EspH utilizes phosphoinositide and Rab binding domains to interact with plasma membrane infection sites and Rab GTPases*
Ipsita Nandi,
Rachana Pattani Ramachandran,
Deborah E. Shalev,
Dina Schneidman-Duhovny,
Raisa Shtuhin-Rahav,
Naomi Melamed-Book,
Efrat Zlotkin-Rivkin,
Alexander Rouvinski,
Ilan Rosenshine,
Benjamin Aroeti
Affiliations
Ipsita Nandi
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus – Givat Ram, Jerusalem, Israel
Rachana Pattani Ramachandran
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus – Givat Ram, Jerusalem, Israel
Deborah E. Shalev
The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, The Edmond J. Safra Campus – Givat Ram, Jerusalem, Israel
Dina Schneidman-Duhovny
The Rachel and Selim Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel
Raisa Shtuhin-Rahav
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus – Givat Ram, Jerusalem, Israel
Naomi Melamed-Book
Bioimaging Unit, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
Efrat Zlotkin-Rivkin
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus – Givat Ram, Jerusalem, Israel
Alexander Rouvinski
Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University-Hadassah Medical School, of Jerusalem, Jerusalem, Israel
Ilan Rosenshine
Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University-Hadassah Medical School, of Jerusalem, Jerusalem, Israel
Benjamin Aroeti
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus – Givat Ram, Jerusalem, Israel
Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen’s benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.
