A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells.
The Legionella pneumophila Dot/Icm 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 Dot/Icm system. In this study, the mechanism of Dot/Icm-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 Dot/Icm 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 Dot/Icm system, most other Dot/Icm 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 Dot/Icm 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.
Nagai
Hiroki
H
Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, Room 354b, 295 Congress Avenue, New Haven, CT 06511, USA.
Cambronne
Eric D
ED
Kagan
Jonathan C
JC
Amor
Juan Carlos
JC
Kahn
Richard A
RA
Roy
Craig R
CR
D037361:Adenylate Cyclase Toxin / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D016952:Legionella pneumophila / Q000737:chemistry / Q000472:pathogenicity*; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D020662:Guanine Nucleotide Exchange Factors; C432954:IcmR protein, Legionella pneumophila; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; C497516:RalF protein, Legionella pneumophila; D011993:Recombinant Fusion Proteins