Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+Escherichia coli on intestinal cells.
BACKGROUND
The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698Tis able to reduce the enterotoxigenic F4+Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698Tprotects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698Tcell wall components in the protection against F4+ETEC infection using the intestinal Caco-2 cell line.


METHODS
Purified cell wall fragments (CWF) from L. amylovorus DSM 16698Twere used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4+ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4+ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4+ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot.


RESULTS
We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4+ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4+ETEC, inhibited membrane damage and prevented NF-kB activation.


CONCLUSIONS
The results indicate that the various components of L. amylovorus DSM 16698Tcell wall may counteract the damage caused by F4+ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4+ETEC infection. U-CWF are not able to defend the cells when they are infected with F4+ETEC but may activate protective mechanisms before pathogen infection.
CREA-NUT, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Food and Nutrition Research Center, Via Ardeatina 546, 00178, Rome, Italy. marianna.roselli@crea.gov.it.
Marianna
M
Roselli
CREA-NUT, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Food and Nutrition Research Center, Via Ardeatina 546, 00178, Rome, Italy.
Alberto
A
Finamore
Department of Veterinary Biosciences, Division of Microbiology and Epidemiology, University of Helsinki, P.O. Box 66, 00014, Helsinki, Finland.
Ulla
U
Hynönen
Department of Veterinary Biosciences, Division of Microbiology and Epidemiology, University of Helsinki, P.O. Box 66, 00014, Helsinki, Finland.
Airi
A
Palva
CREA-NUT, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Food and Nutrition Research Center, Via Ardeatina 546, 00178, Rome, Italy.
Elena
E
Mengheri
Cell wall; L. amylovorus; Membrane damage; NF-kB activation; S-layer proteins
J Appl Microbiol. 2002;92(3):396-403
11872114
J Microbiol Biotechnol. 2014 Aug;24(8):1051-8
24786530
J Appl Bacteriol. 1995 Aug;79(2):230-6
7592119
Gastroenterology. 2001 Sep;121(3):580-91
11522742
PLoS One. 2015 Apr 27;10(4):e0125717
25915861
J Appl Microbiol. 2014 Aug;117(2):485-97
24807045
BMC Microbiol. 2014 Jul 28;14:199
25070625
Anim Health Res Rev. 2005 Jun;6(1):17-39
16164007
Am J Physiol Gastrointest Liver Physiol. 2014 Jun 1;306(11):G1021-32
24742987
Br J Nutr. 2006 Jun;95(6):1177-84
16768842
J Biol Chem. 2007 Apr 13;282(15):11221-9
17287208
PLoS One. 2014 Apr 14;9(4):e94891
24733511
Curr Nutr Food Sci. 2013 May 1;9(2):99-107
24353483
Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19474-9
19047644
Gut. 2003 Mar;52(3):439-51
12584232
Nat Clin Pract Gastroenterol Hepatol. 2005 Sep;2(9):416-22
16265432
Antonie Van Leeuwenhoek. 2011 Jun;100(1):43-53
21327475
J Allergy Clin Immunol. 2009 Jul;124(1):3-20; quiz 21-2
19560575
Proc Natl Acad Sci U S A. 2011 Mar 15;108 Suppl 1:4623-30
21282652
Appl Microbiol Biotechnol. 2013 Jun;97(12):5225-43
23677442
Genes Nutr. 2011 Aug;6(3):261-74
21499799
Microbiology. 2013 Nov;159(Pt 11):2269-82
24002751
Biomed Res Int. 2015;2015:505878
25793197
J Immunol. 2010 Apr 1;184(7):3505-13
20190136
Cell Biol Toxicol. 2005 Jan;21(1):1-26
15868485
J Dairy Res. 2009 Feb;76(1):111-6
19121231
EMBO J. 2015 Apr 1;34(7):881-95
25666591
Microbiology. 2011 Sep;157(Pt 9):2639-46
21700664
Proc Natl Acad Sci U S A. 2012 Jun 26;109(26):10462-7
22689992
Appl Environ Microbiol. 2013 Feb;79(4):1221-31
23220964
Nutr Res Rev. 2010 Jun;23(1):37-46
20403231
Int J Syst Evol Microbiol. 2006 Jan;56(Pt 1):29-32
16403862
FEMS Microbiol Ecol. 2008 Dec;66(3):599-607
18537838
Nutr Rev. 2009 Sep;67(9):546-50
19703261
Int J Food Microbiol. 2007 Sep 30;118(3):264-73
17719671
J Nutr. 2007 Dec;137(12):2709-16
18029488
Cell Microbiol. 2007 Feb;9(2):356-67
16925785
BMC Microbiol. 2008 Oct 01;8:165
18828902
J Cell Sci. 2000 Mar;113 ( Pt 6):985-96
10683147
Am J Physiol Gastrointest Liver Physiol. 2000 Nov;279(5):G851-7
11052980
Vet Microbiol. 2007 Oct 6;124(3-4):264-73
17544232
PLoS One. 2009;4(5):e5601
19440307