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Microbial Proteolipids and Lipopeptides

A number of bacterial and fungal species produce proteolipids (lipoproteins) and lipopeptides (or peptidolipids), most of which have biological functions of vital importance to each species in that they provide structural integrity to bacterial cell walls and are essential for their interaction with host organisms. These are amphiphilic molecules that consist of short linear chains or cyclic structures of amino acids, and including depsipeptides, i.e. peptides in which one or more of the amide bonds is replaced by an ester bond (or lactone in cyclic structures), and they are linked via an ester or an amide bond to a fatty acid, which can vary in chain length and in the presence or absence of substituents of various kinds. Depsipeptide ester linkOften the amino acids are of the D- rather than the usual L-configuration, presumably to resist the action of proteases. A single species can produce many structural variants or 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. In many species, biosynthesis is by linear non-ribosomal peptide synthetases rather than by the conventional ribosomal route (see below).

As such lipopeptides often have surfactant, antibacterial, antifungal, insecticide or haemolytic properties, they have attracted considerable interest from the agricultural, chemical, food and pharmaceutical industries. As a generality, Gram-positive bacteria, and especially the genus Bacillus, are a rich source of antimicrobial cyclic lipopeptides, while Gram-negative bacteria are better known for rhamnolipid and glycolipid biosurfactants. Overuse of broad-spectrum antibiotics to control human and plant pathogens has greatly accelerated the development of antibiotic resistance among bacteria and fungi. Those lipopeptides with anti-bacterial activities (bacteriocins) are now of particular interest as part of the search for novel antibiotics and an important challenge for medicinal chemistry. Many bacteriocins have been shown to have a broad spectrum of activity, kill bacteria rapidly, and show synergy with established antibiotics, although problems remain in transferring research findings to clinical practice.

A variety of different mechanisms may be involved in the antibiotic activities of lipopeptides, both linear and cyclic, as they are very diverse amphiphilic agents that not only interact electrostatically with the charged head groups of membrane lipids, but also with the hydrophobic region of lipid bilayers. This can result in electrostatic and mechanical changes, reduction in surface tension, promotion of metal ion sequestration, and a disturbance of the structures of lipid bilayers in bacterial and fungal membranes. Often, there appears to be a general tendency to induce pore formation.

Note that the terms ‘lipoprotein’, ‘lipopeptide’ and ‘proteolipid’ are used interchangeably for these compounds in the literature. To avoid confusion, I would prefer to reserve the term ‘lipoprotein’ for the non-covalently linked protein-lipid complexes in plasma, but as the wider usage has been in place for 50 years, it is unlikely to change. Simple fatty acid amino acid conjugates or lipoamino acids, such as the ornithine lipids, are discussed on a separate web page as are the Eukaryotic proteolipids.

1.  Bacterial Triacyl Proteolipids (Lipoproteins)

Formula of a bacterial proteolipidAll bacteria contain large numbers of proteins with a unique and distinctive post-translational lipid modification with three fatty acyl groups (more than 2000 have been identified), and this appears to be essential for their efficient function and even for their pathogenesis via host-pathogen interactions. The lipid components consist of N-acyl- and S‑diacylglycerol groups attached to an N-terminal cysteine, i.e. it contains a thio ether bond. In Mycobacterium bovis, for example, positions sn-1 and 2 of the glycerol moiety are linked to palmitic and tuberculostearic acids, respectively, and either fatty acid can be the N-acyl moiety. Proteomic analysis of Staphylococcus aureus revealed 63 different proteolipids of this type.

As with other proteolipids, the lipid moieties act as an anchor to hold the protein tightly to a hydrophobic cellular membrane while permitting it to operate in an aqueous environment in such important activities as transport, signalling, adhesion, digestion and growth; they have an important role in nutrient and ion acquisition, enabling pathogenic species to better survive in the host. They are important constituents of the outer leaflet of the cytoplasmic membrane of Gram-positive bacteria and of the outer leaflet of the cytoplasmic and the inner leaflet of the outer membranes of Gram-negative bacteria. Like the endotoxins (lipopolysaccharides) of Gram-negative bacteria, they are potent stimulants of the human immune system, eliciting pro-inflammatory immune responses by functioning as ligands for specific receptors, especially the Toll-like receptor 2. They are thus responsible for much of the virulence of the organisms and have the potential to be used in vaccines.

