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Tolaasin I-II
General info
Chemical properties
Introduction
Tolaasin, a cyclic lipodepsipeptide (CLiP) produced by Pseudomonas tolaasii, is the causal agent for brown blotch disease in mushrooms. (Wong, 1979) The CLiP features 18 amino acids, whereby the N-terminus is capped by a (3R)-hydroxy fatty acid moiety. The C terminus forms a macrocycle by formation of an ester bond to a side chain hydroxyl function.
Tolaasin I is the namesake CLiP of the Tolaasin (18:5) group that consists of tolaasin I-II (Nutkins, 1991), tolaasin A-E (Bassarello, 2004), tolaasin F (Scherlach, 2013) and sessilin (D’Aes, 2014).
Tolaasin has undergone the most extensive testing among all Pseudomonas CLiPs, particularly with regards to its antimicrobial properties against fungi and both Gram-positive and Gram-negative bacteria. (Geudens, 2018)
Chemical structure
The structure of tolaasin was elucidated in 1991 by means of mass spectrometry, amino acid sequencing and 1H NMR spectroscopy. Tolaasin I is a relatively large CLiP, containing 18 amino acids, 5 of which form a lactone macrocycle. The peptide cyclization involves an ester bond between the C-terminus and the alcohol side-chain function of D-aThr14. Unnatural amino acids, such as homoserine (Hse), 2,4-diaminobutyric acid (Dab) and 2,3-didehydroaminobutyric acid (Dhb) are common in the lipopeptides of the Tolaasin group, while they do not occur in the Viscosin, Orfamide or Amphisin group, indicating a more complex assembly process. The N-terminus of tolaasin is capped by a 3R-hydroxy octanoic acid (3R-OH C8:0). Its stereochemistry was determined by means of chiral gas chromatography. Summarizing, the sequence of tolaasin I is 3-OH C8:0 – zDhb – D-Pro – D-Ser – D-Leu – D-Val – D-Ser – D-Leu – D-Val – L-Val – D-Gln – L-Leu – D-Val – zDhb – D-aThr – L-Ile – L-Hse – D-Dab – L-Lys. The structure bears two positive charges (Dab, Lys) at physiologic pH.
P. tolaasii also produces a minor tolaasin I-like compound, that features a Gly16 instead of L-Hse16. This minor compound was named tolaasin II.
Structurally speaking, tolaasin is a peculiar Pseudomonas CLiP, sitting on the edge of certain structural features. Pseudomonas CLiPs with less than 18 amino acids are typically amphipathic (featuring both hydrophobic and polar amino acids throughout the structure) and negatively charged, while larger CLiPs (>18 amino acids) are hydrophobic (mostly composed of hydrophobic amino acids) and positively charged. Strikingly, tolaasin features properties of both, being amphipathic and positively charged.
Three-dimensional structure
Larger CLiPs, such as tolaasin, are mostly unstructured in aqueous environments. However, while the long exocyclic chain is unstructured in water, CD and NMR studies shows it forms a left-handed α-helix in the presence of SDS micelles in water. (Jourdan, 2003) These conditions can be considered as simple but suitable mimics for prokaryotic membranes. Of note, the left-handedness of the exocyclic helix imposed by the majority of D-amino acids is not significantly perturbed by the presence of two L-amino acids and both zDhbs. The 13-residue helix is ended by the protruding 5-residue macrocycle giving rise to the so-called ‘golf club’ motif. The distribution over the 3D structure of the non-polar and polar side-chains along the sequence generates an amphipathic surface, as expected for a membrane perturbing peptide.
