Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

Abstract

Intracellular bacteria use a variety of strategies to evade degradation and create a replicative niche. Legionella pneumophila is an intravacuolar pathogen that establishes a replicative niche through the secretion of more than 300 effector proteins. The function of most effectors remains to be determined. Toxicity in yeast has been used to identify functional domains and elucidate the biochemical function of effectors. A library of L. pneumophila effectors was screened using an expression plasmid that produces low levels of each protein. This screen identified the effector SdeA as a protein that confers a strong toxic phenotype that inhibits yeast replication. The toxicity of SdeA was suppressed in cells producing the effector SidJ. The effector SdeA is a member of the SidE family of L. pneumophila effector proteins. All SidE orthologs encoded by the Philadelphia isolate of Legionella pneumophila were toxic to yeast, and SidJ suppressed the toxicity of each. We identified a conserved central region in the SidE proteins that was sufficient to mediate yeast toxicity. Surprisingly, SidJ did not suppress toxicity when this central region was produced in yeast. We determined that the amino-terminal region of SidE was essential for SidJ-mediated suppression of toxicity. Thus, there is a genetic interaction that links the activity of SidJ and the amino-terminal region of SidE, which is required to modulate the toxic activity displayed by the central region of the SidE protein. This suggests a complex mechanism by which the L. pneumophila effector SidJ modulates the function of the SidE proteins after translocation into host cells.L egionella pneumophila is a Gram-negative, facultative intravacuolar pathogen that is the causative agent of Legionnaires' disease in humans (1-3). L. pneumophila is phagocytosed by host cells and creates a vacuole that supports replication by inhibiting the fusion of host endocytic vesicles and subverting the transport of secretory vesicles (4-7). The bacterially encoded Dot/Icm type IV secretion system (T4SS) is essential for the creation of this Legionella-containing vacuole (LCV) (8, 9). Proteins secreted by the T4SS, known as effectors, have been shown to both decorate the LCV and modify the location and function of host proteins, especially Rab GTPases (8,10,11). Determining the function of these effector proteins has been complicated by the observation that deletion of a single effector or groups of effectors does not typically result in a strong replication defect within host cells (12)(13)(14).The use of the yeast Saccharomyces cerevisiae as a system to study L. pneumophila effector proteins has been successful in previous studies (15)(16)(17)(18)(19). Examples include studies on the effector protein RalF, which was shown to inhibit yeast replication when overproduced, and the effector proteins YlfA and YlfB were both identified in an early yeast toxicity screen (16). Effector toxicity in yeast has also been used to study effector activities. The L. pneumophila effector AnkX is capa...

Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination

“…S3D) likely reflects an intricate interplay between the effectors of L. pneumophila to achieve spatiotemporal control of host cell processes, underscored by a previous observation that SidJ can remove SdeA from the phagosome after a certain time postinfection (35)(36)(37). The contribution of the catalytic activity of the N-terminal DUB module to SidJ-mediated suppression of SdeA's toxicity (caused by its central domain) adds another layer of complexity to the interplay between these two effectors (Fig.…”

“…It adds another layer of complexity to the regulation of host processes by effectors of opposite biochemical activities exemplified by the reversible AMPylation and phosphorylcholination of the Rab1 small GTPase (33,34). The activity of the SidE family of effectors appears to be regulated by SidJ (35,36), a gene within the gene cluster (37). All members of this family contain the N-terminal DUB module, which appears to redundantly regulate the extent of ubiquitination of the vacuolar surface, but their natural substrates remain to be determined (24).…”

“…All members of this family contain the N-terminal DUB module, which appears to redundantly regulate the extent of ubiquitination of the vacuolar surface, but their natural substrates remain to be determined (24). Clearly, DUB activity is not critical for the role of SidEs in intracellular bacterial replication under laboratory infection conditions; such roles likely are conferred by the activity of other domains of these large proteins (36).…”

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

“…Among these, the putative mART element in SdeA is R 673 -S 827 -E 867 S 868 E 869 , a catalytic motif found in enzymes that transfer the ADP-ribosyl group from NAD to arginine residues 19 . To examine its role in SdeA-mediated yeast toxicity 20,21 , we created the SdeA E/A mutant, in which E 867 and E 869 were mutated to alanine. This mutant has completely lost its toxicity to yeast and was also defective in inhibiting the secretion of the secreted form of the embryonic alkaline phosphatase (SEAP) 22 by mammalian cells ( Fig.…”

“…4c ). The central domain of SdeA remains toxic to yeast 20 , suggesting that it is still biochemically active. Indeed, SdeA 178-1000 robustly ubiquitinates itself and Rab33b in a manner that requires both NAD and the mART motif ( Fig.…”