Orientia tsutsugamushiAnk9 is a multifunctional effector that utilizes a novel GRIP-like Golgi localization domain for Golgi-to-endoplasmic reticulum trafficking and interacts with host COPB2

Abstract

SUMMARY Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that afflicts one million people annually. This obligate intracellular bacterium boasts one of the largest microbial arsenals of ankyrin repeat-containing protein (Ank) effectors, most of which target the endoplasmic reticulum (ER) by undefined mechanisms. Ank9 is the only one proven to function during infection. Here, we demonstrate that Ank9 bears a motif that mimics the GRIP domain of eukaryotic golgins and is necessary and sufficient for its Golgi localization. Ank9 reaches the ER exclusively by retrograde trafficking from the Golgi. Consistent with this observation, it binds COPB2, a host protein that mediates Golgi-to-ER transport. Ank9 destabilizes the Golgi and ER in a Golgi-localization domain (GLD)-dependent manner and induces the ATF4-dependent unfolded protein response. The Golgi is also destabilized in cells infected with O. tsutsugamushi or treated with COPB2 siRNA. COPB2 reduction and/or the cellular events that it invokes, such as Golgi destabilization, benefit Orientia replication. Thus, Ank9/bacterial negative modulation of COPB2 might contribute to the bacterium’s intracellular replication. This report identifies a novel microbial GLD, links Ank9 to the ability of O. tsutsugamushi to perturb Golgi structure, and describes the first mechanism by which any Orientia effector targets the secretory pathway.

Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

“…tsutsugamushi induces the UPR. In O. tsutsugamushi infected cells, the ER is slightly distended and protein secretion is inhibited (34,35). Because these phenomena can indicate ER stress (36), we examined if O. tsutsugamushi infection invokes the UPR.…”

“…During the first 24 to 48 h of O. tsutsugamushi infection, when ERAD is inhibited, xbp1 and xbp1s mRNA levels are increased, but not at 72 h when ERAD is no longer hindered. Ank9, an O. tsutsugamushi effector that traffics in the retrograde direction from the Golgi complex to the ER, invokes the UPR and inhibits the secretory pathway (34). Therefore, the pathogen minimally employs Ank9 to invoke ER stress while also utilizing Ank4 to modulate the ensuing UPR and ERAD to bolster its growth.…”

“…O. tsutsugamushi Ank9, like Ank4 and LegU1, bears a C-terminal F-box (28,72). In addition to binding SKP1 and nucleating the SCF complex in an F-box-dependent manner, Ank9 binds COPB2, which directs in the retrograde direction from the Golgi complex to the ER (28,34). Yet another F-boxcontaining Ank is L. pneumophila AnkX, which exploits SKP1 to mediate ubiquitination and subsequent proteosomal degradation of unknown host cell protein substrates to ultimately yield free amino acids that are essential for the bacterium's intracellular growth (79,80).…”

“…Precipitation of Flag-tagged proteins and Western blot analyses of the resulting eluates were performed exactly as described previously (34).…”

“…When ectopically expressed, 14 of the 20 Anks localize to the ER (32). This finding, together with the bacterium's intracellular tropism for the ER-proximal perinuclear region and its ability to inhibit the host cell secretory pathway (33,34), suggest that O. tsutsugamushi-ER interactions and Ank modulation of ER-associated processes might be critical aspects of the organism's pathobiology.…”