
12 bacteria.
5. Carefully discard the supernatant and store the pellet at À80
C
or use right away for LPS extraction.
3.2 Fast Extraction
for O Antigen Analysis
The fast extraction protocol relies on the incubation of the leptospires with proteinase K to digest proteins and release the lipopolysaccharides from the bacterial membranes. This method is much
faster than the classical hot/water extraction and requires less
bacterial material, but does not allow the study of the immunological properties of the LPS because it is not separated from the rest of
the bacterial cell wall components. Described by Murray et al. [1]
for LPS mutant analyses, this technique has been widely used and
modified. The protocol described hereafter is based on the original
protocol modified by Eshghi and collaborators [7].
1. Resuspend the small bacterial pellet in endotoxin-free DPBS
containing 0.1% SDS at the concentration of 10
9 bacteria/mL.
2. Sonicate the bacteria in ultra-sound bath for 45 s.
3. Incubate the lysate with proteinase K at a final concentration of
30 μg/mL overnight at room temperature.
4. Mix the lysate with Laemmli buffer, denature the sample at
99
C for 10 min, and perform the silver staining analysis
(described in Subheading 3.4.2) using the amount
corresponding to 10
7 bacteria per well on the gel.
3.3 Classical
Purification for
Immunological Studies
3.3.1 Hot Water/Phenol
Extraction
Like other bacterial LPS, leptospiral LPS can be purified by the
classical hot water/phenol extraction described in the literature by
Westphal et al. [6]. Nevertheless, LPS are usually extracted from the
aqueous phase, whereas the leptospiral LPS are essentially obtained
in the phenol phase because of their structural peculiarities (Fig. 2).
1. Resuspend the bacterial pellet in hot endotoxin-free water
(temperature above 68
C) (see Note 8). Add the same volume
of pre-heated 90% phenol (68
C) to the solution (see Note 9).
For 5 Â 10
12 bacterial pellet, add 20 mL of water and 20 mL of
90% phenol.
2. Perform the extraction in a 50 mL endotoxin-free plastic tube
by incubating the bacteria with the water/phenol mixture for
Purification of LPS from Leptospira
57