Quantitative assay of Salmonella adherence to intestinal epithelial cells: A new method for assessing novel intervention products

Alwan, A.; Deignan, Tina; O’Sullivan, M.; Kelly, J; O’Farrelly, Cliona

doi:10.1016/s0167-7012(98)00052-9

Cited by 21 publications

(17 citation statements)

References 24 publications

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Section: Methodsmentioning

confidence: 99%

“…Porcine small intestine epithelial cells were isolated and prepared as described previously (2,40). Microscopic examination of the adherence of F18 fimbrial E. coli to jejunal epithelial cells was performed essentially as described by Alwan et al (2). In order to obtain a semiquantitative estimation of the adhesiveness of E. coli cells, the number of bacteria adhering to 50 randomly chosen epithelial cells was counted after different incubation times (10,30, and 60 min).…”

Section: Methodsmentioning

confidence: 99%

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Mapping the Binding Domain of the F18 Fimbrial Adhesin

et al. 2003

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F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mapping the Binding Domain of the F18 Fimbrial Adhesin

et al. 2003

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“…Binding of bacteria to HT29 cells was determined according to published methods (Alwan et al, 1998). Briefly, 100 ml of bacterial suspension (100 bacteria/cell) was added in triplicate to HT29 cells grown in 96-well plates.…”

Section: Bacterial Binding Assaymentioning

confidence: 99%

Differential expression of gastric MUC5AC in colonic epithelial cells: TFF3-wired IL1 β/Akt crosstalk-induced mucosal immune response againstShigella dysenteriaeinfection

Raja

Murali

Devaraj

et al. 2012

Journal of Cell Science

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SummaryAn understanding of the signaling mechanism(s) that regulate the differential expression of gastric mucin MUC5AC in colonic epithelial cells would contribute significantly to investigations of its role in colonic mucosa infected with the bacterial pathogen Shigella dysenteriae. Here we show that S. dysenteriae-induced expression of interleukin-1b upregulates MUC2 expression and the differential expression of MUC5AC. Differential expression of MUC5AC involves crosstalk between interleukin-1b and Akt, whereby the trefoil factor family peptide TFF3 activates Akt by phosphorylation of EGFR. TFF3 also downregulates E-cadherin expression, causing accumulation of b-catenin in the cytosol. Phosphorylation of GSK-3b (inactivated) by activated Akt inhibits ubiquitylation of b-catenin, leading to its nuclear translocation, which then induces the expression of MUC5AC and cyclin D1. Accumulation of cyclin D1 alters the cell cycle, promoting cell survival and proliferation. Human colon HT29MTX cells, which overexpress MUC5AC, were resistant to adherence and invasion of S. dysenteriae when compared with other mucin-secreting HT29 cell types. Thus, during infection with S. dysenteriae, crosstalk between interleukin-1b and Akt wired by TFF3 induces expression of MUC5AC in colonic epithelial cells. Differentially expressed gastric MUC5AC aids in mucosal clearance of S. dysenteriae, inhibiting adherence and invasion of the pathogen to colonic epithelial cells, which protects the host.

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“…Epithelial cells were isolated from the small intestine (segments of ileum and jejunum) of an 8-week-old pig, and phase-contrast microscopic examination of the adherence of F18 fimbrial E. coli to epithelial cells was performed essentially as described by Alwan et al (1). To obtain a semiquantitative estimation of the level of adhesion, the number of bacteria adhering to 15 randomly chosen epi-thelial cells was counted.…”

mentioning

confidence: 99%

Characterization of the Adhesin of Escherichia coli F18 Fimbriae

et al. 2001

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Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E. coli adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.The operons of many fimbrial adhesins of Escherichia coli are well characterized (4). They contain genes coding for the major subunit protein, molecular chaperone and usher proteins, minor subunits, adhesin, and proteins of unknown function (4,11,12). The genes involved in the biosynthesis of F18 fimbria have been only partially described (5, 6). The major protein of the F18 fimbria, FedA, is not sufficient for recognizing the F18 receptor (5). Two additional genes from the fed gene cluster, fedE and fedF, have been described as essential for fimbrial adhesion and fimbrial length (6). However, so far it has not been possible to assess F18 adhesion function with regard to either of the two gene products.In this study, we sequenced the unknown region of the E. coli fed gene cluster and produced and purified FedF and FedE as fusion proteins with maltose binding protein (MBP) for raising antisera for adhesion studies. Furthermore, using indirect immunofluorescence microscopy and adhesion inhibition tests, we have characterized the FedF proteins as the adhesin of F18 fimbriae.Sequencing of the plasmid pIH120. The entire gene cluster encoding E. coli F18 fimbria was sequenced from the plasmid pIH120 (6) with an ABI 310 sequencer according to the manual of the manufacturer (PE Applied Biosystems). pIH120 was transferred into an E. coli HB101 host, resulting in strain ERF2055. Sequence analyses revealed that the fed gene cluster is composed of five genes. The gene coding for the major protein of F18 fimbria (fedA) and the genes encoding two minor proteins (fedE and fedF) were described earlier (5, 6). Two additional open reading frames were found between fedA and fedE and were designated fedB and fedC. FedB showed the highest similarity (83% identity) to the AfrB protein (AAC28316) from E. coli RDEC-1 (Fig. 1) and significant homology to other usher proteins involved in the biosynthesis of microbial pili (3). The second open reading frame (fedC) overlapped the 3Ј end of fedB, and its product had high identity (82%) with the periplasmic chaperone AfrC (AAC228317) from E. coli RDEC-1. Both FedB and FedC possess a predicted signal peptide for transmembrane secretion with a putative cleavage site for a signal peptidase between amino acids 23 and 24. The calculated molecular masses of the mature FedB and FedC are 86,001 and 23,418 Da, respectively. The fedF gene was also PCR cloned and sequenced from a Finnish E. coli O141 isolate (data not shown) and found to have 99.6% identity with the fedF derived from pIH120. In addition to the previously reported transcription terminator, located downstream of fedA (5), an inverted repeat (⌬G of Ϫ17.3 kcal mol Ϫ1 ) for th...

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Quantitative assay of Salmonella adherence to intestinal epithelial cells: A new method for assessing novel intervention products

Cited by 21 publications

References 24 publications

Mapping the Binding Domain of the F18 Fimbrial Adhesin

Mapping the Binding Domain of the F18 Fimbrial Adhesin

Differential expression of gastric MUC5AC in colonic epithelial cells: TFF3-wired IL1 β/Akt crosstalk-induced mucosal immune response againstShigella dysenteriaeinfection

Characterization of the Adhesin of Escherichia coli F18 Fimbriae

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