MICROBIAL LIPOPEPTIDES


STRUCTURE, OCCURRENCE AND BIOLOGY


A number of bacterial species produce lipopeptides or peptidolipids, most of which have important biological functions. For example, many have surfactant, antibacterial or antifungal properties and have attracted interest from industry. They can consist of short linear chains or cyclic structures of amino acids, linked to a fatty acid via ester or amide bonds or both. Often the amino acids are of the D- rather than the usual L-configuration, presumably to resist the action of proteases. Some representative examples only can be discussed here. Simple fatty acid amino acid conjugates (lipoamino acids), such as the ornithine lipids, are discussed on a separate webpage.


1.   Glycopeptidolipids of Mycobacteria

The glycopeptidolipids or ‘C-mycosides’ from non-tuberculosis Mycobacteria are amongst the best known and most studied of the lipopeptides, as they are both species and type specific. The illustration below is of a typical member of the glycopeptidolipids of the Mycobacterium avium complex, an important human pathogen that is frequently associated opportunistically with acquired immunodeficiency syndrome (AIDS).

Glycopeptidolipid from M. avium

The fatty acid component is often 3-hydroxy-octacosanoate (C28), but it can consist of a range of constituents with an average chain-length of C30 and with variable numbers of double bonds. 3-Methoxy fatty acids are also seen on occasion. The fatty acid is linked to the N-terminus of a tripeptide of hydrophobic amino acids of the D-configuration (produced from the L-forms by the action of a racemase) and thence to L-alaninol and dimethyl-rhamnose. A complex oligosaccharide is linked to the peptide via a disaccharide (deoxy-talose−rhamnose).

Mycobacterial glycopeptidolipids can be classified within two groups – polar and non-polar. Within the M. avium complex, all have in common an N-acylated lipopeptide core attached to a rhamnosylated alaninyl C-terminus. The two groups differ in the structure of the oligosaccharide attached to the allo-threonine residue, which can carry additional O-acyl moieties at undefined locations. In other species of Mycobacteria, the basic structure of the lipopeptide unit does not vary appreciably, but the nature of the carbohydrate moieties does differ importantly in the degree of substitution of the deoxy-talose and rhamnose units by methyl or acetyl groups. It is the complex and highly variable oligosaccharide component that carries most of the antigenicity and type (serovar) specificity.

In M. smegmatis, the glycolipopeptides consist of C26-C34 fatty acyl chains, with rather unusually either hydroxyl or methoxyl groups in position 5, linked to the same tetrapeptide as before (Phe-Thr-Ala-alaninol), in which the hydroxyl groups of threonine and the terminal alaninol are glycosylated.

Glycolipopeptide from M. smegmatis

M. xenopi produces serine-containing glycopeptidolipids with a C12 fatty acyl group, and those from M fortuitum have a somewhat different oligosaccharide and peptide structure.

Glycopeptidolipids are variable, distinctive and highly antigenic molecules, which play a significant role in pathogenesis by activating the host immune response. They are located on the external membrane of the organisms, which contains an assortment of extracellular polysaccharides and lipids. The lipid components include phthiocerol dimycocerosates, triacylglycerols and acylated trehaloses, which are common to most species of Mycobacteria, and the glycopeptidolipids, which are variable in structure and are specific to each species. A number of models have been put forward to describe the associations of these various components within the membrane, but one of the more popular places the lipopeptides in the outermost region of the layer, where they interact with the mycolic acids via hydrophobic attractions.


2.   Lipopeptides from Bacillus species

Bacteria of the Gram-positive genus Bacillus produce a number of cyclic lipopeptides, many of which have appreciable antibacterial or antifungal properties. There is considerable structural diversity as a consequence of differences in the nature of the fatty acid component, for example in chain-length (C6-C18) and often the presence of hydroxyl groups and/or iso- or anteiso-methyl branches, as well as in the type, number and configuration of the amino acids in the peptide chain. For example, various strains of B. subtilis produce more than twenty different molecules with antibiotic activity including many lipopeptides. Surfactin (illustrated) in addition to its antibiotic properties is one of the most powerful biosurfactants known; it can lower the surface tension of water from 72 mN/m to 27 mN/m at concentrations as low as 20 M. Surfactin is composed of seven different amino acids of both the D- and L-configurations, which form a cyclic structure incorporating a fatty acid such as 3-hydroxy-13-methyl-tetradecanoic acid.

Formula of surfactin

Very similar molecules are produced by many other Bacillus species, and the various isoforms have been described under different synonyms, such as bacircine, halo- and isohalobactin, lichenysin, daitocin and pumilacidin. Only a few of these can be discussed here. In addition to the rare D-amino acids, these can contain unusual β-amino acids, and hydroxy- or N-methylated amino acids. The peptide moiety is linked to a β-hydroxy fatty acid (C12–C16) with a linear structure or iso- or anteiso-methyl branches. Ring closure is between the β-hydroxy fatty acid and the C-terminal peptide.

