The E Block motif is associated withLegionella pneumophilatranslocated substrates

Abstract

SummaryLegionella pneumophila promotes intracellular growth by moving bacterial proteins across membranes via the Icm/Dot system. A strategy was devised to identify large numbers of Icm/Dot translocated proteins, and the resulting pool was used to identify common motifs that operate as recognition signals. The 3′ end of the sidC gene, which encodes a known translocated substrate, was replaced with DNA encoding 200 codons from the 3′ end of 442 potential substrate-encoding genes. The resulting hybrid proteins were then tested in a high throughput assay, in which translocated SidC antigen was detected by indirect immunofluorescence. Among translocated substrates, regions of 6–8 residues called E Blocks were identified that were rich in glutamates. Analysis of SidM/DrrA revealed that loss of three Glu residues, arrayed in a triangle on an α-helical surface, totally eliminated translocation of a reporter protein. Based on this result, a second strategy was employed to identify Icm/Dot substrates having carboxyl terminal glutamates. From the fusion assay and the bioinformatic queries, carboxyl terminal sequences from 49 previously unidentified proteins were shown to promote translocation into target cells. These studies indicate that by analysing subsets of translocated substrates, patterns can be found that allow predictions of important motifs recognized by Icm/Dot.

Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal

“…As indicated in the introductory section, it has previously been shown that a sequence containing several residues of glutamic acids (called "E-block") is important for effector translocation (19). This motif was also found to be present in the OSS (Fig.…”

“…Later, an analysis of the C-terminal region of effectors indicated that there are amino acids (e.g., glutamic acid, isoleucine, leucine, valine, serine, threonine) that are enriched and/ or depleted in different regions of the secretion signal (18). Finally, the importance of glutamic acids (termed "E-block motif") for effector translocation was described (19). Our current model suggests that the previous results published concerning the secretion signal were all parts of the puzzle that constitutes the secretion signal of the Icm/Dot effectors.…”

“…In addition, serine and threonine were enriched at positions −10 to −5 (18). A more recent study (19) described the importance of the glutamic acid stretch (E-block) for effector translocation and used this feature to identify several novel effectors. Notably, all these studies were performed only on L. pneumophila.…”

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

“…1A) (29,31). Second, the C terminus of CvpA contains a distribution of charged and hydrophobic amino acids reminiscent of a characterized Dot/Icm T4B secretion signal (19,22,35) (Fig. 1B).…”

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

“…Oomycetes (4) Plants ( Moreover, there is evidence for the presence of a secretion signal in the carboxy terminal of the Dot/Icm substrates (Nagai et al 2005;Kubori et al 2008;Huang et al 2010); therefore, acquisition of this signal is also a challenge for these proteins to be secreted. Thus, although we start to discern how these eukaryotic proteins have appeared in the Legionella genomes, many questions with respect to the detailed mechanism of acquisition and integration remain unanswered.…”