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Identification of the host cell intimin receptor

"Attaching and Effacing" (A/E) activity of AEEC requires a bacterial outer membrane protein called intimin that mediates intimate adhesion between the bacteria and the host epithelium potential interaction. The aim of this work package is to identify host-cell intimin receptors using a yeast two-hybrid system.

Major finding during the project:

While remaining extracellular EHEC and EPEC establish direct links with the cytoskeleton of the target epithelial cell leading to formation of actin rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins.

Although binding of intimin to Tir is an essential step in colonisation and disease, large body of evidence point to the possibility that intimin also binds to a receptor encoded by the host cell. In order to identify the putative host intimin receptor, we employed the yeas two hybrid system, an established tool used to screen for protein:protein interactions. To this end, we have cloned eae (encoding intimin) in the yeast two-hybrid bait plasmid and used an epithelial cDNA library that was cloned in the yeast two-hybrid prey plasmid. We used Tir, cloned into the bait plasmid as a positive control. Unfortunately, we did not identify any putative host cell intimin receptor using intimin as the bait clone. However, using Tir as bait we identified cytokeratin 18 as a novel Tir partner protein.

In order to determine if the observed Tir:CK18 protein interaction is relevant to infection, cytoplasmic extracts of infected HeLa cells were immuno-precipitated (IP) with mouse anti-CK18. Specific co-IP of Tir with the CK18 antiserum observed. In a reciprocal experiment Tir antiserum was used to IP soluble cytoplasmic and membrane extracts of WT-infected cells. Probing the IP content with antibodies specific for cytoskeletal components revealed that in addition to CK18, CK8 was co-IP in both soluble cytoplasmic and membrane extracts of WT- infected HeLa cells but not with the tir mutant.

We examined if CK18 is recruited to the pedestals. HeLa and intestinal epithelial INT407 cells were infected with WT bacteria and triple stained with either anti-bacterial/CK18/Arp3 antibodies or with anti-bacterial/CK18/Tir antibodies and examined by confocal microscopy. In both cell lines concentrated CK18 was found to be co-localised with Tir and Arp3 beneath adherent bacteria. CK8 is a partner protein of CK18 in IF expressed by epithelial cells. Moreover, CK8 was co-IP with anti-Tir following infection. Consistent with these, immuno-staining revealed that CK8 is also recruited to the pedestals.

In order to determine the functional significance of recruitment of CK18 to the pedestal, we used the small interfering RNAs, which provide a sequence-specific, post-transcriptional, gene silencing mechanism. HeLa cells were transfected with a CK18-specific RNA duplex, infected with WT EPEC then tripled stained using anti-bacterial/CK18/Arp3 antibodies; Arp3 staining was used as a marker for pedestal formation. Staining for CK18 revealed a transfection efficiency of less than 50%; silencing was confirmed by Western blotting. In transfected cells exhibiting reduced or no CK18 staining, pedestals were observed in 29 +/- 1.5% of the adherent bacteria whereas 63 +/- 3.6% of adherent bacteria were associated with Arp3 in un-transfected cells. This study is the first to implicate intermediate filaments in microfilament reorganisation following EPEC infection.

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