Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus

Abstract

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone alpha-D-GalpA-(1-->4)-beta-D-DAG-(1-->6)-alpha-D-DAG-(1-->1)-alpha-D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[Delta(5)-12:1(3-OH)]] and 12:0[3-O(Delta(5)-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3-OH) and 12:0[3,6-(OH)( 2)] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF-alpha inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.

Occurrence of an Unusual Hopanoid-containing Lipid A Among Lipopolysaccharides from Bradyrhizobium Species

“…earlier in Mesorhizobium huakuii (20) and Mesorhizobium loti strains (43), which contained a non-reducing trisaccharide backbone comprising a ␤-(136)-D-GlcpN3N disaccharide partly substituted with phosphate at C-4Ј and an ␣-(131)-linked D-GalpA in stoichiometric amounts. Other microorganisms that produce lipid A substituted with ␣-(131)-D-GalpA include A. pyrophilus (48), the stalk-forming bacterium Caulobacter crescentus (51), and the plant-growth promoting bacterium Azospirillum lipoferum (52). In the last case, the lipid A backbone constituted a GlcpN-disaccharide.…”

Characterization of Galacturonosyl Transferase Genes rgtA, rgtB, rgtC, rgtD, and rgtE Responsible for Lipopolysaccharide Synthesis in Nitrogen-fixing Endosymbiont Rhizobium leguminosarum

“…Analysis of the lipid A from mutant EL206 (rgtD Ϫ ) demonstrated the absence of GalA from the 4Ј-position and, therefore, showed that rgtD encodes the GalAT that transfers GalA to this position of the lipid A. Bacteria that contain 4Ј-galacturonosylated lipid A include R. leguminosarum and R. etli strains, which are members of the Rhizobiales (1), the stalk forming Caulobacter crescentus (34), and the hyperthermophile Aquifex aeolicus (35). Indeed, strains C. crescentus CB15 and A. aeolicus VF5 contain RgtD homologues (supplemental Fig.…”

Development of an HIV-1 Specific Microbicide Using Caulobacter crescentus S-Layer Mediated Display of CD4 and MIP1α

“…It can be cultivated readily to high densities on defined growth media consisting only of glucose and essential inorganic ions. Although a gram negative organism, its unusual lipopolysaccharide structure appears to have a much reduced sepsis potential, relative to enteric bacteria [9]. With these characteristics we expect the possibility that engineered strains can be formulated to be applied as stabilized killed organisms, designed to be applied to vaginal or other mucosal tissues at relevant times, such prior to sexual acts or childbirth, roughly comparable to spermicide use.…”