Lipid hexaacyl

Fig. 11.—Chemical structure of the two preponderant lipid A forms of Pseudomonas aeruginosa. (A) Pentaacyl lipid A (major lipid A fraction 75%, w/w). (B) Hexaacyl lipid A (minor lipid A fraction, 25%, w/w). Dashed lines indicate nonstoichiometric a-hydroxylation of 12 0. A lipid A species having 2 mol 12 0(2-OH)/mol lipid A was not detected (77).

Like other amphiphilic molecules, LPS aggregate and build up clusters in aqueous solutions. This process occurs only above a critical aggregate concentration (CAC) which has been established for a few lipids, but not, however, for LPS owing to extreme experimental difficulties. Estimations based on comparisons to values of other lipids led to the assumption of a CAC of < 10 M for hexaacyl-lipid A (O Fig. 16). For the lipid A precursor IVa (O Fig. 16) which represents a tetraacyl-lipid A, a CAC of < 10 has been reported [78]. 

Chemical stnictures of hexaacyl-lipid A from Escherichia coii (left) and precursor IVA from Escherichia coii (right)... 

Evidence to support the hypothesis that MsbA may be involved in lipid A transport is growing. htrB mutants accumulate tetraacylated lipid A in the inner membrane at the non-permissive temperature of 42 °C [41, 63). However, when mshA is provided in trans on a low copy plasmid, the tetraacylated lipid A is transported to the outer membrane [41]. In cells lacking mshA, hexaacylated lipid A accumulates in the inner membrane indicating that the transport of lipid A has been affected by the lack of mshA [41]. Taken together this strongly suggests that mshA plays a role in the movement of lipid A from the inner to the outer membrane, but direct biochemical assays remain to be developed. 

A pentaacyl lipid A is also present in P. aeruginosa (77) (Fig. 11 A). Here, the main lipid A species contains a total of five fatty acids, and a minor hexaacyl species (Fig. 1 IB) corresponds structurally to lipid A of C. viola-ceum (Fig. 10). The prominent pentaacyl component, which makes up approximately 75% (w/w) of P. aeruginosa lipid A, encompasses three structural forms that all possess the same /H1 — 6)-linked GlcpN backbone, but with only three (primary) 3-hydroxy fatty acids attached to positions 3, 2, 3, and 2 (Fig. 11A). These structural forms differ from each other by the 3-0-acylation of each of the two amide-linked 12 0(3-OH) residues by the secondary acyl groups 12 0 or 12 0(2-OH), as indicated by the dashed lines. Of the four conceivable structural types, the one bearing two 12 0(2-OH) residues is not present. 

Single crystals of free lipid A or LPS are as yet not available. Therefore, the most promising approach to obtain molecular models is to perform theoretical calculations. After the chemical structures of enterobacterial lipid A had been elucidated, this methodology was successfully applied with heptaacyl S. minnesota lipid A (220) and hexaacyl E. coli Re LPS (221). As an example, Fig. 13 shows the atomic model of the E. coli lipid A molecule, as calculated by Kastowsky et al. (221) using energy-minimization techniques. 

On the other hand, Boons et al. demonstrated the synthesis of Rhizobium sin-1 lipid A derivatives [96, 97], E. coli hexaacylated and Salmonella typhimurium hep-taacylated lipid A [98], tetraacylated derivatives of lipid A from Porphyromonas gingivalis [99], and lipid A derivatives and KDO-linked lipid A of Neisseria meningitidis [100],... 

The completed lipid A-core oligosaccharide structure is transported across the plasma membrane by the ATP-dependent transporter MsbA. In vitro experiments with E. coli MsbA suggest that the ATPase recognizes only fully hexaacylated Kd02-lipid A, but the transporter appears to be slightly less selective in vivo [209]. Crystal structures have been reported for MsbA homologues from . coli and Vibrio cholera [210,211,212]. However, these structures... 

---