Poison domains block transit of translocated substrates via the Legionella pneumophila Icm/Dot system.

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system.

The E Block motif is associated with Legionella pneumophila translocated substrates.

Legionella 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.