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Box , Durham, North Carolina The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most Gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes.
Following attachment of the core oligosaccharide, lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of lipid A may occur during its transit from the outer surface of the inner membrane to the outer membrane.
Lipid A modification enzymes are therefore reporters for LPS trafficking within the bacterial cell envelope. Modification systems are highly variable and are often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate the virulence of some Gram-negative pathogens. Heterologous expression of the genes encoding the lipid A modification enzymes in diverse bacteria facilitates the re-engineering of lipid A structures and should enable the development of new vaccines.
Lipid A endotoxin , the hydrophobic anchor of lipopolysaccharide LPS , is a glucosamine-based saccharolipid 1 that makes up the outer monolayer of the outer membranes of most Gram-negative bacteria 2 - 4.
There are approximately 10 6 lipid A residues, 10 7 phospholipids and 10 5 undecaprenyl phosphate-sugar molecules in an E. In wild-type strains, additional core and O-antigen sugars are present Fig. These complex glycoforms are not needed for growth but protect bacteria from antibiotics and complement-mediated lysis.
The core and O-antigen domains are required for virulence, and consequently are present in most clinical and environmental isolates 3.
The structures and biosynthesis of core and O-antigen sugars are reviewed elsewhere 2 , 3. Here, we focus on the biosynthesis of lipid A and its modification during transport to the outer membrane. The mechanisms of lipid A transport are covered in greater detail by Tommassen et al.
The structure and biosynthesis of LPS 2 , 3 , peptidoglycan , membrane-derived oligosaccharides , , lipoproteins and phospholipids , are reviewed elsewhere. Strains of E. The Kdo 2 -lipid A region of LPS the topic of this review usually represent the minimal substructure required for growth of gram-negative bacteria.
Exceptions include some spirochetes and strains of Sphinogmonas , in which the lipid A biosynthesis genes are absent, and Neisseria meningitidis in which lpxA knockouts lacking LPS are viable These strains still make the tetra-acylated precursor lipid IV A , which is nevertheless required for growth 8. The red phospholipids represent phosphatidylethanolamine and the yellow phosphatidylglycerol.
Each enzyme of the constitutive lipid A pathway is encoded by a single structural gene 2 , The glucosamine disaccharide backbone of lipid A is blue. The Kdo disaccharide is black. The distal enzymes of the pathway, starting with LpxK, are integral inner membrane proteins, the active sites of which face the cytoplasm 2.
The red numbers specify the glucosamine ring positions of lipid A and its precursors. The black numbers indicate the predominant fatty acid chain lengths found in E. Additional minor acyl chain variants can be detected by high-resolution mass spectrometry Most Gram-negative bacteria synthesize lipid A molecules resembling those made by E.
Lipid A furthermore activates the production of co-stimulatory molecules required for adaptive immunity 20 , With mononuclear and endothelial cells, lipid A stimulates tissue factor production 22 , All these events are desirable for clearing local infections. When overproduced systemically during sepsis, however, the inflammation caused by some of these proteins damages small blood vessels and can precipitate Gram-negative septic shock 24 , LPS, or even synthetic E.
The characteristic structural features of E. The lipid A biosynthetic pathway may be viewed as having a conserved and a variable component. The conserved constitutive enzymes Fig.
In contrast, the lipid A modification enzymes, discussed below, are mostly extra-cytoplasmic and vary from organism to organism. In many instances, the lipid A modification systems are induced or repressed by growth conditions, such as changes in pH, divalent cation concentrations or the presence of anti-microbial peptides 41 - Most modification enzymes reside either on the periplasmic surface of the inner membrane or in the outer membrane 46 - They are excellent markers for following the translocation of nascent LPS from its initial site of biosynthesis on the inner surface of the inner membrane to the outer surface of the outer membrane 55 - 60 Fig.
The systematic elucidation of the constitutive pathway for lipid A biosynthesis Fig. The discovery of lipid X 61 , 62 coincided with the correct structure determination 64 , 65 and chemical synthesis of lipid A Recognition of the existence of an acylated monosaccharide 62 representing a precursor to the proximal right subunit of lipid A Fig. The nine enzymes of the constitutive lipid A pathway and the single-copy genes encoding them Fig.