The first of these to be discovered and termed "Braun's lipoprotein" is one of the most abundant membrane proteins in the cell walls of Gram-negative bacteria such as Escherichia coli. It has a molecular weight of only 5.8 kDa and folds into a trimeric helical structure. Uniquely, much of it is covalently attached by the ε-amino group of the C-terminal lysine to the carboxyl group of a meso-diaminopimelic acid residue in the peptidoglycan of the cell wall to provide the only covalent connection between the inner and outer membranes of the cell wall. It is embedded in the outer membrane by its hydrophobic head and provides a tight link between the two layers, giving structural integrity to the outer membrane; it also fixes the distance between the inner and outer membranes.

In the main secretory pathway, proteins destined to become lipidated have N-terminal signal peptides containing a motif known as a lipobox with an invariant cysteine residue, which directs them to the lipoprotein biogenesis machinery after transport mainly in an unfolded state. The three fatty acyl groups and the glycerol component responsible for binding to the membrane surface are derived from bacterial phospholipids, especially phosphatidylglycerol. Three enzymes are involved in the biosynthetic pathway, which occurs in the cytoplasmic (inner) membrane. The first (Lgt; phosphatidylglycerol:prolipoprotein diacylglyceryl transferase) attaches the diacylglycerol group from phosphatidylglycerol to the thiol of cysteine, the first amino acid after a signal peptide in the pro-lipoprotein. In E. coli, for example, there is an 18- to 36-amino-acid-long signal peptide, which is distinguished by a C-terminal lipobox comprising a conserved three-amino-acid sequence in front of an invariable cysteine.

Proteolipid biosynthesis in bacteria

A second enzyme (Lsp; prolipoprotein signal peptidase) then removes the signal peptide, leaving the cysteine as the new amino-terminal residue of the protein component. The third enzyme (Lnt; apolipoprotein N-acyltransferase) acylates the N-terminal amine group of the modified cysteine with a fatty acid from position sn-1 of whatever phospholipid is available (the resulting lysophospholipid is flipped back across the membrane and re-esterified). This last step always occurs in Gram-negative bacteria, but is found only rarely in Gram-positive bacteria (Lgt and Lsp are essential to all bacteria). However, other species have related Lnt-like enzymes that can acylate, or alternatively acetylate or add a peptide unit to the N-terminal cysteinyl residue. Most of the proteolipids are then transferred to the inner leaflet of the outer membrane by a complex mechanism involving five proteins (Lol pathway), which sort and translocate them via specific signal residues located C-terminally to the diacylglyceryl-cysteine so that the acyl chains are within the membrane. In the outer membrane, proteolipids undergo topology changes that govern the biogenesis and integrity of the membrane.

In Gram-positive bacteria, lipoprotein maturation and processing are not vital to the organism, but they are essential to their pathogenicity. It is now evident that the degree of proteolipid acylation in these species has a substantial influence on the immune response. Thus, exposure of the skin to diacylated proteolipid induces immune suppression, while exposure to triacylated proteolipid does not. In addition, the fatty acid composition of the lipid moiety influences the pro-inflammatory response.

2.   Glycopeptidolipids of Mycobacteria

An unusual post-translation O-acyl modification of specific proteins by mycolic acids is one means of targeting them for assembly in the outer membrane (mycomembrane) in bacteria of the order Corynebacteriales; a short linear amino acid motif for O-acylation of proteins has been revealed that seems to be preserved throughout the kingdoms of life.

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.

3.   Lipopeptides from Bacillus and Paenibacillus species

Bacteria of the Gram-positive genus Bacillus produce a large number of cyclic lipopeptides, many of which have appreciable antibacterial or antifungal properties, although only the more important of these can be discussed here. 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, produced by B. subtilis and B. licheniformis strains, 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 ยตM. The form illustrated 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, and it essential for biofilm formation and root colonization.

Formula of surfactin

Very similar molecules are produced by many other Bacillus species, and various isoforms have been described and given different names, such as bacircine, halo- and isohalobactin, lichenysin, daitocin and pumilacidin. 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. The last 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. A surfactin variant produced by Bacillus amyloliquefaciens, WH1fungin, induces apoptosis in fungal cells by a mitochondria-dependent pathway.

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.

Iturins: 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 (amino acids?), which form part of a cyclic heptapeptide structure. They are important constituents of many Bacillus strains that have been commercialized as biological control agents against fungal plant pathogens and as plant growth promoters. By interacting with sterol components in fungal membrane, iturins create a pore that leads to increased loss of K+ and other cellular constituents and eventually to cell death.

