A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

Abstract

The Legionella pneumophila Dot͞Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot͞Icm system. In this study, the mechanism of Dot͞Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot͞Icm system. A leucine (L372) residue at the ؊3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot͞Icm system, most other Dot͞Icm substrates were found to have amino acid residues with similar physical properties at their ؊3 or ؊4 C-terminal positions. These data demonstrate that the Dot͞Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides.

Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium

“…This finding is in line with the results described by Nagai et al (36) in the accompanying manuscript that indicate that the translocation signal for recruitment of the RalF effector protein by the L. pneumophila Dot͞Icm system is disordered, and is thus probably flexible. Besides, a long ␣-helix upstream of the sequence that contains the transport signal may be required to project the signal for optimal interaction with the coupling protein and the T4SS (36).…”

“…Interestingly, in the accompanying article, Nagai et al (36) performed a detailed analysis of the Dot͞Icm-translocated Legionella pneumophila RalF protein, and showed that a Leu residue at the Ϫ3 position in the C terminus is critical for transfer. Similarly, in the A. tumefaciens VirF protein, the Arg residue at the Ϫ3 position is very important for transfer.…”

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…The interaction is mediated by the C-terminal domain of VirE2 (Atmakuri et al, 2003). The C-termini of several T4SS-translocated substrates have been identiWed as the signals mediating secretion by their associated secretion systems (Nagai et al, 2005;Schulein et al, 2005;Simone et al, 2001;Vergunst et al, 2000Vergunst et al, , 2003Vergunst et al, , 2005. It therefore seems probable that substrate recognition by the CPs, as a general rule, occurs via speciWc interactions between the CPs and the C-termini of their associated substrates.…”

“…Finally, other type IV secretion systems, which are ancestrally related to the conjugative Mpf/CP machinery, are found to secrete protein substrates without any indication for concomitant DNA transfer (Cascales and Christie, 2003;Schröder et al, 2005). The existence of a C-terminal signal mediating translocation of dedicated T4SS protein substrates has been discovered in three diVerent T4SS: the VirB/VirD4 systems of B. henselae and A. tumefaciens, and the Dot/Icm system of L. pneumophila (Atmakuri et al, 2003;Nagai et al, 2005;Schulein et al, 2005;Simone et al, 2001;Vergunst et al, 2000Vergunst et al, , 2003Vergunst et al, , 2005. Evidence is accumulating that these secretion systems have evolved from conjugation systems and that the C-terminal secretion signal of their secreted protein substrates originates from a relaxase ancestor (Schulein et al, 2005).…”

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.…”