<i>Anaplasma phagocytophilum</i> effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

Abstract

The obligatory intracellular bacterium <i>Anaplasma phagocytophilum</i> causes human granulocytic anaplasmosis, an emerging zoonosis. <i>Anaplasma</i> has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, <u>E</u>R-<u>G</u>olgi <u>e</u>xit site protein of <i><u>A</u>naplasma</i> (EgeA). In cells infected by <i>Anaplasma</i>, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to <i>Anaplasma-</i>containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the <i>Anaplasma</i> inclusion surface. Furthermore, knockdown of <i>Anaplasma</i> EgeA or either host TANGO1 or SCFD1 significantly reduced <i>Anaplasma</i> infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into <i>Anaplasma</i> inclusions, and several ER stress marker genes were not up-regulated in <i>Anaplasma-</i>infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates <i>Anaplasma</i> nutrient acquisition and reduces ER stress for <i>Anaplasma</i> survival and proliferation.