Abstract
A limited number of bacterial strains usually grown under nutrient limitation secrete rhamnolipids (RLs), which are recorded as virulence factors that are implicated in the pathogenicity of a microorganism. The non-pathogenic T. thermophilus HB8 produces extracellular rhamnolipids (TthRLs) under defined cultivation conditions using sunflower seed oil and sodium gluconate as carbon sources. In particular, the secreted TthRLs have been isolated, purified and identified with ATR–FTIR. Their effects on the cells’ viability were examined when they were supplemented in a culture of human skin fibroblasts. Purified TthRLs triggered a sequence of rapid and pronounced morphological alterations characterized by transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation, rounding up, distortion of nuclei and loss of lamellar processes, and finally disruption of membrane. The addition of TthRLs in the cultured fibroblasts caused cytotoxicity, in contrast to that of rhamnose that stimulated viability, as it was assessed by MTT test. These results revealed that among the constituents of RLs that are implicated in the cytotoxicity, it has to be attributed to the lipidic chain variation and not to the carbohydrate part. TthRLs cytotoxicity on fibroblasts is comparable, and provoked similar effects, to that caused by saponin white, a known surfactant. TthRLs secretion might be a crucial point for the transformation of a non-pathogenic bacterium to a pathogenic one under certain environmental conditions favoring their secretion. RLs secretion in the microorganism’s world might be a general route for the passage in the pathogenicity to ensure their survival under nutrient limitation conditions.
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Abbreviations
- RLs:
-
Rhamnolipids
- TthRLs:
-
Thermus thermophilus rhamnolipids, TthRL1 and TthRL2, Thermus thermophilus rhamnolipids produced in the presence of sunflower seed oil and sodium gluconate, respectively
- HAAs:
-
3-(3-hydroxyalkanoyloxy)alkanoic acids
References
Baumann E, Stoya G, Völkner A, Richter W, Lemke C, Linss W (2000) Hemolysis of human erythrocytes with saponin affects the membrane structure. Acta Histochem 102:21–35
Caiazza NC, Shanks RM, O’Toole GA (2005) Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa. J Bacteriol 187:7351–7361
Chwalek M, Lalun N, Bobichon H, Plé K, Voutquenne-Nazabadioko L (2006) Structure-activity relationships of some hederagenin diglycosides: haemolysis, cytotoxicity and apoptosis induction. Biochim Biophys Acta 1760:1418–1427
El-Mawgoud AMA, Lépine F, Déziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86:1323–1336
Glauert AM, Dingle JT, Lucy JA (1962) Action of saponin on biological cell membranes. Nature 8:953–955
Guerra-Santos L, Käppeli O, Fiechter A (1984) Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. Appl Environ Microbiol 48:301–305
Guerra-Santos L, Kappeli O, Fiechter A (1986) Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. Appl Microbiol Biotechnol 24:443–448
Haruma M, Tanaka M, Sugimoto T, Kojima R, Suzuki Y, Konoshima T et al (1995) Alteration of Na+ permeability in human erythrocytes as studied by 23Na-NMR and inhibition of the kidney Na+, K+-ATPase activities with saponins: interaction of gleditsia saponins with human erythrocyte membranes. Bioorg Med Chem Lett 5(8):827–830
Häussler S, Nimtz M, Domke T, Wray V, Steinmetz I (1998) Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei. Infect Immun 66:1588–1593
Häussler S, Rohde M, von Neuhoff N, Nimtz M, Steinmetz I (2003) Structural and functional cellular changes induced by Burkholderia pseudomallei rhamnolipid. Infect Immun 71:2970–2975
Hori K, Marsudi S, Unno H (2002) Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa. Biotechnol Bioeng 78:699–707
