Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

Abstract

Background Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. Previously, a large number of genes that encode proteins containing eukaryotic protein-protein interaction motifs such as ankyrin-repeat (Ank) domains were identified in the O. tsutsugamushi genome. However, little is known about the Ank protein function in O. tsutsugamushi. Methodology/Principal FindingsTo characterize the function of Ank proteins, we investigated a group of Ank proteins containing an F-box–like domain in the C-terminus in addition to the Ank domains. All nine selected ank genes were expressed at the transcriptional level in host cells infected with O. tsutsugamushi, and specific antibody responses against three Ank proteins were detected in the serum from human patients, indicating an active expression of the bacterial Ank proteins post infection. When ectopically expressed in HeLa cells, the Ank proteins of O. tsutsugamushi were consistently found in the nucleus and/or cytoplasm. In GST pull-down assays, multiple Ank proteins specifically interacted with Cullin1 and Skp1, core components of the SCF1 ubiquitin ligase complex, as well as the eukaryotic elongation factor 1 α (EF1α). Moreover, one Ank protein co-localized with the identified host targets and induced downregulation of EF1α potentially via enhanced ubiquitination. The downregulation of EF1α was observed consistently in diverse host cell types infected with O. tsutsugamushi.Conclusion/SignificanceThese results suggest that conserved targeting and subsequent degradation of EF1α by multiple O. tsutsugamushi Ank proteins could be a novel bacterial strategy for replication and/or pathogenesis during mammalian host infection.

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

“…The data presented here complement a recent study by Min et al that examined nine Anks from O. tsutsugamushi strain Boryong and identified F-boxes within Ank1A2, Ank1F1, Ank1U5, Ank1U9, Ank1U4, and Ank1B1 (46). These Boryong proteins share sequence identity (shown as percent identity) to Ikeda Ank10_01 (83%), Ank12_01 (91%), Ank2 (84%), Ank20 (90%), Ank6_02 (66%), and Ank8 (93%), respectively, which are reported to have F-boxes in this study.…”

“…These Boryong proteins share sequence identity (shown as percent identity) to Ikeda Ank10_01 (83%), Ank12_01 (91%), Ank2 (84%), Ank20 (90%), Ank6_02 (66%), and Ank8 (93%), respectively, which are reported to have F-boxes in this study. Boryong Ank1E2 was also noted as having an F-box (46), though its ORF is annotated as a pseudogene and it does not have an Ikeda protein homolog (44). Of note, Boryong Ank1D1 and Ank1C14 do not interact with SKP1 (46), though their Ikeda homologs, Ank4_01 (63%) and Ank3_11 (74%), respectively, interacted with SKP1 and other SCF1 components in this study.…”

“…Several ectopically expressed Boryong F-box-containing Anks examined by Min and colleagues were suggested to interact with and promote ubiquitination and proteasomal destruction of eukaryotic elongation factor 1␣ (46). Despite numerous attempts, however, we were unable to recapitulate these phenomena with any ectopically expressed Ikeda Ank, even when the proteasome was pharmacologically inhibited.…”

“…A previous scan of 22 bacterial species for putative F-box proteins predicted that only one O. tsutsugamushi strain Boryong candidate, Ank1U9 (OTBS_2178), possessed a C-terminal F-box downstream of its ankyrin repeats (22). Further examination of Boryong Anks revealed that Ank1U9 and at least six other Anks possessed F-boxes and, when ectopically expressed in recombinant form, were capable of binding SCF1 components in pulldown assays (46), underscoring the necessity for manual sequence evaluation in searching for putative F-box proteins. Here, 16 O. tsutsugamushi strain Single copy a Pseudogenes were excluded.…”

“…To further infer the functional importance of O. tsutsugamushi Ank F-box residues, alignments were performed with two Anks from O. tsutsugamushi strain Boryong that are known not to interact with SKP1 (46). These Anks have homologs in the Ikeda genome that were demonstrated in this study to have functional F-boxes.…”