The only exceptions are organisms like Sphingomonas , which make bioactive sphingolipids instead of lipid A The sequences of the lipid A genes are easily recognized when Gram-negative genomes are compared Their active sites are presumed to face the cytoplasmic surface of the inner membrane, given that their water-soluble co-substrates are cytoplasmic molecules Fig.
Interestingly, higher plants like Arabidopsis thaliana encode significant orthologues of the constitutive enzymes within their nuclear genomes 2 , 83 , but lipid A-like molecules have yet been identified in plants by mass spectrometry or NMR spectroscopy It does not recognize R hydroxymyristoyl-coenzyme A. The active site of E. In Pseudomonas aeruginosa , the LpxA ruler is reset to incorporate C10 chains 86 , 87 , while in Neisseria meningitidis and Leptospira interrogans it measures C12 chains 88 , The rest of the lipid A molecule is unchanged Single amino acid substitutions can switch P.
The crystal structure of LpxA 91 - 93 reveals that it is a homo-trimer Fig. All hexad repeat-containing proteins studied to date are helical homo-trimers.
The three identical active sites of LpxA, which were first proposed based on site-directed mutagenesis, are located at the subunit interfaces 93 , A recent X-ray structure of E. In addition to validating the proposed locations of the LpxA active sites 94 , these studies provide a structural explanation for the extraordinary chain length selectivity of these enzymes. The LpxA homotrimer was solved at 2.
Each subunit has its own color. The top-down view of this complex right reveals the location of the active site and the positioning of the acyl chain, consistent with previous proposals based on site-directed mutagenesis and NMR studies 94 , Many bacteria, including L. LpxA of L. This remarkable selectivity accounts for the fact that L. A significant number of the Gram-negative bacteria sequenced to date contain GnnA and GnnB orthologues.
The presence of additional N -linked acyl chains may increase the stability of lipid A to base hydrolysis or may prevent its degradation by lipases. A crystal structure of L. LpxA from L. It displays no sequence similarity to other deacetylases or amidases. It is an excellent target for the development of novel antibiotics 10 , , Slow, tight-binding inhibitors of LpxC with low nM affinity have recently been reported Fig.
These compounds are N -aroyl-L-threonine hydroxamates Fig. They possess antibiotic activity comparable to ciprofloxacin Clinical applications would include the treatment of cystic fibrosis patients infected with multi-drug resistant P.
The slow, tight-binding inhibitor CHIR inhibits diverse LpxC orthologues in the low nM range and displays potent antibiotic activity against many gram-negative bacteria The substrate mimetic TU inhibits E. This ribbon diagram is based on the NMR studies of Coggins et al. The recent crystal structure of the same complex is similar, except for slight differences in the orientation of the tetrahydropyran ring LpxC levels increase five- to ten-fold in cells treated with sub-lethal doses of LpxC inhibitors Induction is not associated with increased transcription but may be due to reduced LpxC turnover when lipid A biosynthesis is curtailed.
LpxC induction is also seen in temperature-sensitive LpxA mutants in the absence of LpxC inhibitors Although the signaling mechanisms controlling LpxC induction are unknown, two amino acids at the C-terminus of LpxC are critical for this regulation The FtsH protease is partially responsible for regulating LpxC turnover in vivo but additional processes cannot yet be excluded.
The X-ray structure of LpxD W. Hunter, personal communication shows that it, like LpxA, is a homotrimer constructed around multiple contiguous hexad repeats. LpxH is unusual in that it is missing in about one third of the Gram-negative genomes. An alternative pyrophosphatase of this kind must exist in these strains, since all of them contain LpxD and LpxB Fig.
This enzyme is distantly related to the phosphoprotein phosphatase family. Its structure is not yet available. Like LpxH, LpxB is a peripheral membrane protein. LpxB is very useful for the chemo-enzymatic synthesis of lipid A analogues , Its crystal structure has not been reported. A second LpxB orthologue of unknown function is present in strains of Legionella , where it is required for growth inside of Acanthamoeba Each protein contains one predicted membrane-spanning segment at its N -terminus.
The active sites likely face the cytoplasm. This important precursor is an excellent endotoxin antagonist in human cells, but an agonist of reduced potency in the mouse
LIPID A MODIFICATION SYSTEMS IN GRAM-NEGATIVE BACTERIA