Orthosipon stamineus extract exerts inhibition of bacterial adhesion and chaperon-usher system of uropathogenic Escherichia coli—a transcriptomic study

  • Shabnam Sarshar Beydokhti
  • Christoph Stork
  • Ulrich Dobrindt
  • Andreas HenselEmail author
Applied microbial and cell physiology


Specific recognition and bacterial adhesion to host cells by uropathogenic Escherichia coli (UPEC) are the first steps towards infection of epithelial tissue of the human urogenital system. Therefore, targeting of UPEC virulence factors, relevant for adhesion, is a promising approach for prevention of recurrent urinary tract infections (UTI). A fully characterized plant-derived aqueous extract from the leaves of Orthosiphon stamineus (OWE), a plant traditionally used in clinical practice in Europe and Asia for UTI, has been shown to exert strong antiadhesive effects under in vitro and in vivo conditions. For improved understanding of the underlying mechanisms, transcriptome analysis of OWE-treated UPEC strain UTI89 by Illumina sequencing and cross-validation of these data by qPCR indicated significant downregulation of bacterial adhesins (curli, type 1-, F1C-, and P fimbriae) and of the chaperone-mediated protein folding/unfolding and pilus assembly process; in contrast, flagellar and motility-related genes were upregulated. We conclude that OWE transforms the sessile lifestyle of bacteria into a motile one and therefore disables bacterial attachment to the host cell. Additionally, the extract inhibited gene expression of multiple iron-acquisition systems (ent, fep, feo, fhu, chu, sit, ybt). The present study explains the antiadhesive and anti-infective effect of the plant extract by pinpointing specific biochemical and molecular targets.


Orthosiphon stamineus Adhesion Transcriptome Uropathogenic E. coli Chaperone–usher Iron uptake 



The help of Mr. Michael Holtkamp, Institute of Analytical and Inorganic Chemistry, University of Münster for determination of iron content in the extracts by TXRF is greatly acknowledged.

Funding information

The work of CS and UD was supported by the German Research Foundation (SFB1009, TP B05).

Compliance with ethical standards

Ethical approval

This article does not contain studies with human participants performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10120_MOESM1_ESM.pdf (432 kb)
ESM 1 (PDF 432 kb)