Biological activity
More than 100 years ago, the Gram-negative Pseudomonas tolaasii Paine bacterium was revealed as being the organism responsible for causing brown-blotch disease on cultivated mushrooms, effectively making these unappealing and ruining their consumption. Since then, its detrimental effect on agricultural crops has also been reported for other crops, including strawberries, cauliflower and tobacco. The bacterium is endemic to the compost ground used for cultivation where it is non-pathogenic. Under the influence of multifactorial conditions where temperature and relative humidity are known to play a decisive role, P. tolaasii switches to a pathogenic state, where it secretes several virulence factors, i.e. secondary metabolites that effect the colonization and infection at the molecular level. Tolaasin, an 18 residue cyclic lipopeptide, is the main virulence factor of P. tolaasii. Upon secretion by the bacterium, it damages the mushroom cellular membranes, thereby initiating a series of intracellular events that are well documented and ultimately lead to formation of pits in the mushroom caps with brown colored discoloration resulting from the production of melanin. (Soler-Rivas, 1999) In addition to antifungal activity, activity against both Gram-positive and Gram-negative bacteria has been described. (Geudens, 2018)
Genomic characterization
Tolaasin produced by so-called Non-Ribosomal Peptide Synthetases (NRPSs). These are large modular enzymes, whereby each module consists of several domains. Each domain has a dedicated function for the introduction of a single amino acid in the growing (lipo)peptide chain. Consequently, the number of modules in the NRPS is equal to the number of amino acids (co-linearity rule). In case of Pseudomonas, NRPSs can also readily introduce D-amino acids, through the action of a dual activity E/C domain that epimerizes an amino acid and forms a peptide bond with the following amino acid in the sequence. The unnatural amino acids in the tolaasin structure are not recruited from the medium. Rather, L-threonine acts as a precursor for 2,3-dehydroaminobutyric acid while homoserine and 2,4-diaminobutyric originate from L-aspartic acid. (Bender, 1999).
References
Bassarello, et al. “Tolaasins A-E, five new lipodepsipeptides produced by Pseudomonas tolaasii.” Journal of Natural Products67, 5 (2004) https://doi.org/10.1021/np0303557.
Bender, et al. “Pseudomonas syringae phytotoxins: Mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.” Microbiology and Molecular Biology Reviews63, 2 (1999).
D’Aes, et al. “To settle or to move? The interplay between two classes of cyclic lipopeptides in the biocontrol strain Pseudomonas CMR12a.” Environmental Microbiology16, 7 (2014): https://dx.doi.org/10.1111/1462-2920.12462.
Geudens, et al. “Cyclic lipodepsipeptides from Pseudomonas spp. – Biological Swiss-Army Knives.” Frontiers in Microbiology9, 1867 (2018): https://dx.doi.org/10.3389/fmicb.2018.01867.
Hutchison, et al. “Evidence for the involvement of the surface active properties of the extracellular toxin tolaasin in the manifestation of brown blotch disease symptoms by Pseudomonas tolaasii on Agaricus bisporus.” Physiological and Molecular Plant Pathology42, 5 (1993): https://dx.doi.org/10.1016/S0885-5765(05)80013-X.
Jourdan, et al. “A left-handed alfa-helix containing both L- and D-amino acids: The solution structure of the antimicrobial lipodepsipeptide Tolaasin.” Proteins: Structure, Function and Genetics52 (2003).
Mortishire-Smith, et al. “Determination of the structure of an extracellular peptide produced by the mushroom Saprotroph Pseudomonas reactans.” Tetrahedron47, 22 (1991): https://dx.doi.org/10.1016/s0040-4020(01)80877-2.
Nutkins, et al. “Structure determination of tolaasin, an extracellular lipodepsipeptide produced by the mushroom pathogen, Pseudomonas tolaasii Paine.” Journal of the American Chemical Society113, 7 (1991) https://doi.org/10.1021/ja00007a040.
Scherlach, et al. “Biosynthesis and mass spectrometric imaging of tolaasin, the virulence factor of brown blotch mushroom disease.” ChemBioChem14, 18 (2013): https://dx.doi.org/10.1002/cbic.201300553.
Soler-Rivas, et al. “Biochemical and physiological aspects of brown blotch disease of Agaricus bisporus.” FEMS Microbiology Reviews23, 5 (1999): https://dx.doi.org/10.1111/j.1574-6976.1999.tb00415.x.
Wong, et al. “Identification of Pseudomonas tolaasi: the white line in agar and mushroom tissue block rapid pitting tests.” Journal of Applied Bacteriology47 (1979).