Using Genomic Deletion Mutants to Investigate Effector-Triggered Immunity During Legionella pneumophila Infection.

Legionella pneumophila is an intracellular bacterial pathogen that uses a type IV secretion system (T4SS), termed Dot/Icm, to secrete more than 330 virulence effector proteins into the infected host cell. Many Dot/Icm effectors are involved in biogenesis of the Legionella-containing vacuole (LCV), which allows intracellular bacterial replication in environmental amoebae and alveolar macrophages. Through their activity, some effectors trigger the mammalian host immune response in a phenomenon termed effector-triggered immunity (ETI). Here, we describe a protocol to create and use L. pneumophila genome deletion mutants to identify effector(s) that alter pro-inflammatory cytokine production and bacterial clearance in the lungs of mice.

Structural and Functional Characterization of Legionella pneumophila Effector MavL.

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose "reader" proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.

Genome-wide genetic screen identifies host ubiquitination as important for Legionella pneumophila Dot/Icm effector translocation.

The Dot/Icm system of Legionella pneumophila is essential for virulence and delivers a large repertoire of effectors into infected host cells to create the Legionella containing vacuole. Since the secretion of effectors via the Dot/Icm system does not occur in the absence of host cells, we hypothesised that host factors actively participate in Dot/Icm effector translocation. Here we employed a high-throughput, genome-wide siRNA screen to systematically test the effect of silencing 18,120 human genes on translocation of the Dot/Icm effector, RalF, into HeLa cells. For the primary screen, we found that silencing of 119 genes led to increased translocation of RalF, while silencing of 321 genes resulted in decreased translocation. Following secondary screening, 70 genes were successfully validated as 'high confidence' targets. Gene set enrichment analysis of siRNAs leading to decreased RalF translocation, showed that ubiquitination was the most highly overrepresented category in the pathway analysis. We further showed that two host factors, the E2 ubiquitin-conjugating enzyme, UBE2E1, and the E3 ubiquitin ligase, CUL7, were important for supporting Dot/Icm translocation and L. pneumophila intracellular replication. In summary, we identified host ubiquitin pathways as important for the efficiency of Dot/Icm effector translocation by L. pneumophila, suggesting that host-derived ubiquitin-conjugating enzymes and ubiquitin ligases participate in the translocation of Legionella effector proteins and influence intracellular persistence and survival.

The Salmonella Effector SseK3 Targets Small Rab GTPases.

During infection, Salmonella species inject multiple type III secretion system (T3SS) effector proteins into host cells that mediate invasion and subsequent intracellular replication. At early stages of infection, Salmonella exploits key regulators of host intracellular vesicle transport, including the small GTPases Rab5 and Rab7, to subvert host endocytic vesicle trafficking and establish the Salmonella-containing vacuole (SCV). At later stages of intracellular replication, interactions of the SCV with Rab GTPases are less well defined. Here we report that Rab1, Rab5, and Rab11 are modified at later stages of Salmonella infection by SseK3, an arginine N-acetylglucosamine (GlcNAc) transferase effector translocated via the Salmonella pathogenicity island 2 (SPI-2) type III secretion system. SseK3 modified arginines at positions 74, 82, and 111 within Rab1 and this modification occurred independently of Rab1 nucleotide binding. SseK3 exhibited Golgi localization that was independent of its glycosyltransferase activity but Arg-GlcNAc transferase activity was required for inhibition of alkaline phosphatase secretion in transfected cells. While SseK3 had a modest effect on SEAP secretion during infection of HeLa229 cells, inhibition of IL-1 and GM-CSF cytokine secretion was only observed upon over-expression of SseK3 during infection of RAW264.7 cells. Our results suggest that, in addition to targeting death receptor signaling, SseK3 may contribute to Salmonella infection by interfering with the activity of key Rab GTPases.