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Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.

ABSTRACT: Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY(458)) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF(U), and N-WASP form a complex, but neither EspF(U) nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF(U) identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF(U), and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY(458) sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.

SUBMITTER: Vingadassalom D

PROVIDER: S-EPMC2672544 | biostudies-other | 2009 Apr

REPOSITORIES: biostudies-other

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Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.

Vingadassalom Didier D Kazlauskas Arunas A Skehan Brian B Cheng Hui-Chun HC Magoun Loranne L Robbins Douglas D Rosen Michael K MK Saksela Kalle K Leong John M JM

Proceedings of the National Academy of Sciences of the United States of America 20090406 16

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment ...[more]

PMID: 19366662

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Project description:Two lineages of enterohemorrhagic (EHEC) Escherichia coli O157:H7 (EDL933, Stx1+ and Stx2+) and 86-24 (Stx2+) were investigated in regards to biofilm formation on an abiotic surface. Strikingly, EDL933 strain formed a robust biofilm while 86-24 strain formed no biofilm on either a polystyrene plate or a polyethylene tube. To identify the genetic mechanisms of different biofilm formation in two EHEC strains, DNA microarrays were first performed and phenotypic assays were followed. In the comparison of the EDL933 strain versus 86-24 strain, genes (csgBAC and csgDEFG) involved in curli biosynthesis were significantly induced while genes (trpLEDCB and mtr) involved in indole signaling were repressed. Additionally, a dozen of phage genes were differentially present between two strains. Curli assays using a Congo red plate and scanning electron microscopy corroborate the microarray data as the EDL 933 strain produces a large amount of curli, while 86-24 forms much less curli. Also, the indole production in the EDL933 was 2-times lower than that of 86-24. It was known that curli formation positively regulates and indole negatively regulates biofilm formation of EHEC. Hence, it appears that less curli formation and high indole production in the 86-24 strain are majorly responsible for no biofilm formation. For the microarray experiments, E. coli O157:H7 EDL933 and 86-24 were inoculated in 25 ml of LB in 250 ml shake flasks with overnight cultures that were diluted 1:100. Cells were shaken at 250 rpm and 37°C for an absorbance of 4.0 at 600 nm. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).

Dataset Information

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.

Publications

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.

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