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

Samual C Allgood,Rebecca R Noll,Colleen Pike,Sean Lein,Barbara P Romero Dueñas,M Ramona Neunuebel

doi:10.3389/fcimb.2017.00397

Samual C Allgood, Rebecca R Noll + Show 4 more

Open Access

https://doi.org/10.3389/fcimb.2017.00397

Copy DOI

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.

Highlights

Legionella pneumophila is a Gram-negative, facultative intracellular bacterium that is regarded as an important cause of hospital- and communityacquired pneumonia (CDC, 2011; Viasus et al, 2013)
AnkX did not localize at the Golgi where Rab1 is predominantly found (Mukherjee et al, 2011), and we reasoned that perhaps AnkX is co-opted at cellular locations where Rab35 is present, namely the plasma membrane and endosomes
We determined that GFP-AnkX was associated with the plasma membrane, nascent endosomes, and tubular compartments at the periphery of the cell, reminiscent of the subcellular localization of recycling endosomes (Figure 1, Supplementary Figures 1, 2)

Summary

Introduction

Legionella pneumophila (strain Philadelphia-1, hereafter Legionella) is a Gram-negative, facultative intracellular bacterium that is regarded as an important cause of hospital- and communityacquired pneumonia (CDC, 2011; Viasus et al, 2013). Legionella remains enclosed in a plasma membrane-derived compartment known as the Legionella-containing vacuole (LCV) that undergoes drastic remodeling into a compartment resembling the endoplasmic reticulum (Tilney et al, 2001) This process is dependent on the Dot/Icm specialized type IV secretion system (T4SS) that translocates over 300 bacterial effector proteins into the host cytosol (Berger and Isberg, 1993; Segal and Shuman, 1999; Luo and Isberg, 2004; Segal, 2013). The Legionella ankX mutant is impaired in its ability to escape phagosome maturation, and more than 50% of the LCVs in ankX–infected macrophage fuse with the lysosomal marker LAMP-1 (Pan et al, 2008) This is a dramatic effect considering that functional redundancy among Legionella effectors often masks phenotypes caused by the deletion of single genes. Existing data indicate that AnkX prevents microtubule-dependent vesicular transport (Pan et al, 2008), the precise mechanism through which AnkX inhibits LCV fusion with lysosomes has not yet been defined

Methods

Results

Conclusion