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Microbial Lipopeptides

A number of bacterial species produce lipopeptides or peptidolipids, most of which have important biological functions. For example, they may have surfactant, antibacterial, antifungal, insecticide or haemolytic properties, and in consequence, they have attracted considerable interest from the agricultural, chemical, food and pharmaceutical industries. They are amphiphilic molecules that 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. A single organism can produce several isoforms differing in the nature of one or more of the amino acids and of the fatty acid component, and a few 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 web page.

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, while 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, where an assortment of extracellular polysaccharides and lipids are located. 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. While a number of models have been put forward to describe the associations of these various components within the membrane, 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.

Other than the Mycobacteria, a Gram-negative bacterium Myxococcus sp. produces distinctive glycopeptidolipids, termed myxotyrosides, with a normal or an iso-branched fatty acid amide-linked to a tyrosine-derived structure and thence to rhamnose. Cystobacter fuscus produces lipopeptides (cystomanamides) containing N-glycosylated 3-amino-9-methyldecanoic acid, a fatty acid that is rare in nature and was first found in the iturins (see below).

2.   Lipopeptides from Bacillus and Paenibacillus 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 µM. 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, but 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. Surfactins and lichenysins contain the chiral sequence LLDLLDL. The peptide moiety is linked to a β-hydroxy fatty acid (C12–C16) with a linear structure or with iso- or anteiso-methyl branches; ring closure is between the β-hydroxy fatty acid and the C-terminal peptide.

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 while also explaining its antibiotic activity. For the latter, 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 amino 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, limiting pharmaceutical use, although modified synthetic analogues are less toxic.

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 related 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, which forms part of a cyclic heptapeptide structure. They have potential as biocontrol agents towards plant pathogens.

Formula of iturnin A

In the fengycins, the decapeptide ring structure is formed by an ester bond between a tyrosine residue at position 3 in the peptide sequence and the C-terminal residue, and they have a 3-hydroxy fatty acid tail. They inhibit the growth of a number of filamentous fungi.

B. 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.

Formula of a polymyxinIn addition, this organism and other Bacillus or Paenibacillus species produce basic lipopeptides with antibiotic actions termed ‘polymyxins’. These consist 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. Six of the amino acids in polymyxin B are the uncommon L-2,4-diaminobutyric acid, which make the molecule cationic. Polymixins act by binding to the lipid A moiety of the lipopolysaccharides of the anionic outer membrane of Gram-negative bacteria and are responsible for much of the virulence; they displace competitively the calcium and magnesium bridges that 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 because of a potential to cause severe injury to the kidney, they (and novel synthetic analogues) are now finding application as a last-line therapy against multi-drug-resistant Gram-negative bacilli. Octapeptins, i.e. naturally occurring truncated polymyxins, together with cationic polypeptins also have anti-microbial activities.

The tridecaptins, isolated from strains of Paenbacillus polymyxa, are linear cationic tridecapeptides with a combination of L- and D-amino acids that are acylated with β-hydroxy fatty acids. They show strong activity against Gram-negative bacteria, exerting their bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. They are considered strong candidates for therapeutic use.

Fusaricidins are cyclic lipopeptides from Paenibacillus spp. that are distinctive in that they contain the unique 15-guanidino-3-hydroxypentadecanoic acid as the fatty acid component linked to six-membered cyclic peptides. As with most lipopeptides, a family of structural variants (more than twenty) is now known to exist. There are three main families, mainly differing in position 3 of the peptide chain, with a fourth having an additional alanine attached to the hydroxyl group of threonine in position 4 via an ester bond.

15-Guanidino-3-hydroxypentadecanoic acid and the fusaricidins

Paenibacterin produced by Paenibacillus thiaminolyticus consists of 13 amino acids and a C15 fatty acyl chain; it is attracting interest as it binds to negatively charged Gram negative endotoxins in vitro and inhibits drug-resistant P. aeruginosa in vivo. As an example of a linear lipopeptide, tauramadine from Brevibacillus laterosporus consists of five amino acids linked to iso-methyl-octadecanoic acid (7-methyloctanoyl-Tyr-Ser-Leu-Trp-Arg). It strongly inhibits pathogenic Enterococcus species.

The mixture of D- and L-amino acids in these lipopeptides results in enhanced stability to proteolytic enzymes from target organisms as well as to proteases in human plasma. 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 coordinate growth and differentiation. All of these peptidolipids are also 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.

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. In these and related molecules, the positioning of the D-amino acids is conserved 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 (see below).

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 by Gram-positive bacteria in 2003, and as a last resort for methicillin-resistant S. aureus (MRSA) infections of the bloodstream in 2006. The probable mechanism of action involves permeabilization of the cytoplasmic membrane through the formation of membrane-associated oligomers; calcium-dependent binding of the lipophilic tail of daptomycin to the bacterial plasma membrane in conjunction with an interaction with phosphatidylglycerol 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, while 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). Again, 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 Cyanobacteria

Increasing numbers of cyanobacteria species, especially those of marine origin, are being found to produce lipopeptides and glycolipopeptides with novel structures. For example, several molecular forms of 'hassilidins' have been isolated from Hassallia sp., 'puwainaphycins' have been characterised from Cylindrospermum alatosporum and 'anabaenolysins' from Anabaena sp. They are often distinctive in that the fatty acid component (C12 to C18) contains a hydroxyl group in position 2 and an amine group in position 3 (c.f. the iturins above); usually the fatty acid chain is saturated, but at least one C18 fatty acid has six double bonds (two groups of three in conjugation), while others contain methyl branches and methoxyl groups. Dragomide E from Lyngbya majuscule (a marine cyanobacterial species) has five amino acids in a linear peptide linked to an acetylenic C8 fatty acid. Although the biological properties of these lipopeptides have barely been explored, some are known to have anti-fungal actions or cytolytic activities against mammalian cell lines.

5.   Lipopeptides from Pseudomonas and Other Bacterial 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

Formula of syringomycin

The viscosin group, which has 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.

6.  Biosynthesis

Rather than the normal ribosomal mechanism of protein synthesis, bacterial lipopeptides are produced by a linear non-ribosomal peptide synthetase, such as the surfactin synthetase, which is a large multi-enzyme complex consisting of four modular building blocks, i.e. a 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), which catalyses N-acylation of the first amino acid of the lipopeptide molecule thereby linking the lipid moiety to the oligopeptide, and a thioesterase domain (te domain) to ensure the cleavage of the thio ester bond to the nascent peptide and usually to bring about cyclization. In addition, there are enzymes within the synthetase complex that effect epimerization of amino acids before formation of a peptide linkage with the next amino acid.

Identification of the genes involved in lipopeptide synthesis in different organisms together with an understanding of their organization within the genome holds immense promise for functional manipulation and the development of new biologically active molecules.

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