many human pathogens including Salmonella [5], Shigella species
[6], Vibrio cholerae [7], and Escherichia coli [8].
Lipopolysaccharide (LPS) is the major component of the surface of most gram-negative bacteria. It is constituted by a polysaccharide chain of repeating units, the O-antigen, linked to a core
region containing 10–12 sugar units, that, in turn, is covalently
bound to lipid A through the 2-keto-3-deoxyoctonate (KDO).
Lipid A is highly conserved and exerts endotoxic activity, while
the O-antigen chain differs between serovars and is a major contributor to the serological specificity of bacteria.
In most published protocols for producing O-antigen-based
vaccines, O-antigen purification is carried out after LPS extraction
and hydrolysis. Traditional methods for O-antigen extraction
involve sedimentation of the bacteria, inactivation of the culture
by formalin fixation, hot phenol extraction of the LPS [9], and
treatment of the extracted LPS with acetic acid or anhydrous
hydrazine [10] for LPS hydrolysis prior to O-antigen purification,
usually performed by treatments with enzymes and size-exclusion
chromatography to remove contaminants such as proteins and
nucleic acids. The traditional process for O-antigen isolation and
purification overall is complex and time consuming and involves
manipulation of large volumes of hazardous phenol and toxic
intermediate LPS.
Here, we describe a simplified method for O-antigen isolation,
which employs acetic acid hydrolysis taking place directly in the
bacterial culture, avoiding operator exposure to pathogenic bacteria. This treatment cleaves the labile linkage between KDO, at the
proximal end of the core oligosaccharide, and lipid A, releasing the
O-antigen chain attached to the core sugars (Fig. 1). The hydrolyzed O-antigen chain and core sugars are indicated here as OAg for
simplicity. We also describe the steps for OAg purification. The
process described has been built based on the structural characteristics of Salmonella OAg [11] and can be generally applied to OAg
with neutral sugar chains. The extraction procedure is general and
applicable to any LPS.
We also describe a conjugation protocol for linkage of the OAg
to an appropriate carrier protein, based on the sequential insertion
of adipic acid dihydrazide (ADH) and adipic acid bis(N-hydroxy
succinimide) ester (SIDEA) as linkers, using the terminus KDO,
thus leaving the repeating units of the O-antigen chain unmodified
[12–14]. This protocol is generic and independent from O-antigen
chain structural features. The complete panel of analytical methods
for purified OAg, conjugate and intermediates characterization is
also described.
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Francesca Micoli et al.
[6], Vibrio cholerae [7], and Escherichia coli [8].
Lipopolysaccharide (LPS) is the major component of the surface of most gram-negative bacteria. It is constituted by a polysaccharide chain of repeating units, the O-antigen, linked to a core
region containing 10–12 sugar units, that, in turn, is covalently
bound to lipid A through the 2-keto-3-deoxyoctonate (KDO).
Lipid A is highly conserved and exerts endotoxic activity, while
the O-antigen chain differs between serovars and is a major contributor to the serological specificity of bacteria.
In most published protocols for producing O-antigen-based
vaccines, O-antigen purification is carried out after LPS extraction
and hydrolysis. Traditional methods for O-antigen extraction
involve sedimentation of the bacteria, inactivation of the culture
by formalin fixation, hot phenol extraction of the LPS [9], and
treatment of the extracted LPS with acetic acid or anhydrous
hydrazine [10] for LPS hydrolysis prior to O-antigen purification,
usually performed by treatments with enzymes and size-exclusion
chromatography to remove contaminants such as proteins and
nucleic acids. The traditional process for O-antigen isolation and
purification overall is complex and time consuming and involves
manipulation of large volumes of hazardous phenol and toxic
intermediate LPS.
Here, we describe a simplified method for O-antigen isolation,
which employs acetic acid hydrolysis taking place directly in the
bacterial culture, avoiding operator exposure to pathogenic bacteria. This treatment cleaves the labile linkage between KDO, at the
proximal end of the core oligosaccharide, and lipid A, releasing the
O-antigen chain attached to the core sugars (Fig. 1). The hydrolyzed O-antigen chain and core sugars are indicated here as OAg for
simplicity. We also describe the steps for OAg purification. The
process described has been built based on the structural characteristics of Salmonella OAg [11] and can be generally applied to OAg
with neutral sugar chains. The extraction procedure is general and
applicable to any LPS.
We also describe a conjugation protocol for linkage of the OAg
to an appropriate carrier protein, based on the sequential insertion
of adipic acid dihydrazide (ADH) and adipic acid bis(N-hydroxy
succinimide) ester (SIDEA) as linkers, using the terminus KDO,
thus leaving the repeating units of the O-antigen chain unmodified
[12–14]. This protocol is generic and independent from O-antigen
chain structural features. The complete panel of analytical methods
for purified OAg, conjugate and intermediates characterization is
also described.
268
Francesca Micoli et al.