Hostettmann K, Marston A (1995) Saponins, chemistry and pharmacology of natural products. Cambridge Univ. Press, Cambridge
Jacob MC, Favre MJC, Bensa JC (1991) Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry. Cytometry 12:550–558
Jensen PO, Bjarnsholt T, Phipps R, Rasmussen TB, Calum H, Christoffersen L, Moser C, Williams P, Pressler T, Givskov M, Hoiby N (2007) Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. Microbiol Sgm 153:1329–1338
Johnson MK, Boese-Marazzo D (1980) Production and properties of heat-stable extracellular hemolysin from Pseudomonas aeruginosa. Infect Immunol 29:1028–1033
Kipnis E, Sawa T, Wiener-Kronish J (2006) Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect 36:78–91 Review
Koch AK, Kappeli O, Ficher A, Reiser J (1991) Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants. J Bacteriol 173:4212–4219
Lang S, Wullbrandt D (1999) Rhamnose lipids—biosynthesis, microbial production and application potential. Appl Microbiol Biotechnol 51:22–32
Lazdunski A (1998) Les facteurs de virulence de Pseudomonas aeruginosa et leur regulation. Med Mal Infect 28:109–118
Leitermann F, Syldatk C, Hausmann R (2008) Fast quantitative determination of microbial rhamnolipids from cultivation broths by ATR–FTIR spectroscopy. J Biol Eng 2:13
Maier RM, Soberon-Chavez G (2000) Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl Microbiol Biotechnol 54:625–633
Majno G, Joris I (1995) Apoptosis, oncosis and necrosis. An overview of cell death. Am J Pathol 146:3–15
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Mulligan CN, Gibbs BF (1989) Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa. Appl Environ Microbiol 55:3016–3019
Nakamura T, Inoue K, Nojima S, Sankawa U, Shoji J, Kawasaki T et al (1979) Interaction of saponins with red blood cells as well as with the phosphatidylcholine liposomal membranes. J Pharm Dyn 2:374–382
Nietzsche M, Costa S, Contiero J (2005) Rhamnolipid surfactants: an update on the general aspects of these remarkable biomolecules. Biotechnol Prog 21:1593–1600
Ochsner UA, Hembach T, Fiechter A (1996) Production of rhamnolipid biosurfactants. Adv Biochem Eng Biotechnol 53:89–118
Pantazaki AA, Karagiorgas AA, Liakopoulou-Kyriakides M, Kyriakidis DA (1998) Hyperalkaline and thermostable phosphatase in Thermus thermophilus. Appl Biochem Biotechnol 75:249–259
Pantazaki AA, Tambaka MG, Langlois V, Guerin P, Kyriakidis DA (2003) Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase. Mol Cell Biochem 254:173–183
Pantazaki AA, Dimopoulou MI, Simou OM, Pritsa AA (2010) Sunflower seed oil and oleic acid utilization for the production of rhamnolipids by Thermus thermophilus HB8. Appl Microbiol Biotechnol 88:939–951
Passeri A, Lang S, Wagner F, Wray V (1991) Marine biosurfactants, II. Production and characterisation of an anionic trehalose tetraester from the marine bacterium Arthrobacter sp. EK 1. Zeitscrift für Naturfosch 46c:204–209
Pearson JP, Pesci EC, Iglewski BH (1997) Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179:5756–5767
Pesci EC, Pearson JP, Seed PC, Iglewski BH (1997) Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179:3127–3132
Piljac G, Piljac V (1995a) Immunological activity of rhamnolipids. US Patent 5466675
Piljac G, Piljac V (1995b) Pharmaceutical preparation based on rhamnolipid. US Patent 5455232
Ron EZ, Rosenberg E (2001) Natural roles of biosurfactants. Environ Microbiol 3:229–236
Schenk T, Schuphan I, Schmidt B (1995) High-performance liquid chromatographic determination of the rhamnolipids produced by Pseudomonas aeruginosa. J Chromatogr A 693:7–13
Seeman P (1974) Ultrastructure of membrane lesions in immune lysis, osmotic lysis and drug-induced lysis. Fed Proc 33:2116–2124
Segal R, Milo-Goldzweig I (1978) The susceptibility of cholesterol-depleted erythrocytes to saponin and sapogenin hemolysis. Biochim Biophys Acta 512:223–226
