Fatty acid analysis of lipid A from seven immunotypes of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a pathogenic gram-negative bacterium, resistant to antibiotics, that proliferates in immunologically compromised individuals leading to lethal sepsis. The toxic principle in the outer cell membrane is a lipopolysaccharide (LPS) and a vaccine that combines LPS from several strains of the organism shows therapeutic promise. The lipid component (Lipid A) obtained from LPS comprises a C-1 phosphorylated beta-(1→6)-linked glucosamine disaccharide extensively O- and N-acylated by long-chain fatty acyl groups. This report describes the identification by GC-FID and CG-MS these acyl groups in the lipid A of seven immunotypes of P. aeruginosa (Fisher classification) from a proposed heptavalent LPS vaccine prepared from whole cells by extraction with trichloroacetic acid (Boivin procedure). Acyl groups identified were decanoic acid, dodecanoic acid, 2-hydroxydecanoic acid, and 3-hydroxydodecanoic acid as anticipated from literature reports, along with saturated fatty acids hexanoic acid, octanoic acid, nonanoic acid, undecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, tetracosanoic acid, eicosanoic acid, and docosanoic acid. In addition, branched and unsaturated fatty acids 14-methylpentadecanoic acid, 9-octadecenoic acid (Z), 12-octadecadienoic acid (Z,Z), and 9,12,15-octadecatrienoic acid (Z,Z,Z) were also detected. ESI-MS analysis of the original free lipid A samples and GC-MS analysis of acyl groups isolated from the partition of the Boivin LPS between phenol and water showed that the acyl groups other than decanoic acid, dodecanoic acid, 2-hydroxydecanoic acid, and 3-hydroxydodecanoic acid identified are not associated with the free lipid A structure and appear to be the result of the cell culture process and subsequent trichloroacetic acid extraction (Boivin procedure) of LPS from the cell culture medium.