Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

Abstract

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.

Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

“…The reason for this difference in amino acid profile between effectors and noneffectors is currently not known, but a possible explanation is that the requirement of effectors to unfold during translocation by the Icm/Dot transporter (66) and to refold with their C-terminal ends exposed generated evolutionary constraints that affect the amino acid composition of the effector proteins. The newly identified C. burnetii effectors are expressed during infection.…”

Structural insights into the mechanism and inhibition of transglutaminase-induced ubiquitination by the Legionella effector MavC

“…It has been reported that in bacteriological medium, the Lpg2149 expression only becomes detectable in the early exponential phase, suggesting that it functions when the bacteria began to replicate 40 . Because proteins being translocated by the Dot/Icm system are in their unfolded forms 46 , Lpg2149 may inhibit the activity of MavC and MvcA by preventing their translocation through forming protein complexes in bacterial cells or by blocking the activity of translocated proteins in host cells. Yet, the exact time and the extent of its inhibition of MavC and MvcA still needs further investigation.…”

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

“…As many effectors seem to be of low abundancy, several assays employ overexpression and exploit that the T4SS tolerates reporter domains fused to the N-terminus of effectors, if these do not fold rapidly into rigid structures (Amyot et al, 2013 ). One or multiple epitope tags [e.g., M45 (Weber et al, 2006 ), 4xHemagglutinin (HA) (Dolezal et al, 2012 ), 13xMyc (Viner et al, 2012 ) or 3xFlag (Isaac et al, 2015 )] were employed to detect translocated effectors.…”