Bacterial usurpation of the OTU deubiquitinase fold

Abstract

The extensive cellular signalling events controlled by posttranslational ubiquitination are tightly regulated through the action of specialized proteases termed deubiquitinases. Among them, the OTU family of deubiquitinases can play very specialized roles in the regulation of discrete subtypes of ubiquitin signals that control specific cellular functions. To exert control over host cellular functions, some pathogenic bacteria have usurped the OTU deubiquitinase fold as a secreted virulence factor that interferes with ubiquitination inside infected cells. Herein, we provide a review of the function of bacterial OTU deubiquitinases during infection, the structural basis for their deubiquitinase activities and the bioinformatic approaches leading to their identification. Understanding bacterial OTU deubiquitinases holds the potential for discoveries not only in bacterial pathogenesis but in eukaryotic biology as well.

Keywords: OTU; bacteria; deubiquitinase; infection; protein prediction; structure; ubiquitin.

Fig. 1. The scheme of LCV biogenesis and functions of the LCV-associated bacterial Ub ligases and DUBs.

Upon infection, L. pneumophila delivers various effector proteins (colored circles) into host cell cytosol to modulate cellular systems. Reversible Ub modification of host proteins is a relevant example of actions of the effector proteins. Just after internalization, the Legionella phagosome is morphologically converted to the ER-like compartment [–81]. This conversion is thought to be a result of fusion between the phagosome and ER-derived vesicles which are captured by intercepting ER-to-Golgi traffic [47]. At this stage of infection, SidE- and SidC-family effector proteins, having distinctive enzymatic activities as Ub ligases, contribute toward conjugation of Ub (red dots) on LCV-associated substrates. At later stages, the Ub chains can be cleaved by L. pneumophila OTU DUBs, LotA, LotB and LotC, according to their linkage specificities (see text), reducing the level of Ub on the LCV. The function of LotD on the LCV-associated Ub chains has not been analyzed yet. The functional interplay between the bacterial Ub ligases and the DUBs is apparently correlated with the recruitment of key host players in membrane fusion, like Rab GTPases and SNARE proteins, to the LCV. This suggests that the effector-mediated regulation of Ub chain assembly is closely connected to the establishment of the LCV replicative niche, although the exact roles of the bacterial enzymes on this scheme have not been fully elucidated yet. This image was created with BioRender.com.

Fig. 2. Manipulation of host proteins by the functions of LotB and LotC.

(A) Upon internalization, L. pneumophila acquires plasma membrane-localized t-SNAREs including Stx3 on its early phagosome. It then promotes the recruitment of ER-derived vesicles to the LCV by intercepting vesicle traffic. This results in noncanonical pairing of ER-derived v-SNARE Sec22b with Stx3. L. pneumophila also induces polyubiquitination of Sec22b in a T4SS-dependent manner at the initial stage of infection. LotB is delivered to the cell cytosol via the T4SS and localizes to the LCV depending on its TM domains. The polyUb chains on Sec22b are cleaved by the DUB activity of LotB, leading to the dissociation of Stx3 from Sec22b residing on the LCV. (B) The Legionella E3 Ub ligase SidC induces Rab10 ubiquitination and promotes the recruitment of Rab10 to the LCV. The overexpression of LotC in bacteria leads to reduction in ubiquitinated Rab10 as well as the level of Rab10 on LCVs, demonstrating that LotC can reverse the activity of SidC on the LCV. These images were created with BioRender.com.

Fig. 3. Structural comparison of bacterial S1 Ub-binding sites.

(A) Schematic of Ub-binding regions mapped onto the structure of CCHFV vOTU (PDB 3PHW [82]) as a prototypical OTU fold. Catalytic triad residues forming the active site are shown in ball-and-stick. The S1 Ub-binding site is composed of variable regions (VR) highlighted in blue. (B) As in (A), for the structure of Escherichia albertii EschOTU (PDB 6W9S [43]). Variable regions and a VR-1 sequence permutation are annotated. (C) As in (A), for the AlphaFold2 model of Chlamydia pneumoniae ChlaOTU [66]. Variable regions and a large VR-1 insertion domain are annotated. (D) As in (A), for the structure of Wolbachia pipientis wMelOTU (PDB 6W9R [43]). Variable regions are annotated. All structure figures were generated using PyMOL (www.pymol.org).

Fig. 4. Structural analysis of Legionella pneumophila LOT-class DUBs.

(A) Structures of all LOT-class DUBs. Active sites within the core OTU domain are annotated, along with adaptive α-1,2 regions of the A-UBDs inserted into VR-1. (B) Active site views of all LOT-class DUBs. Catalytic triad residues are shown in ball-and-stick, with hydrogen bonds indicated by dashed lines. Residues missing from the crystal structure (either due to insufficient electron density or construct boundaries) are indicated by asterisks. Configurations consistent with active or inactive catalytic triads are indicated. (C) A-UBD structures for all LOT-class DUBs. Topologies of the 4-helix A-UBD sub-structure are indicated with numbered arrows. The distinct α-1,2 regions responsible for Ub binding are highlighted. All structure figures were generated using PyMOL (www.pymol.org).