Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli

Georgina L. Pollock; Andrey M. Grishin; Cristina Giogha; Jiyao Gan; Clare V. Oates; Paul J. McMillan; Isabella Gaeta; Matthew J. Tyska; Jaclyn S. Pearson; Nichollas E. Scott; Miroslaw Cygler; Elizabeth L. Hartland

doi:10.1073/pnas.2204332119

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli

Georgina L. Pollock, Andrey M. Grishin, Cristina Giogha, Jiyao Gan, Clare V. Oates, Paul J. McMillan, Isabella Gaeta, Matthew J. Tyska, Jaclyn S. Pearson, Nichollas E. Scott, Miroslaw Cygler^*, Elizabeth L. Hartland^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY"segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Original language	English
Article number	e2204332119
Pages (from-to)	1-11
Number of pages	11
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	119
Issue number	34
Early online date	17 Aug 2022
DOIs	https://doi.org/10.1073/pnas.2204332119
Publication status	Published - 23 Aug 2022

Keywords

EPEC
Eps8
Kinase
Microvillus
T3SS

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Pollock, G. L., Grishin, A. M., Giogha, C., Gan, J., Oates, C. V., McMillan, P. J., Gaeta, I., Tyska, M. J., Pearson, J. S., Scott, N. E., Cygler, M., & Hartland, E. L. (2022). Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli. Proceedings of the National Academy of Sciences of the United States of America, 119(34), 1-11. Article e2204332119. https://doi.org/10.1073/pnas.2204332119

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title = "Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli",

abstract = "Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical {"}proline-rich{"} motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal {"}DY{"}segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.",

keywords = "EPEC, Eps8, Kinase, Microvillus, T3SS",

author = "Pollock, {Georgina L.} and Grishin, {Andrey M.} and Cristina Giogha and Jiyao Gan and Oates, {Clare V.} and McMillan, {Paul J.} and Isabella Gaeta and Tyska, {Matthew J.} and Pearson, {Jaclyn S.} and Scott, {Nichollas E.} and Miroslaw Cygler and Hartland, {Elizabeth L.}",

note = "Funding: This work was supported by National Health and Medical Research Council of Australia project grants awarded to E.L.H. (APP1175976). N.E.S. was supported by an Overseas (Biomedical) Fellowship (APP1037373) and is currently the recipient of an Australian Research Council Future Fellowship (FT200100270). G.L.P. was the recipient of an Australian Postgraduate Award. J.G. was supported by a China Scholarship Council-University of Melbourne PhD scholarship. I.G. was supported by the Vanderbilt Cellular, Biochemical and Molecular Sciences training grant 5T32GM 008554-25. M.J.T. was supported by National Institutes of Health grants R01-DK111949 and R01-DK095811. The crystallography diffraction dataset described in this paper was collected from beamline Canadian Macromolecular Crystallography Facility-Bending Magnet at the Canadian Light Source, a national research facility of the University of Saskatchewan, which is supported by the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council, the National Research Council, the Canadian Institutes of Health Research, the Government of Saskatchewan, and the University of Saskatchewan.",

year = "2022",

month = aug,

day = "23",

doi = "10.1073/pnas.2204332119",

language = "English",

volume = "119",

pages = "1--11",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

issn = "0027-8424",

publisher = "NATL ACAD SCIENCES",

number = "34",

}

Pollock, GL, Grishin, AM, Giogha, C, Gan, J, Oates, CV, McMillan, PJ, Gaeta, I, Tyska, MJ, Pearson, JS, Scott, NE, Cygler, M & Hartland, EL 2022, 'Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli', Proceedings of the National Academy of Sciences of the United States of America, vol. 119, no. 34, e2204332119, pp. 1-11. https://doi.org/10.1073/pnas.2204332119

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli. / Pollock, Georgina L.; Grishin, Andrey M.; Giogha, Cristina et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 119, No. 34, e2204332119, 23.08.2022, p. 1-11.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli

AU - Pollock, Georgina L.

AU - Grishin, Andrey M.

AU - Giogha, Cristina

AU - Gan, Jiyao

AU - Oates, Clare V.

AU - McMillan, Paul J.

AU - Gaeta, Isabella

AU - Tyska, Matthew J.

AU - Pearson, Jaclyn S.

AU - Scott, Nichollas E.

AU - Cygler, Miroslaw

AU - Hartland, Elizabeth L.

N1 - Funding: This work was supported by National Health and Medical Research Council of Australia project grants awarded to E.L.H. (APP1175976). N.E.S. was supported by an Overseas (Biomedical) Fellowship (APP1037373) and is currently the recipient of an Australian Research Council Future Fellowship (FT200100270). G.L.P. was the recipient of an Australian Postgraduate Award. J.G. was supported by a China Scholarship Council-University of Melbourne PhD scholarship. I.G. was supported by the Vanderbilt Cellular, Biochemical and Molecular Sciences training grant 5T32GM 008554-25. M.J.T. was supported by National Institutes of Health grants R01-DK111949 and R01-DK095811. The crystallography diffraction dataset described in this paper was collected from beamline Canadian Macromolecular Crystallography Facility-Bending Magnet at the Canadian Light Source, a national research facility of the University of Saskatchewan, which is supported by the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council, the National Research Council, the Canadian Institutes of Health Research, the Government of Saskatchewan, and the University of Saskatchewan.

PY - 2022/8/23

Y1 - 2022/8/23

N2 - Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY"segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

AB - Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY"segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

KW - EPEC

KW - Eps8

KW - Kinase

KW - Microvillus

KW - T3SS

U2 - 10.1073/pnas.2204332119

DO - 10.1073/pnas.2204332119

M3 - Article

C2 - 35976880

AN - SCOPUS:85136909827

SN - 0027-8424

VL - 119

SP - 1

EP - 11

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 34

M1 - e2204332119

ER -

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli

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Keywords

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