Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

Abstract

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.

Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase

“…More than 400 hypothetical proteins were found to possess this motif. Among these candidates, 29 proteins have been identified as substrates of the Dot/Icm transporter (22) (Table S1). Some of these proteins were then expressed, purified, and examined for in vitro PI phosphatase activities by a malachite green-based assay (23).…”

Genome Dynamics in Legionella: The Basis of Versatility and Adaptation to Intracellular Replication

“…The main secretion system of Legionella, essential for intracellular growth, is the type IVB Dot/Icm secretion system (Marra and Shuman 1992;Berger and Isberg 1993). Using many dif-ferent methods it has been shown that at least 275 L. pneumophila proteins are secreted by this system (Campodonico et al 2005;de Felipe et al 2005;Shohdy et al 2005;de Felipe et al 2008;Burstein et al 2009;Heidtman et al 2009;Zhu et al 2011), which represents nearly 10% of the protein-coding genes predicted in the L. pneumophila genomes. At least 75 of these are eukaryotic-like proteins or carry eukaryotic domains and, for certain of these, it has also been shown experimentally that they interfere with host-cell signaling pathways ( Table 2).…”

The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling

“…Another feature of the LCV is enrichment of polyubiquitin conjugates around the vacuolar membrane (8). This sophisticated membrane remodeling process is achieved by the coordinated activity of effector proteins delivered into the host through the Dot/Icm apparatus (9,10). However, the mechanism underlying these processes is still not well established.…”