  1. Aberg V, Fällman E, Axner O, Uhlin BE, Hultgren SJ, Almqvist F (2007) Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod. Mol BioSys 3:214–218CrossRefGoogle Scholar
  2. Abraham SN, Miao Y (2015) The nature of immune responses to urinary tract infections. Nat Rev Immunol 15:655–663CrossRefGoogle Scholar
  3. Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, Hultgren SJ (2003) Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301:105–107CrossRefGoogle Scholar
  4. Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. Accessed Aug 2018
  5. Barnett ME, Zolkiewska A, Zolkiewski M (2000) Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains. J Biol Chem 275:37565–37571CrossRefGoogle Scholar
  6. Berger CN, Billker O, Meyer TF, Servin AL, Kansau I (2004) Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC). Mol Microbiol 52:963–983CrossRefGoogle Scholar
  7. Beydokthi SS, Sendker J, Brandt S, Hensel A (2017) Traditionally used medicinal plants against uncomplicated urinary tract infections: hexadecyl coumaric acid ester from the rhizomes of Agropyron repens (L.) P. Beauv. with antiadhesive activity against uropathogenic E. coli. Fitoterapia 117:22–27CrossRefGoogle Scholar
  8. Bien J, Sokolova O, Bozko P (2012) Role of uropathogenic Escherichia coli virulence factors in development of urinary tract infection and kidney damage. Int J Nephrol 2012:681473CrossRefGoogle Scholar
  9. Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman W-H, Pagès F, Trajanoski Z, Galon J (2009) ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25:1091–1093CrossRefGoogle Scholar
  10. Bombarrdelli E, Bonati A, Gabetta B, Mustich G (1972) Flavonoid constituents of Orthosiphonis stamineus Benth. Fitoterapia 35–40Google Scholar
  11. Busch A, Waksman G (2012) Chaperone-usher pathways: diversity and pilus assembly mechanism. Philos Trans R Soc B 367:1112–1122CrossRefGoogle Scholar
  12. Castanié-Cornet M-P, Bruel N, Genevaux P (2014) Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane. Biochim Biophys Acta 1843:1442–1456CrossRefGoogle Scholar
  13. Cegelski L, Marshall GR, Eldridge GR, Hultgren SJ (2008) The biology and future prospects of antivirulence therapies. Nat Rev Microbiol 6:17–27CrossRefGoogle Scholar
  14. Chen SL, Hung C-S, Xu J, Reigstad CS, Magrini V, Sabo A, Blasiar D, Bieri T, Meyer RR, Ozersky P, Armstrong JR, Fulton RS, Latreille JP, Spieth J, Hooton TM, Mardis ER, Hultgren SJ, Gordon JI (2006) Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. Proc Natl Acad Sci U S A 103:5977–5982CrossRefGoogle Scholar
  15. Connell I, Agace W, Klemm P, Schembri M, Mărild S, Svanborg C (1996) Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract. Proc Natl Acad Sci U S A 93:9827–9832CrossRefGoogle Scholar
  16. Dobrindt U, Hacker J (2010) Uropathogens and virulence factors. In: Naber KG, Schaeffer AJ, Heyns CF, Matsumoto T, Shoskes DA, Bherklunc Johansen TE (eds) Urogenital infections. European Association of Urology, Anhem, pp 4–22Google Scholar
  17. European Medicines Agency (2010) Herbal medicines for human use: The official European Union Herbal Monographs of the Comittee on Herbal Medicinal Products (HMPC). Orthosiphonis folium. Accessed 20 Aug 2018
  18. European Medicines Agency EMA (2014) Note for guidance on validation of analytical procedures: text and methodology (CPMP (ICVH/381/95). Accessed 22 Aug 2018
  19. European Scientific Cooperative on Phytotherapy (ed) (2003) ESCOP monographs. Orthosiphonis folium, 2nd edn. Stuttgart, ThiemeGoogle Scholar
  20. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 13:269–284CrossRefGoogle Scholar
  21. Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, Lin J, Minguez P, Bork P, von Mering C, Jensen LJ (2013) STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res 41:15Google Scholar
  22. Fronzes R, Remaut H, Waksman G (2008) Architectures and biogenesis of non-flagellar protein appendages in gram-negative bacteria. EMBO J 27:2271–2280CrossRefGoogle Scholar
  23. Genest O, Hoskins JR, Camberg JL, Doyle SM, Wickner S (2011) Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling. Proc Natl Acad Sci U S A 108:8206–8211CrossRefGoogle Scholar
  24. Green ER, Mecsas J (2016) Bacterial secretion systems: an overview. Microbiol Spectr 4(1).
  25. Greene SE, Hibbing ME, Janetka J, Chen SL, Hultgren SJ (2015) Human urine decreases function and expression of type 1 pili in uropathogenic Escherichia coli. mBio 6:e00820CrossRefGoogle Scholar
  26. Guignot J, Peiffer I, Bernet-Camard MF, Lublin DM, Carnoy C, Moseley SL, Servin AL (2000) Recruitment of CD55 and CD66e brush border-associated glycosylphosphatidylinositol-anchored proteins by members of the Afa/Dr diffusely adhering family of Escherichia coli that infect the human polarized intestinal Caco-2/TC7 cells. Infect Immun 68:3554–3563CrossRefGoogle Scholar
