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

Tir 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 Tyr474, 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 Tyr474 phosphorylation to transduce signals.

Functional analysis of EspB from enterohaemorrhagic Escherichia coli.

In enterohaemorrhagic Escherichia coli (EHEC), the type III secretion protein EspB is translocated into the host cells and plays an important role in adherence, pore formation and effector translocation during infection. The secretion domain of EspB has been mapped previously. To define the other functional determinants of EspB, several plasmids encoding different fragments of EspB were created and analysed to see which of them lost the functions of the full-length molecule. One finding was that residues 118-190 of EspB were required for both efficient translocation of EspB and interaction of EspB with EspA. Additionally, the segment consisting of residues 217-312 was necessary for bacterial adherence. Furthermore, a predicted transmembrane domain (residues 99-118) was found to be critical for EHEC to cause red blood cell haemolysis, presumably by forming pores in the cell membrane. The same segment was also important for actin accumulation induced beneath the bacterial-attachment site. Taken together, these data indicate that the EspB protein (312 residues in total) has functions associated with its different regions. These regions may interact with each other or with other components of the type III system to orchestrate the intricate actions of EHEC during infection.

Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7.

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118-190 or residues 191-282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.