Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner

Abstract

The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella-containing vacuole (LCV) with lysosomes.

The recycling endosome and bacterial pathogens

“…Thus, it would be expected that during infection, the Rab35 pool accessed by AnkX remains locked in an inactive GDP-bound conformation, which presumably contributes to inhibition of transferrin recycling. In support of this notion, transferrin recycling was inhibited in macrophages infected with the ΔankX mutant complemented with wild-type AnkX but not when complemented with the catalytically inactive AnkX H229A (Allgood et al, 2017). This directly links post-translational modification of host proteins to an inhibitory effect on recycling endocytosis, but it is unclear if this effect was mediated by targeting other recycling Rabs in addition to Rab35.…”

“…Though more is known about how L. pneumophila effectors exploit endoplasmic‐reticulum‐to‐Golgi traffic, how other membrane trafficking pathways might be manipulated is less clear. In a recent study using a pulse‐chase approach, transferrin recycling was found to be drastically diminished in U937 macrophages infected with wild‐type L. pneumophila but not in those infected with a translocation‐deficient mutant (Allgood et al, ). This is consistent with the notion that L. pneumophila disrupts one or more host endocytic recycling pathway because TfR is recycled through both the “fast” and “slow” recycling pathways.…”

The Function of Rab35 in Development and Disease

“…Another L. pneumophila effector protein is AnkX, a phosphocholine transferase that covalently links a phosphocholine moiety to Rab35 [82]. Fluorescent protein-tagged AnkX introduced to mammalian cells was found to localize at the plasma membrane and tubular membrane compartments by transmission electron microscopy [83]. Consistent with its targeting of the endocytic recycling pathway, AnkX co-localized with Rab35 and a Rab35 cargo, the major histocompatibility class I protein (MHCI) [83].…”

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts byLegionella pneumophila

“…Zhu et al, 2011). Effectors are responsible for preventing lysosome fusion, recruitment of ER-derived vesicles to the LCV, evasion of the innate immune response, and modulation of a plethora of other host processes to promote intracellular survival and replication of L. pneumophila (Allgood et al, 2017; Bärlocher, Welin, & Hilbi, 2017; Fontana et al, 2011; Horwitz, 1983; Horwitz & Maxfield, 1984; Isberg, O’Connor, & Heidtman, 2009; ubori, Bui, Hubber, & Nagai, 2017; Luo, 2011; C. Price et al, 2017; C. T. Price, Al-Quadan, Santic, Rosenshine, & Abu Kwaik, 2011; Qiu & Luo, 2017; Swanson & Isberg, 1995a, 1995b).…”