  27. Haiko J, Westerlund-Wikström B (2013) The role of the bacterial flagellum in adhesion and virulence. Biology 2:1242–1267CrossRefGoogle Scholar
  28. Holtkamp M, Elseberg T, Wehe CA, Sperling M, Karst U (2013) Complexation and oxidation strategies for improved TXRF determination of mercury in vaccines. J Anal At Spectrom 28:719CrossRefGoogle Scholar
  29. Hunstad DA, Justice SS (2010) Intracellular lifestyles and immune evasion strategies of uropathogenic Escherichia coli. Ann Rev Microbiol 64:203–221CrossRefGoogle Scholar
  30. Justice SS, Hung C, Theriot JA, Fletcher DA, Anderson GG, Footer MJ, Hultgren SJ (2004) Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc Natl Acad Sci U S A 101:1333–1338CrossRefGoogle Scholar
  31. Kansau I, Berger C, Hospital M, Amsellem R, Nicolas V, Servin AL, Bernet-Camard M-F (2004) Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion. Infect Immun 72:3733–3742CrossRefGoogle Scholar
  32. Krachler AM, Orth K (2013) Targeting the bacteria-host interface: strategies in anti-adhesion therapy. Virulence 4:284–294CrossRefGoogle Scholar
  33. Kumar CMS, Mande SC, Mahajan G (2015) Multiple chaperonins in bacteria - novel functions and non-canonical behaviors. Cell Stress Chaperones 20:555–574CrossRefGoogle Scholar
  34. Lane MC, Lockatell V, Monterosso G, Lamphier D, Weinert J, Hebel JR, Johnson DE, Mobley HLT (2005) Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract. Infect Immun 73:7644–7656CrossRefGoogle Scholar
  35. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Meth 9:357–359CrossRefGoogle Scholar
  36. Malterud KE, Hanche-Olsen IM, Smith-Kielland I (1989) Flavonoids from Orthosiphon spicatus. Planta Med 55:569–570CrossRefGoogle Scholar
  37. Mayer MP, Rüdiger S, Bukau B (2000) Molecular basis for interactions of the DnaK chaperone with substrates. Biol Chem 381:877–885CrossRefGoogle Scholar
  38. Messing J, Thöle C, Niehues M, Shevtsova A, Glocker E, Borén T, Hensel A (2014) Antiadhesive properties of Abelmoschus esculentus (okra) immature fruit extract against Helicobacter pylori adhesion. PLoS One 9:e84836CrossRefGoogle Scholar
  39. Mulvey MA (2002) Adhesion and entry of uropathogenic Escherichia coli. Cell Microbiol 4:257–271CrossRefGoogle Scholar
  40. Mydock-McGrane LK, Hannan TJ, Janetka JW (2017) Rational design strategies for FimH antagonists: new drugs on the horizon for urinary tract infection and Crohn's disease. Expert Opin Drug Discovery 12:711–731CrossRefGoogle Scholar
  41. Nguyen MTT, Awale S, Tezuka Y, Chien-Hsiung C, Kadota S (2004) Staminane- and isopimarane-type diterpenes from Orthosiphon stamineus of Taiwan and their nitric oxide inhibitory activity. J Nat Prod 67:654–658CrossRefGoogle Scholar
  42. Olsén A, Jonsson A, Normark S (1989) Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature 338:652–655CrossRefGoogle Scholar
  43. Pinkner JS, Remaut H, Buelens F, Miller E, Aberg V, Pemberton N, Hedenström M, Larsson A, Seed P, Waksman G, Hultgren SJ, Almqvist F (2006) Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria. Proc Natl Acad Sci U S A 103:17897–17902CrossRefGoogle Scholar
  44. Plançon L, Du Merle L, Le Friec S, Gounon P, Jouve M, Guignot J, Servin A, Le Bouguénec C (2003) Recognition of the cellular beta1-chain integrin by the bacterial AfaD invasin is implicated in the internalization of afa-expressing pathogenic Escherichia coli strains. Cell Microbiol 5:681–693CrossRefGoogle Scholar
  45. Prieto C, de Las Rivas J (2006) APID: agile protein interaction data analyzer. Nucleic Acids Res 34:302CrossRefGoogle Scholar
  46. Qin X, Hu F, Wu S, Ye X, Zhu D, Zhang Y, Wang M (2013) Comparison of adhesin genes and antimicrobial susceptibilities between uropathogenic and intestinal commensal Escherichia coli strains. PLoS One 8:e61169CrossRefGoogle Scholar
  47. Rafsanjany N, Lechtenberg M, Petereit F, Hensel A (2013) Antiadhesion as a functional concept for protection against uropathogenic Escherichia coli: in vitro studies with traditionally used plants with antiadhesive activity against uropathognic Escherichia coli. J Ethnopharmacol 145:591–597CrossRefGoogle Scholar
  48. Rafsanjany N, Sendker J, Lechtenberg M, Petereit F, Scharf B, Hensel A (2015a) Traditionally used medicinal plants against uncomplicated urinary tract infections: are unusual, flavan-4-ol- and derhamnosylmaysin derivatives responsible for the antiadhesive activity of extracts obtained from stigmata of Zea mays L. against uropathogenic E. coli and Benzethonium chloride as frequent contaminant faking potential antibacterial activities? Fitoterapia 105:246–253CrossRefGoogle Scholar