Shryock TR, Silver SA, Banschbach MW, Kramer JC (1984) Effect of Pseudomonas aeruginosa rhamnolipid on human neutrophil migration. Curr Microbiol 10:323–328
Siegmund I, Wagner F (1991) New method for detecting rhamnolipids excreted by Pseudomonas aeruginosa species during growth on minimal agar. Biotechnol Tech 5:265–268
Smith RS, Iglewski BH (2003) P. aeruginosa quorum-sensing systems and virulence. Curr Opin Microbiol 6:56–60
Smith TJ, Wang HS, Hogg MG, Henrikson RC, Keese CR, Giaever I (1994) Prostaglandin E2 elicits a morphological change in cultured orbital fibroblasts from patients with Graves’s ophthalmopathy. Proc Natl Acad Sci USA 91:5094–5098
Soberón-Chávez G (2004) Biosynthesis of rhamnolipids. In: Ramos JL (ed) Pseudomonas. Kluwer Academic/Plenum Publishers, New York, pp 173–189
Soberón-Chávez G, Lépine F, Déziel E (2005) Production of rhamnolipids by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 68:718–725
Soell M, Lett E, Holveck F, Scholler M, Wachsmann D, Klein JP (1995) Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-release. J Immunol 154:851–860
Sotirova AV, Spasova DI, Galabova DN, Karpenko E, Shulga A (2008) Rhamnolipid-biosurfactant permeabilizing effects on gram-positive and gram-negative bacterial strains. Curr Microbiol 56:639–644
Spoecker S, Wray V, Nimtz M, Lang S (1999) Glycolipids of the smut ungus Ustilago maydis from cultivation on renewable resources. Appl Microbiol Biotechnol 51:33–39
Stipcevic T, Piljac T, Isseroff RR (2005) Di-rhamnolipid from Pseudomonas aeruginosa displays differential effects on human keratinocyte and fibroblast cultures. J Dermatol Sci 40:141–143
Syldatk C, Lang S, Matulovic V, Wagner F (1985) Production of four interfacial active rhamnolipids from n-alkanes or glycerol by resting cells of Pseudomonas species DSM 2847. Z Naturforsch 40c:61–67
Takechi M, Tanaka Y (1995) Haemolytic time course differences between steroid and triterpenoid saponins. Planta Med 61:76–77
Tamariz E, Grinnell F (2002) Modulation of fibroblast morphology and adhesion during collagen matrix remodeling. Mol Biol Cell 13:3915–3929
Trouillas P, Corbière C, Liagre B, Duroux JL, Beneytout JL (2005) Structure–function relationship for saponin effects on cell cycle arrest and apoptosis in the human 1547 osteosarcoma cells: a molecular modeling approach of natural molecules structurally close to diosgenin. Bioorg Med Chem 13:1141–1149
Van Gennip M, Christensen LD, Alhede M, Phipps R, Jensen PO, Christophersen L, Pamp SJ, Moser C, Mikkelsen PJ, Koh AY, Tolker-Nielsen T, Pier GB, Hoiby N, Givskov M, Bjarnsholt T (2009) Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear leukocytes. Apmis 117:537–546
Voutquenne L, Lavaud C, Massiot G, LeMen-Olivier L (2002) Structure–activity relationships of haemolytic saponins. Pharm Biol 40:253–262
Wang F, Lee SY (1997) Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli. Appl Environ Microbiol 63:4765–4769
Winzer K, Williams P (2001) Quorum sensing and the regulation of virulence gene expression in pathogenic bacteria. Int J Med Microbiol 291:131–143
Zulianello L, Canard C, Köhler T, Caille D, Lacroix JS, Meda P (2006) Rhamnolipids are virulence factors that promote early infiltration of primary human airway epithelia by Pseudomonas aeruginosa. Infect Immun 74:3134–3147
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Pantazaki, A.A., Choli-Papadopoulou, T. On the Thermus thermophilus HB8 potential pathogenicity triggered from rhamnolipids secretion: morphological alterations and cytotoxicity induced on fibroblastic cell line. Amino Acids 42, 1913–1926 (2012). https://doi.org/10.1007/s00726-011-0917-z
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DOI: https://doi.org/10.1007/s00726-011-0917-z