Indispensable Role for the Eukaryotic-Like Ankyrin Domains of the Ankyrin B Effector ofLegionella pneumophilawithin Macrophages and Amoebae

Abstract

The Dot/Icm-translocated ankyrin B (AnkB) effector of Legionella pneumophila exhibits molecular mimicry of eukaryotic F-box proteins and is essential for intracellular replication in macrophages and protozoa. In addition to two eukaryotic-like ankyrin (ANK) domains, AnkB harbors a conserved eukaryotic F-box domain, which is involved in polyubiquitination of proteins throughout the eukaryotic kingdom. We have recently shown that the F-box domain of the AnkB effector is essential for decoration of the Legionella-containing vacuole (LCV) with polyubiquitinated proteins within macrophages and protozoan hosts. To decipher the role of the two ANK domains in the function of AnkB, we have constructed in-frame deletion of either or both of the ANK domain-encoding regions (ankB⌬A1, ankB⌬A2, and ankB⌬A1A2) to trans-complement the ankB null mutant. Deletion of the ANK domains results in defects in intracellular proliferation and decoration of the LCV with polyubiquitinated proteins. Export of the truncated variants of AnkB was reduced, and this may account for the observed defects. However, while full-length AnkB ectopically expressed in mammalian cells trans-rescues the ankB null mutant for intracellular proliferation, ectopic expression of AnkB⌬A1, AnkB⌬A2, and AnkB⌬A1A2 fails to trans-rescue the ankB null mutant. Importantly, ectopically expressed full-length AnkB is targeted to the host cell plasma membrane, where it recruits polyubiquitinated proteins. In contrast, AnkB⌬A1, AnkB⌬A2, and AnkB⌬A1A2 are diffusely distributed throughout the cytosol and fail to recruit polyubiquitinated proteins. We conclude that the two eukaryotic-like ANK domains of AnkB are essential for intracellular proliferation, for targeting AnkB to the host membranes, and for decoration of the LCV with polyubiquitinated proteins.

Secreted Effectors Encoded within and outside of the Francisella Pathogenicity Island Promote Intramacrophage Growth

“…Ankyrin domains, localized to the C-terminus of OpiB, mediate protein–protein interaction and, similarly, have diverse targets. For example, the ankyrin repeats of L. pneumophila AnkB target the protein to membranes, while those of AnkX are involved in disrupting microtubule-dependent vesicle trafficking (Ge and Shao, 2011; Pan et al, 2008; Price et al, 2010). …”

Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

“…Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacterial pathogens (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). These effectors contain one or more ankyrin repeats, each of which consists of a 33-amino-acid motif that mediates proteinprotein interactions (15).…”

ThANKs for the repeat

“…AnkB's ankyrin repeats are predicted to direct binding to a host protein that then is targeted for ubiquitination and potential degradation via activity of the F-box domain in the SCF complex. This prediction is supported by experimental evidence showing the Ank domains 29 and F-box region 28 of AnkB are needed for optimal Legionella intracellular replication and decoration of the Legionella-containing vacuole with ubiquitinated proteins. AnkB recruits at least one protein, parvin B, but does not increase ubiquitination or degradation of the protein.…”