Comparison of Tir from enterohemorrahgic and enteropathogenic Escherichia coli strains: two homologues with distinct intracellular properties

Abstract

SummaryTir of enteropathogenic Escherichia coli (EPEC) or enterohemorrahgic E. coil (EHEC) is translocated by a type III secretion system to the host cell membranes where it serves as a receptor for the binding of a second bacterial membrane protein. In response to the binding, EPEC Tir is phosphorylated at Tyr 474 , and this phosphorylation is necessary for the signaling of pedestal formation. Tir of EHEC has no equivalent phosphorylation site but it is similarly needed for cytoskeleton rearrangement. How these two Tir molecules achieve their function by apparently different mechanisms is not completely clear. To examine their intrinsic differences, the two Tirs were expressed in HeLa cells and compared. Actin in complexes could be pelleted down from the lysate of cells expressing EHEC Tir but not EPEC Tir. By immunostaining, neither Tir molecule was found in phosphorylated state. In the cytoplasm, EHEC Tir was frequently found in fibrous structures whereas EPEC Tir was observed completely in a diffusive form. The determinant critical for the EHEC Tir fibrous formation was mapped to the C-terminal region of the molecule that deviates from the EPEC counterpart. This region may play a role in taking an alternative route different from Tyr 474 phosphorylation to transduce signals.

Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspFU-mediated actin assembly and harbours N-terminal sequences that influence pedestal length

“…Whereas EPEC Tir exhibits virtually no colocalization with F‐actin following transfection, EHEC Tir prominently colocalizes with endogenous cell surface projections, suggesting that EHEC Tir possesses an inherently greater ability to associate with F‐actin than its EPEC counterpart. EHEC Tir, but not EPEC Tir, is also capable of triggering actin aggregation into abnormally thick cables in the cytoplasm of a subset of transfected cells, similar to results from a recent study (Chuang et al ., 2006). These activities of EHEC Tir appear to involve the N‐ or C‐terminal domains, as constructs containing either one of these cytoplasmic segments are capable of colocalizing with F‐actin and causing filament aggregation (Fig.…”

“…In addition, a small fraction ( < 10%) of cells expressing high levels of Tir caused aggregation of F-actin into structures resembling abnormally thick and bundled cables that partially colocalized with TirFL ( Fig. S1), similar to observations presented in an independent study examining transfected Tir (Chuang et al ., 2006). These results are not simply an artefact of overexpressing a bacterial protein in mammalian cells, because HA-tagged EPEC Tir did not colocalize with F-actin or trigger ectopic actin aggregation in cells transfected in parallel (see Fig.…”

Effect of combination of taurine and azelaic acid on antimelanogenesis in murine melanoma cells

“…Ampicillin (100 μg/ml), cholorampenical (34 μg/ml), tetracycline (10 μg/ml) or kanamycin (50 μg/ml) was added in the media when needed. Mutant strains Δ tir , Δ espB and Δ grlA have been previously described [16]. …”

Vignette for V13N1 issue

“…EHEC Tir was frequently found in fibrous structures whereas EPEC Tir was observed completely in a diffuse form. Tir fibrous formation was mapped to the C-terminal region of EHEC Tir that deviates from the EPEC counterpart, which utilizes an alternative route different from Tyr474 phosphorylation to transduce signals [15].…”