mpidb The Microbial Protein Interaction Database Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol. Microbiol. (0950-382X) 1999 imex curation Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. jkaper@umaryland.edu 23-Feb-2010: Contacted by JYOTI. Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. jkaper@umaryland.edu Accepted 2009-Not a month!-3 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Mol. Microbiol. (0950-382X) 1999 Only protein-protein interactions imex curation Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol. Microbiol. (0950-382X) 1999 imex curation Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. jkaper@umaryland.edu 23-Feb-2010: Contacted by JYOTI. Only protein-protein interactions ecoli Escherichia coli (strain K12) crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. Mutant strain SE896 was induced to express truncated Tir and His6CesT, treated with DSP (Dithiobis[succinimidyl propionate]) and the His-tagged proteins in cell lysates collected on a Ni2+-NTA column. jkaper@umaryland.edu Accepted 2009-Not a month!-3 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Mol. Microbiol. (0950-382X) 1999 Only protein-protein interactions imex curation Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol. Microbiol. (0950-382X) 1999 imex curation Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. jkaper@umaryland.edu 23-Feb-2010: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. jkaper@umaryland.edu Accepted 2009-Not a month!-3 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Mol. Microbiol. (0950-382X) 1999 Only protein-protein interactions imex curation Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol. Microbiol. (0950-382X) 1999 imex curation Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. jkaper@umaryland.edu 23-Feb-2010: Contacted by JYOTI. Only protein-protein interactions ecoli Escherichia coli (strain K12) crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Elliott SJ., Hutcheson SW., Dubois MS., Mellies JL., Wainwright LA., Batchelor M., Frankel G., Knutton S., Kaper JB. E2348/69 was grown in MEM and treated with the thiol cleavable cross-linker DSP (Dithiobis[succinimidyl propionate]). Antibodies raised against Tir were bound to Protein A-Sepharose beads and mixed with a cytoplasmic preparation obtained from the treated E2348/69 jkaper@umaryland.edu Accepted 2009-Not a month!-3 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Mol. Microbiol. (0950-382X) 1999 Only protein-protein interactions imex curation tir_eco27 Translocated intimin receptor Tir tir Secreted effector protein Tir espE E2348C_3941 protein protein eco27 Escherichia coli O127:H6 (strain E2348/69 / EPEC) MPIGNLGNNVNGNHLIPPAPPLPSQTDGAARGGTGHLISSTGALGSRSLFSPLRNSMADSVDSRDIPGLPTNPSRLAAATSETCLLGGFEVLHDKGPLDILNTQIGPSAFRVEVQADGTHAAIGEKNGLEVSVTLSPQEWSSLQSIDTEGKNRFVFTGGRGGSGHPMVTVASDIAEARTKILAKLDPDNHGGRQPKDVDTRSVGVGSASGIDDGVVSETHTSTTNSSVRSDPKFWVSVGAIAAGLAGLAATGIAQALALTPEPDDPTTTDPDQAANAAESATKDQLTQEAFKNPENQKVNIDANGNAIPSGELKDDIVEQIAQQAKEAGEVARQQAVESNAQAQQRYEDQHARRQEELQLSSGIGYGLSSALIVAGGIGAGVTTALHRRNQPAEQTTTTTTHTVVQQQTGGNTPAQGGTDATRAEDASLNRRDSQGSVASTHWSDSSSEVVNPYAEVGGARNSLSAHQPEEHIYDEVAADPGYSVIQNFSGSGPVTGRLIGTPGQGIQSTYALLANSGGLRLGMGGLTSGGESAVSSVNAAPTPGPVRFV DIP protein B7UM99 original sequence version: 16 19DD08A9BE9251CB cest_eco27 Tir chaperone cesT E2348C_3940 protein protein eco27 Escherichia coli O127:H6 (strain E2348/69 / EPEC) MSSRSELLLDRFAEKIGVGSISFNENRLCSFAIDEIYYISLSDANDEYMMIYGVCGKFPTDNPNFALEILNANLWFAENGGPYLCYESGAQSLLLALRFPLDDATPEKLENEIEVVVKSMENLYLVLHNQGITLENEHMKIEEISSSDNKHYYAGR 1E9F7ED2B9D5F1DF cest_tir 1 5 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association Table 1 The ability of CesT to interact with Tir was tested using the yeast two-hybrid assay. cest_tir-2 3 6 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association Fig. 9 the ability of immobilized CesT to specifically bind Tir in crude protein preparations was tested cest_tir-3 2 6 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 5 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Fig. 11 The coelution of CesT and truncated Tir from the matrix indicates the presence of a CesT binding domain in the 233 amino acid N-terminus of Tir similar to the N-terminal chaperone binding domain seen in other type III secreted proteins. cest_tir-4 4 5 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) physical association physical association Fig. 10 Antibodies against Tir coprecipitate CesT. Intact bacteria were incubated with the thiol-cleavable cross-linker DSP and then lysed and processed to isolate cytoplasmic proteins