The intracellular pathogen Legionella pneumophila subverts vesicle traffic in eukaryotic host cells to create a vacuole that supports replication. The dot/icm genes encode a protein secretion apparatus that L. pneumophila require for biogenesis of this vacuole. Here we show that L. pneumophila produce a protein called RalF that functions as an exchange factor for the ADP ribosylation factor (ARF) family of guanosine triphosphatases (GTPases). The RalF protein is required for the localization of ARF on phagosomes containing L. pneumophila. Translocation of RalF protein through the phagosomal membrane is a dot/icm-dependent process. Thus, RalF is a substrate of the Dot/Icm secretion apparatus.

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.

When bacteria cells are exposed to higher temperature, a set of heat-shock proteins (hsps) is induced rapidly and transiently to cope with increased damage in proteins. The mechanism underlying induction of hsps has been a central issue in the heat-shock response and studied intensively in Escherichia coli. Immediately upon temperature upshift, the cellular level of sigma 32 responsible for transcription of heat-shock genes increases rapidly and transiently. The increase in sigma 32 results from both increased synthesis and stabilization of sigma 32, which is ordinarily very unstable. A clue to further understanding of early regulatory events came from recent analysis of translational induction and subsequent shut-off of sigma 32 synthesis. Whereas a 5'-coding region of mRNA for sigma 32 is involved in the induction mediated by the mRNA secondary structure, a distinct segment of sigma 32 polypeptide further downstream is involved in the DnaK/DnaJ-mediated shut-off and destabilization of sigma 32 that may be mutually interconnected.

SummaryLegionella pneumophila has a Dot/Icm type IV secretion system used to translocate a number of 'effector proteins' which subvert host cell functions. In this study, we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A BLAST analysis revealed that one of the substrates, which we named LubX (LegionellaU-box protein), contains two domains that have a remarkable similarity to the U-box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induced upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2-like kinase 1). We demonstrate that one of the U-boxes (U-box 1) is critical to the ubiquitin ligation, and the other U-box (U-box 2) mediates interaction with Clk1. Thus, the two U-boxes of LubX have distinct functions, and U-box 2 plays a non-canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated. Together, these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.