Rather than the normal ribisomal mechanism of protein synthesis, they are produced by a linear non-ribosomal peptide synthetase, surfactin synthetase, which is a large multi-enzyme complex consisting of four modular building blocks, i.e. the multicarrier thio-template mechanism. Such enzyme systems typically contain an enzyme component that activates the initial substrate, one that tethers the covalent intermediates as an enzyme-bound thioester (peptidyl-carrier-protein), an enzyme that carries out peptide bond formation (condensation or C-domain), and a thioesterase domain (te domain) to ensure the cleavage of the thio ester bond to the nascent peptide and usually to effect cyclization.

Scottish thistleThe amino acids glutamic acid and asparagine are the main polar components that counterbalance the fatty acyl moiety and give the molecule its amphiphilic character, also explaining its antibiotic activity. Thus, various mechanisms have been proposed, all of which depend on the fact that the hydrocarbon tail of the molecule can insert itself readily into the membranes of both Gram-positive and Gram-negative bacteria where it forms associations with the hydrophilic fatty acid chains of the phospholipids. One suggestion is that the two acid residues are arranged spatially so that they can stabilize divalent cations, such as Ca2+. The proximity of this to the polar head group of the phospholipids in the membrane causes the complex to cross the lipid bilayer via a flip-flop mechanism, delivering the cation into the intracellular medium. Alternatively, self association of surfactin molecules on both sides of an uncharged membrane may create a pore through which cations can pass. A third hypothesis is that such self association of surfactin molecules leads to the formation of mixed micelles and ultimately causes disruption of the bilayer. These effects are non-specific so do not produce resistant strains of bacteria. Indeed, at high concentrations surfactin can disrupt most membranes including those of erythrocytes.

Surfactin is distinctive in that it also has antiviral properties, causing disintegration of enveloped viruses, including both the viral lipid envelope and the capsid, through ion channel formation. However, it only affects cell-free viruses and not those within cells. Because of its detergent properties, surfactin has been investigated as a potential bio-remediation agent to assist in the degradation of oil spills and to mop up heavy metals from contaminated soils.

B. subtilis produces two further families of lipopeptide antibiotics, the iturins (bacillomycins, iturins and mycosubtilins) and fengycins (plipastatins). The iturins especially are unusual in that they contain long-chain fatty acids (C14 to C17) with an amine group in position 3. Such fatty acids are rare in nature, but 3-amino-9-methyldecanoic acid has been detected in lipopeptides from myxobacteria.

Formula of iturnin A

In the fengycins, the ring structure is formed by an ester bond between a tyrosine residue at position 3 in the peptide sequence and the C-terminal residue.

All of these peptidolipids are under investigation as agents for the control of plant diseases. Not only do they have the potential to act against phytopathogens, including bacteria, fungi and oomycetes, but they also stimulate defence mechanisms in the plant hosts.

Formula of a polymyxinB. brevis produces a family of linear pentadecapeptides (gramicidins) with alternating L- and D-amino acids. They enter membranes readily and form ion channels specific for monovalent cations.

In addition, this organism and other Bacillus species produce basic lipopeptides with antibiotic actions, consisting of decapeptides (7-membered cyclic peptides attached to a linear peptide) linked to a fatty acid such as 6-methyl-octanoic or 6-methyl-heptanoic acids, and termed ‘polymyxins’. Six of the amino acids in polymyxin B are the uncommon L-2,4-diaminobutyric acid. Polymixins act by binding to the lipid A moiety of the lipopolysaccharides of the anionic outer membrane of Gram-negative bacteria, competitively displacing calcium and magnesium bridges which stabilize the outer leaflet of the outer membrane. They are used to treat a variety of infections including those caused by pseudomonads, enterobacteria and Acinetobacter species in topical applications such as wound creams and eye or ear drops. While polymixins were once considered to be too toxic to be used as systemic antibiotics, they (and synthetic analogues) are now finding application as a last-line therapy against multi-drug-resistant Gram-negative bacilli.

In general terms, the main natural functions of lipopeptides from Bacillus species are believed to be control of other microorganisms, motility and attachment to surfaces, although they may also have a signalling function to coordinated growth and differentiation.