Formula of iturnin A

In the related fengycins, a 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.

Polymyxins: Formula of a polymyxinVarious strains of Paenibacillus polymyxa, which are efficient plant growth promoting rhizobacteria that protect plants from phytopathogenic microorganisms in the soil, together with other species of Bacillales produce a variety of linear and cyclic lipopeptides. In particular, Paenibacillus species produce at least four families of basic lipopeptides with potent antibiotic actions of which the most studied are the polymyxins (15 variants). 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 (DAB), which give the molecule a positive charge. 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 bind to the anionic phosphate and pyrophosphate groups to displace the calcium and magnesium bridges that stabilize the outer leaflet of the outer membrane leading to disruption of the permeability barrier.

Polymixins 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 they were once considered to be too toxic to be used as systemic antibiotics because of a potential to cause severe injury to the kidney, the use of polymyxin B or polymyxin E ('colistin') is now permitted as a last-line therapy against multi-drug-resistant Gram-negative bacilli. Novel synthetic analogues in which changes have been made to specific amino acids or the fatty acyl group are under development. Unfortunately, some strains of Gram-negative bacteria have developed resistance to polymyxins by remodelled their lipid A by addition of palmitate to the R-2-hydroxymyristate residue, increasing the hydrophobicity of the outer membrane to hinder the diffusion of the lipopeptide through it, while others add further phosphoethanolamine or 4-amino-arabinose residues to block anionic binding sites. On the other hand, there is an encouraging development in that polymyxin derivatives that lack any notably direct antibacterial activity can sensitize bacteria to other antibiotics by damaging and increasing the permeability of their outer membranes.

Octapeptins, i.e. naturally occurring truncated polymyxins, together with cationic polypeptins also have anti-microbial activities. For example, octapeptin A is not only active against Gram-negative bacteria but also against Gram-positive bacteria and fungi. The N-terminal fatty acyl group carries a 3(R)‑hydroxyl group, and it varies in chain length from C8 to C10 and can be linear or branched. As battacin (octapeptin B5), isolated from Paenibacillus tianmuensis, exhibits antimicrobial activity against multidrug-resistant E. coli and P. aeruginosa and is threefold less toxic than polymyxin B, octapeptins are considered strong candidates for therapeutic use. Tridecaptins, isolated from strains of P. 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.

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. Fusaricidins are effective against a number of plant fungal pathogens, but in mammalian cells, they are toxic to mitochondria and induce apoptosis in consequence of their ion channel-forming properties.

15-Guanidino-3-hydroxypentadecanoic acid and the fusaricidins

Paenibacterin produced by P. thiaminolyticus consists of a cyclic 13-residue peptide with a C15 fatty acyl chain at the N-terminus; it is attracting interest as it binds to negatively charged Gram negative endotoxins in vitro and inhibits drug-resistant P. aeruginosa in vivo. Similarly, Paenibacillus sp. OSY-N produces cyclic and linear lipopeptides ('paenipeptins'), and both types show antimicrobial activity against Gram-negative and Gram-positive bacteria by binding to lipopolysaccharides and lipoteichoic acid to disrupt the cytoplasmic membrane.

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. Others include cerexins and tridecaptins. Kurstakins are lipoheptapeptides displaying antifungal activities and were first classified as linear molecules, though cyclic forms are now known. Linear lipopeptides are of pharmaceutical interest in that they are more accessible by chemical synthesis, although to date cyclic lipopeptides have greater antibacterial potency and greater oral bioavailability.

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 and indeed the organisms per se, especially strains of P. polymyxa, 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 and promote plant growth.

4.   Lipopeptides from Actinomycetes

The Actinomycetes in general and the genus Streptomyces in particular are sources 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-Glc 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 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. Some resistant strains of the target organism synthesise lysyl-phosphatidylglycerol, a cationic metabolite of phosphatidylglycerol, which changes the membrane charge from negative to positive and limits the ability of daptomycin to bind to the membrane in a calcium-dependent manner.

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. For example, many of these lipopeptides incorporate piperazic (diazinane-3-carboxylic) and pipecolic (piperidine-2-carboxylic) acids, which are important structural units of many other natural products of microbial origin. 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). Friulimicin B is undergoing clinical trials. Ramoplanin is a glycolipodepsipeptide antibiotic (i.e. with carbohydrate, ester and amide bonds in the molecule) obtained from fermentation of Actinoplanes sp. ATCC 33076 that is active against multi-drug-resistant, Gram-positive pathogens including Enterococcus sp., Staphylococcus aureus (MRSA), and Clostridium difficile. It acts by disrupting bacterial cell walls by sequestering the peptidoglycan intermediate lipid II. While it is unsuitable for intravenous use in humans because of side effects, it is being trialed for oral use against gastrointestinal infections. 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 infections by Gram-positive bacteria but with fewer side effects.

