Chapter 6
Purification of LPS from Leptospira
Delphine Bonhomme and Catherine Werts
Abstract
Leptospira species are one of the few spirochetes to possess a lipopolysaccharide (LPS) embedded in their
outer membrane. Two protocols are currently available to extract and/or purify the leptospiral lipopolysaccharides: the rapid proteinase K method and the classical hot water/phenol extraction. The first method
allows to get a quick overview of the LPS O antigen structure, whereas the second method is fitted to study
the immunological properties of the leptospiral LPS. These two methods will be detailed in this chapter.
Methodologies to assess the quality of the purification, such as the modified silver staining coloration, will
also be reviewed. Both advantages and limitations of the different analyses will be described.
Key words Leptospira ssp., Lipopolysaccharide/endotoxin purification, Proteinase K, Hot water/
phenol extraction, Silver staining
1 Introduction
Unlike most other spirochetes, Leptospira species possess surfaceexposed lipopolysaccharides (LPS). These LPS, also referred to as
“endotoxins,” are one of the many Microbe-associated molecular
patterns (MAMPs) that are recognized by the innate immune
system and play an important role in the bacterial detection by
the infected host. They are also essential components of the outer
membrane structure and are known to play a key role in the
leptospiral virulence [1].
Leptospiral LPS are composed of (1) a conserved lipidic part
called lipid A and (2) a variable polysaccharide part, highly antigenic
(Fig. 1). This part is composed of a succession of sugars divided
between the core section and the O antigen (responsible for the
antigenicity of the LPS). The lipid A is composed of a hexa-acylated
lipidic part attached to a disaccharide and insures the docking of the
LPS in the outer membrane. The lipid A is the part recognized by
the innate immune system and often referred to as the “endotoxin.” Unlike other bacteria, the lipid A of L. interrogans serovars
Icterohaemorrhagiae, Lai, and Manilae possesses a peculiar
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020
53
Purification of LPS from Leptospira
Delphine Bonhomme and Catherine Werts
Abstract
Leptospira species are one of the few spirochetes to possess a lipopolysaccharide (LPS) embedded in their
outer membrane. Two protocols are currently available to extract and/or purify the leptospiral lipopolysaccharides: the rapid proteinase K method and the classical hot water/phenol extraction. The first method
allows to get a quick overview of the LPS O antigen structure, whereas the second method is fitted to study
the immunological properties of the leptospiral LPS. These two methods will be detailed in this chapter.
Methodologies to assess the quality of the purification, such as the modified silver staining coloration, will
also be reviewed. Both advantages and limitations of the different analyses will be described.
Key words Leptospira ssp., Lipopolysaccharide/endotoxin purification, Proteinase K, Hot water/
phenol extraction, Silver staining
1 Introduction
Unlike most other spirochetes, Leptospira species possess surfaceexposed lipopolysaccharides (LPS). These LPS, also referred to as
“endotoxins,” are one of the many Microbe-associated molecular
patterns (MAMPs) that are recognized by the innate immune
system and play an important role in the bacterial detection by
the infected host. They are also essential components of the outer
membrane structure and are known to play a key role in the
leptospiral virulence [1].
Leptospiral LPS are composed of (1) a conserved lipidic part
called lipid A and (2) a variable polysaccharide part, highly antigenic
(Fig. 1). This part is composed of a succession of sugars divided
between the core section and the O antigen (responsible for the
antigenicity of the LPS). The lipid A is composed of a hexa-acylated
lipidic part attached to a disaccharide and insures the docking of the
LPS in the outer membrane. The lipid A is the part recognized by
the innate immune system and often referred to as the “endotoxin.” Unlike other bacteria, the lipid A of L. interrogans serovars
Icterohaemorrhagiae, Lai, and Manilae possesses a peculiar
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020
53