Quorum Sensing

these proteins and activates p38 and p42/44 MAPK cascades,

causing the dissociation of junction complexes and compromising

barrier integrity [8–10]. Additionally, different concentrations of

3O-C 12 -HSL, such as those found in biofilms, modulate epithelial

Caco-2 cell migration [7].

Communication between the microbiota and mammalian cells

of the mucosa is valuable to better understand both microbe–host

interactions and its relevance to human health and disease. By

investigating the effects of theP. aeruginosaautoinducer 3O-C 12 -

HSL on Caco-2 cell monolayers, we can better appreciate the

complex relationship between bacteria and host cells within the

gastrointestinal environment.

2 Materials

1.N-3-Oxo-dodecanoyl-L-homoserine (3O-C 12 -HSL) (C 16 H 27 -

NO 4 , MW 297.4) is synthesized by Peter Konradsson and Lan

Bui (Department of Organic Chemistry, Linko ̈ping University,

Sweden) as described by Chhabra et al. [14]. These synthetic

analogues are structurally and functionally identical to those

obtained fromP. aeruginosacultures. Synthesized 3O-C 12 -

HSL is also commercially available (seeNote 1). The identity

and purity of the synthesized 3O-C 12 -HSL should be verified

by HPLC and its activity as a quorum sensing molecule can be

confirmed using the lux-basedE. coliJM109 pSB1075 biosen-

sor assay described in detail by Surette et al. [15]. 3O-C 12 -HSL

is prepared as a stock solution in 100% dimethylsulfoxide

(DMSO) and diluted with aqueous buffer of choice dictated

by each independent experiment.

2. Cell lysis buffer: RIPA Buffer (1% NP-40, 1% deoxycholic acid
   sodium salt, 0.1% SDS, 150 mM NaCl, 10 mM Tris pH 7.4,
   10 mM EDTA pH 8.0 dissolved in 1
   phosphate-buffered
   saline solution (PBS)) containing 25 U nulcease, 1 mM phe-
   nyl-methyl-sulfonyl-fluoride, 1 mM Na 3 VaO 4 , 25 mM NaF,
   and other protease inhibitors.


3. Blocking buffer (immunofluorescence imaging): 1
   PBS sup-
   plemented with 1% bovine serum albumin (BSA) and 10 mM
   glycine.


4. Blocking buffer (immunoblotting): 5% nonfat milk in 1
   PBS
   pH 7.6, supplemented with 0.18% Tween 20.


5. Primary Antibodies (Abs) for Fluorescence Staining: anti-ZO-
   3 (Thermo Fisher Scientific, Rockford, IL, USA); anti-JAM-A
   (Thermo Fisher Scientific, Rockford, IL, USA); anti-IQGAP1

Autoinducer Effects on Mammalian Cells 215