Bacillus and Paenibacillus species also produce a wide range of polyketide metabolites, some of which are linked to amino acids, but this would take us into a quite separate aspect of lipid chemistry and biochemistry

5.  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 or anti-parasitic actions or cytolytic activities against mammalian cell lines.

6.   Lipopeptides from Pseudomonas Species

The genus Pseudomonas produces many cyclic lipopeptides (lipodepsipeptides) with surfactant, antibacterial and antifungal properties; some have even been reported to have anti-cancer activity. They are based on a similar structural blue-print and have been have been classified into at least 14 groups with numerous structurally homologous members of which the viscosin, syringomycin, amphisin, putisolvin, tolaasin and syringopeptin groups are the best known. While it is hoped that some will prove to have pharmaceutical use as antibiotics in humans, they also have great potential against plant pathogens of various kinds. 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. Viscosin has been shown to inhibit metastasis of breast and prostate cancer cell lines without causing toxicity, while pseudofactin II, a cyclic lipopeptide biosurfactant isolated from a strain of Pseudomonas fluorescens was found to induced apoptosis of melanoma cells by a specific interaction with the plasma membrane. 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 it appears to be a strong candidate as an antibiotic in pharmaceutical applications.

7.   Proteolipids and Lipopeptides from Other Bacterial Species

Bacteria of the genus Mycoplasma lack a cell wall and are obligate parasites that must obtain all their lipids from the host. Recently, it has been demonstrated that otherwise cytoplasmic proteins, lacking signal peptides, are tethered to the outer membrane by a link from glutamine near the C-terminus of the protein to rhamnose and thence to a phospholipid, presumed for the moment to be phosphatidic acid. Whether other bacteria have a similar mechanism has yet to be determined.

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 in addition to rhamnolipids (glycolipid surfactants). 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.

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. In addition, genome mining of Myxobacteria has found many strains that produce lipopeptides termed myxochromides. 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 above).

8.  Fungal Lipopeptides

More than 30 genera of fungi produce cyclic and linear lipopeptides with antibiotic and antifungal properties, some of which are mycotoxins. These can have from three to thirteen amino acids, often modified from the usual forms, and many different fatty acid constituents. The best known of these appear to be the echinocandins, which are nonribosomal cyclic hexapeptides produced by fungi such as Glarea lozoyensis with potent antifungal properties. There are three forms, echinocandin, pneumocandin A0 pneumocandin B0, and these contain two types of hydroxy-L-prolines linked to a fatty acid that can be linoleate or 10,12-dimethyltetradecananoate. Their fungicidal properties are due to inhibition of the 1,3-β-D-glucan synthase in fungal cell walls, but other comparable fungal metabolites inhibit many different enzyme systems. Other important lipopeptides from fungi include the peptaibols, pleofungins, beauvericins and enniatins.

9.  Biosynthesis of Bacterial Lipopeptides

Bacterial strains produce lipopeptides through either ribosomal or nonribosomal pathways. In the former, a ribonucleoprotein complex generates a linear peptide, as directed by mRNA, and this is released from the ribosome into the cytoplasm, where several post-translational modifications, including epimerization and cyclization, can occur to adapt the peptide to its specific function. In contrast, peptides formed by nonribosomal peptide synthetases are able to self-modify and incorporate D- or other non-proteinogenic amino acids without the need for epimerization, and cyclization takes place as part of this process through macrolactonization or macrolactamization.

The surfactin synthetase is considered typical of the linear non-ribosomal peptide synthetases in that it 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) and 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. Iturins are distinctive in that they are synthesised by a non-ribosomal peptide synthetase complexed with a polyketide synthase.

Identification of the genes involved in lipopeptide synthesis in different organisms ('genome mining') together with an understanding of their organization within the genome holds immense promise for the discovery of new antibiotic lipopeptides. Hopefully, functional manipulation of genes will lead to the development of new biologically active molecules.

Recommended Reading

Further information on bacterial proteolipids is available at a dedicated web site -

Lipid listings Credits/disclaimer Updated: October 3rd, 2019 Author: William W. Christie LipidWeb icon