Abstract
Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.
Highlights
Legionella pneumophila is the causative agent of the lung malady Legionnaires’ disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication
By pairwise comparison of profile hidden Markov models (HHpred) [36], we found that the N-terminal portion of Ceg23, a protein translocated by the Dot/Icm transporter, is distantly similar to members of the ovarian tumor (OTU) protein subfamily
Most deubiquitinating enzymes (DUBs) of the motif interacting with ubiquitin– containing novel DUB family are specific for Lys-48 linkage [42], whereas Jab1/Mov34/Mpr1 Pad1 N-terminalϩ (JAMM) family DUBs often show Lys-63 linkage specificity [43]; characterized ubiquitin-specific protease family DUBs have little or no linkage preference [44]
Summary
Legionella pneumophila is the causative agent of the lung malady Legionnaires’ disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. It is believed that the great majority of proteins are ubiquitinated at least one time prior to being degraded, this post-translational modification regulates virtually all cellular functions, cellular homeostasis, cell cycle, immune responses, vesicle trafficking, and DNA damage responses [2]. Such diversity in function in part is accomplished by the formation of topologically distinct architecture formed by different types of ubiquitin modifications, which dictate the fate of modified proteins. Components of the ubiquitination machinery including E2 and E3 enzymes and ubiquitin itself can be modified by either ubiquitination or other forms of post-translational modifications, which further increases the scope and intricacy of cellular events regulated by ubiquitination [6]
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