Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

Abstract

The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.

Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport

“…LepB displays very high activity (k cat /K m = 3.6 × 10 6 m − 1 s − 1 ; Rothmeier et al, 2013), underlining a common theme in Legionella effectors that interact with Rab proteins, i.e. that their activity or affinity is generally higher than that of human counterparts, allowing effective competition in infected cells.…”

Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors

“…Following several replication cycles and exploitation of the resources in the host cell, L. pneumophila is released into the environment. effector LegG1 (Rothmeier et al 2013), which localizes to the LCV where it stimulates the small GTPase Ran (Fig. 2).…”

“…LegG1 has sequence homology to the Ran GEF RCC1. Although purified LegG1 does not have direct Ran GEF activity in vitro, it induces Ran activation in infection, thus promoting LCV recruitment of the RanGTP-binding protein RanBP1, stabilization of microtubules, LCV motility, and phagocyte migration (Rothmeier et al 2013;Simon et al 2014). The advantages of keeping the LCV mobile are not fully understood; however, it might enable the LCV to leave the default "track" of phagosomes, which would otherwise efficiently ensure the encounter and fusion with endocytic vesicles.…”

“…The advantages of keeping the LCV mobile are not fully understood; however, it might enable the LCV to leave the default "track" of phagosomes, which would otherwise efficiently ensure the encounter and fusion with endocytic vesicles. In the absence of either LegG1 or Ran, intracellular growth of L. pneumophila is compromised (Rothmeier et al 2013). Whether motility of the LCV contributes to the evasion of phagosomallysosomal maturation or the promotion of intracellular growth by facilitating the interaction with other cellular compartments remains to be established.…”

“…In contrast to Rab1 manipulation, RalF is currently the only known effector that modulates ARF1 signaling and has been shown to be essential for the recruitment of ARF1 to the LCV (Nagai et al 2002). However, neither deletion of RalF nor depletion of ARF1 by RNAi cause a pronounced intracellular growth defect, indicating that Legionella ensures the formation of a replication-permissive LCV by additional mechanisms (Nagai et al 2002;Rothmeier et al 2013). Interestingly, in the absence of the effector SidC and its paralogue SdcA, L. pneumophila is not able to recruit ARF1 to the LCV despite the presence of RalF, indicating SidC and SdcA need to prime the LCV for ARF1 recruitment (Horenkamp et al 2014).…”