3.   Antibiotic Lipopeptides from Streptomyces

The genus Streptomyces is the source of a large number of antifungal and antibiotic compounds. Streptomyces roseosporus (Actinobacteria), for example, produces daptomycin, which is an acidic, cyclic lipopeptide consisting of 13 amino acids, which includes three D-amino acid residues (D-asparagine, D-alanine, and D-serine), linked via the N-terminal trypsin to decanoic acid (related lipopeptides contain anteiso-undecanoyl, iso-dodecanoyl or anteiso-tridecanoyl residues). The macrocycle contains ten amino acid residues with a terminal kynurenine connected to the hydroxyl group of threonine to form a macrolactone. The positioning of the D-amino acids is conserved in these and related molecules, as is the Asp-X-Asp-Gly motif, which is a Ca2+ binding region. Like the lipopeptides produced by Bacillus species, daptomycin is synthesised by a non-ribosomal mechanism.

formula of daptomycin

Daptomycin, one of several calcium-dependent antibiotics, was licensed by the FDA in the United States for use against skin and soft tissue infections in 2003, and for methicillin-resistant S. aureus (MRSA) infections of the bloodstream in 2006. The mechanism of action involves calcium-dependent binding of the lipophilic tail of daptomycin to the bacterial plasma membrane, probably in conjunction with an interaction with phosphatidylglycerol, and this results in potassium efflux, membrane disruption, cessation of the synthesis of macromolecules and eventually cell death.

Other species of Streptomyces and Actinomyces contain related antibiotic molecules, including amphomycins, friulimicins, and glycinocins (laspartomycins), in a macrocycle closed with a lactam rather than a lactone bond. Certain of the amino acids are modified during biosynthesis via enzymatic oxidation and methylation to produce new amino acids not found in proteins. The fatty acids are C13 to C16 with iso- or anteiso-methyl branches, and a double bond in position 3 (or in position 2 in the case of the glycinocins). Such lipopeptides are providing biochemists with opportunities for genetic modifications both to the peptide and fatty acid moieties to produce novel compounds with further antibiotic properties against gram-positive infections. Friulimicin B is undergoing clinical trials.


4.   Lipopeptides from Pseudomonas and Other Species

The genus Pseudomonas produces many different cyclic lipopeptides, which have been have been classified into at least six groups, including viscosin, syringomycin, amphisin, putisolvin, tolaasin, and syringopeptin. For example, the phytopathogenic bacterium Pseudomonas syringae pv. syringae produces two classes of necrosis-inducing lipodepsipeptide toxins termed the syringomycins and syringopeptins. Syringomycin form SRE is illustrated; it contains nine amino acids of which three are unusual (Dab = 1,4-diaminobutyric acid; Dhb = 2,3-dehydroamino-butyric acid; 4(Cl)Thr = C-terminal chlorinated threonine residue), while three are of the D-form

syringomycin

The viscosin group, which have antiviral properties, also consists of lipopeptides with nine amino acids, whereas members of the amphisin have eleven in the peptide moiety. The tolaasin group are more varied because of differing lengths of the peptide chains (19–25 amino acids, including 2,3-dehydro-2-aminobutyric acid and homoserine). 3-Hydroxydecanoic acid is usually the lipid moiety in these groups. In contrast, the putisolvins have a hexanoic lipid tail and a peptide moiety of 12 amino acids with a different mode of cyclization.

Plusbacins are produced by a Pseudomonas species also, and they are very similar to tripropeptins, and empedopeptin found in Gram-negative soil bacteria. They are cyclic lipopeptides differing mainly in the first three amino acids and the nature of the fatty acid component. The last of these binds and de-activates lipid II, a key molecule in the biosynthesis of cell wall peptidoglycans in bacteria, and appears to be a strong candidate as an antibiotic in pharmaceutical applications.

A complex mixture of water-soluble lipodepsipeptides is produced by Gram-negative Lysobacter spec. One of these, designated WAP-8294A2 or lotilibcin, is a dodeca-peptide linked to 3-hydroxy-7-methyl-octanoic acid and is a potent antibacterial agent against Gram-positive bacteria, including antibiotic-resistant strains. It functions by interacting with phospholipids, specifically cardiolipin and phosphatidylglycerol, in the bacterial cell membrane, eventually causing cell death.

Serratia marcescens produces at least three surface-active exolipids designated serrawettins W1 to W3. As an example, serrawettin W2 is 3-hydroxydecanoyl-D-leucyl-L-seryl-L-threonyl-D-phenylalanyl-L-isoleucyl lactone. Their function is to reduce the surface tension of thin films of water on solid surfaces, assisting with motility, cellular communication and nutrient accession of the bacteria.

As examples of linear lipopeptides, tauramadine from Brevibacillus laterosporus consists of five amino acids linked to iso-methyl-octadecanoic acid, while dragomide E from Lyngbya majuscule has five amino acids linked to an acetylenic C8 fatty acid.


Recommended Reading


W.W. Christie

James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

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AOCS Credits/disclaimer Updated: May 15th, 2014 Author