  49. Rafsanjany N, Senker J, Brandt S, Dobrindt U, Hensel A (2015b) In vivo consumption of cranberry exerts ex vivo antiadhesive activity against FimH-dominated uropathogenic Escherichia coli: a combined in vivo, ex vivo, and in vitro study of an extract from Vaccinium macrocarpon. J Agric Food Chem 63:8804–8818CrossRefGoogle Scholar
  50. Saito R, Smoot ME, Ono K, Ruscheinski J, Wang P-L, Lotia S, Pico AR, Bader GD, Ideker T (2012) A travel guide to Cytoscape plugins. Nat Methods 9:1069–1076CrossRefGoogle Scholar
  51. Sakarya S, Ertem GT, Oncu S, Kocak I, Erol N, Oncu S (2003) Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence. FEMS Immunol Med Microbiol 39:45–50CrossRefGoogle Scholar
  52. Sarshar S, Brandt S, Asadi Karam MR, Habibi M, Bouzari S, Lechtenberg M, Dobrindt U, Qin X, Goycoolea FM, Hensel A (2017) Aqueous extract from Orthosiphon stamineus leaves prevents bladder and kidney infection in mice. Phytomed 28:1–9CrossRefGoogle Scholar
  53. Sarshar S, Sendker J, Qin X, Goycoolea FM, Asadi Karam MR, Habibi M, Bouzari S, Dobrindt U, Hensel A (2018) Antiadhesive hydroalcoholic extract from Apium graveolens fruits prevents bladder and kidney infection against uropathogenic E. coli. Fitoterapia 127:237–244CrossRefGoogle Scholar
  54. Scharf B, Sendker J, Dobrindt U, Hensel A (2019) Influence of cranberry extract on Tamm-Horsfall protein in human urine and its antiadhesive activity against uropathogenic Escherichia coli. Planta Med 85:126–138CrossRefGoogle Scholar
  55. Schreiber HL, Spaulding CN, Dodson KW, Livny J, Hultgren SJ (2017) One size doesn’t fit all: unraveling the diversity of factors and interactions that drive E. coli urovirulence. Ann Translat Med 5:28CrossRefGoogle Scholar
  56. Schurch NJ, Schofield P, Gierliński M, Cole C, Sherstnev A, Singh V, Wrobel N, Gharbi K, Simpson GG, Owen-Hughes T, Blaxter M, Barton GJ (2016) How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? RNA 22:839–851CrossRefGoogle Scholar
  57. Selvarangan R, Goluszko P, Popov V, Singhal J, Pham T, Lublin DM, Nowicki S, Nowicki B (2000) Role of decay-accelerating factor domains and anchorage in internalization of Dr-fimbriated Escherichia coli. Infect Immun 68:1391–1399CrossRefGoogle Scholar
  58. Servin AL (2014) Pathogenesis of human diffusely adhering Escherichia coli expressing Afa/Dr adhesins (Afa/Dr DAEC): current insights and future challenges. Clin Microbiol Rev 27:823–869CrossRefGoogle Scholar
  59. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504CrossRefGoogle Scholar
  60. Snel B (2000) STRING. A web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res 28:3442–3444CrossRefGoogle Scholar
  61. Spaulding CN, Hultgren SJ (2016) Adhesive pili in UTI pathogenesis and drug development. Pathogens 5:30CrossRefGoogle Scholar
  62. Spurbeck RR, Stapleton AE, Johnson JR, Walk ST, Hooton TM, Mobley HLT (2011) Fimbrial profiles predict virulence of uropathogenic Escherichia coli strains: contribution of ygi and yad fimbriae. Infect Immun 79:4753–4763CrossRefGoogle Scholar
  63. Svensson A, Larsson A, Emtenäs H, Hedenström M, Fex T, Hultgren SJ, Pinkner JS, Almqvist F, Kihlberg J (2001) Design and evaluation of pilicides: potential novel antibacterial agents directed against uropathogenic Escherichia coli. Chembiochem 2:915–918CrossRefGoogle Scholar
  64. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:52CrossRefGoogle Scholar
  65. Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, Franz M, Grouios C, Kazi F, Lopes CT, Maitland A, Mostafavi S, Montojo J, Shao Q, Wright G, Bader GD, Morris Q (2010) The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 38:20CrossRefGoogle Scholar
  66. Werneburg GT, Thanassi DG (2018) Pili assembled by the chaperone/usher pathway in Escherichia coli and Salmonella. EcoSal Plus 8.
  67. Westerlund B, Kuusela P, Risteli J, Risteli L, Vartio T, Rauvala H, Virkola R, Korhonen TK (1989) The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein. Mol Microbiol 3:329–337CrossRefGoogle Scholar
  68. Whitley D, Goldberg SP, Jordan WD (1999) Heat shock proteins: a review of the molecular chaperones. J Vasc Surg 29:748–751CrossRefGoogle Scholar
  69. Wurpel DJ, Beatson SA, Totsika M, Petty NK, Schembri MA (2013) Chaperone-usher fimbriae of Escherichia coli. PLoS One 8:e52835CrossRefGoogle Scholar
  70. Yuliana ND, Khatib A, Link-Struensee AM, Ijzerman AP, Rungkat-Zakaria F, Choi YH, Verpoorte R (2009) Adenosine A1 receptor binding activity of methoxy flavonoids from Orthosiphon stamineus. Planta Med 75:132–136CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Shabnam Sarshar Beydokhti
    • 1
  • Christoph Stork
    • 2
  • Ulrich Dobrindt
    • 2
  • Andreas Hensel
    • 1
    Email author
  1. 1.Institute of Pharmaceutical Biology and PhytochemistryUniversity of MünsterMünsterGermany
  2. 2.Institute of HygieneUniversity Hospital MünsterMünsterGermany

Personalised recommendations