Quorum Sensing pp 213-225 | Cite as

Assessing Pseudomonas aeruginosa Autoinducer Effects on Mammalian Epithelial Cells

  • Jake Everett
  • Rebecca Gabrilska
  • Kendra P. Rumbaugh
  • Elena Vikström
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1673)

Abstract

The human mucosal environment in the gut is rich with interactions between microbiota and mammalian epithelia. Microbes such as the Gram-negative bacterium Pseudomonas aeruginosa may use quorum sensing to communicate with other microorganisms and mammalian cells to alter gene expression. Here, we present methodologies to evaluate the effects of P. aeruginosa N-(3-oxo-dodecanoyl)-l-homoserine lactone (3O-C12-HSL) on Caco-2 cell monolayers. First, we describe a method for assessing barrier function and permeability of epithelial cells when exposed to 3O-C12-HSL by measuring transepithelial electrical resistance (TER) and paracellular flow using fluorescently labeled dextran. Secondly, we detail methods to investigate the effect of 3O-C12-HSL on protein–protein interactions of epithelial junction proteins. Lastly, we will detail imaging techniques to visualize Caco-2 barrier disruption following exposure to 3O-C12-HSL through the use of confocal laser scanning microscopy (CLSM) and a super resolution technique, stimulated emission depletion (STED) microscopy, to achieve nanoscale visualization of Caco-2 monolayers.

Key words

Host pathogen interaction Pseudomonas aeruginosa Quorum sensing Acyl homoserine lactone Transepithelial electrical resistance Paracellular permeability Cell junction IQGAP1 Imaging 

References

  1. 1.
    Holm A, Vikstrom E (2014) Quorum sensing communication between bacteria and human cells: signals, targets, and functions. Front Plant Sci 5:309CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Schuster M, Lostroh CP, Ogi T, Greenberg EP (2003) Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185:2066–2079CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH (2003) Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185:2080–2095CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Shreiner AB, Kao JY, Young VB (2015) The gut microbiome in health and in disease. Curr Opin Gastroenterol 31:69–75CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Joe GH, Andoh M, Nomura M, Iwaya H, Lee JS, Shimizu H et al (2014) Acyl-homoserine lactones suppresses IEC-6 cell proliferation and increase permeability of isolated rat colon. Biosci Biotechnol Biochem 78:462–465CrossRefPubMedGoogle Scholar
  6. 6.
    Eum SY, Jaraki D, Bertrand L, Andras IE, Toborek M (2014) Disruption of epithelial barrier by quorum-sensing N-3-(oxododecanoyl)-homoserine lactone is mediated by matrix metalloproteinases. Am J Physiol Gastrointest Liver Physiol 306:G992–G1001CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Karlsson T, Turkina MV, Yakymenko O, Magnusson KE, Vikstrom E (2012) The Pseudomonas aeruginosa N-acylhomoserine lactone quorum sensing molecules target IQGAP1 and modulate epithelial cell migration. PLoS Pathog 8:e1002953CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Vikstrom E, Bui L, Konradsson P, Magnusson KE (2010) Role of calcium signalling and phosphorylations in disruption of the epithelial junctions by Pseudomonas aeruginosa quorum sensing molecule. Eur J Cell Biol 89:584–597CrossRefPubMedGoogle Scholar
  9. 9.
    Vikstrom E, Bui L, Konradsson P, Magnusson KE (2009) The junctional integrity of epithelial cells is modulated by Pseudomonas aeruginosa quorum sensing molecule through phosphorylation-dependent mechanisms. Exp Cell Res 315:313–326CrossRefPubMedGoogle Scholar
  10. 10.
    Vikstrom E, Tafazoli F, Magnusson KE (2006) Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells. FEBS Lett 580:6921–6928CrossRefPubMedGoogle Scholar
  11. 11.
    Angelis ID, Turco L (2011) Caco-2 cells as a model for intestinal absorption. Curr Protoc Toxicol Chapter 20:Unit20.26. doi:10.1002/0471140856.tx2006s47Google Scholar
  12. 12.
    Hidalgo IJ, Raub TJ, Borchardt RT (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736–749CrossRefPubMedGoogle Scholar
  13. 13.
    Artursson P, Palm K, Luthman K (2001) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev 46:27–43CrossRefPubMedGoogle Scholar
  14. 14.
    Chhabra SR, Harty C, Hooi DS, Daykin M, Williams P, Telford G et al (2003) Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators. J Med Chem 46:97–104CrossRefPubMedGoogle Scholar
  15. 15.
    Surette MG, Miller MB, Bassler BL (1999) Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production. Proc Natl Acad Sci U S A 96:1639–1644CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Katouli M, Ramos NL, Nettelbladt CG, Ljungdahl M, Robinson W, Ison HM et al (2009) Host species-specific translocation of Escherichia coli. Eur J Clin Microbiol Infect Dis 28:1095–1103CrossRefPubMedGoogle Scholar
  17. 17.
    Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefPubMedGoogle Scholar
  18. 18.
    Blom H, Brismar H (2014) STED microscopy: increased resolution for medical research? J Intern Med 276:560–578CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Jake Everett
    • 1
  • Rebecca Gabrilska
    • 1
  • Kendra P. Rumbaugh
    • 1
  • Elena Vikström
    • 2
  1. 1.Department of SurgeryTexas Tech University Health Sciences CenterLubbockUSA
  2. 2.Department of Clinical and Experimental Medicine, Faculty of Medicine and Health SciencesLinköping UniversityLinköpingSweden

Personalised recommendations