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.

SummaryLegionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein± protein interactions. Specific interactions between different Icm proteins were detected by yeast twohybrid and gel overlay analysis. These data support a model in which the IcmQ±IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS±IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.

Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.

When Escherchia coli cells are transferred from 300C to 4?C, transcription from specific promoters recognized by RNA polymerase containing a32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of a!32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-acZ gene fusion. Synthesis of a a!32-iB-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation-namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.Exposure of cells to heat or other stress induces the synthesis of heat shock proteins (1, 2). In Escherichia coli, a shift from 30°C to 42°C induces heat shock genes transiently by activating transcription from promoters specifically recognized by RNA polymerase containing o32 (3,4), encoded by the rpoH (htpR, hin) gene (5, 6). Enhanced transcription from heat shock promoters is caused by a transient increase in the cellular level of &32 (7,8) as a result of increased synthesis and stabilization (8). The increased synthesis of a£32 has been thought to occur primarily at the translational level, because (i) the synthesis rate of 32, but not of rpoH mRNA, increases markedly upon temperature up-shift (9), (it) heat-induced synthesis of a &32-,B-galactosidase fusion protein depends on the translational initiation region of rpoH and not on the promoters (ref. 8; see below), and (iii) the rpoH mRNA level also increases upon temperature up-shift, but this increase is preceded by the increase of a32 synthesis (8, 10).To further substantiate translational control of &32 and gain insight into early events of the heat shock response, we examined expression of deletion and base substitution derivatives of rpoH-lacZ gene fusion during heat shock. We show that expression of gene fusion is transiently activated even in the absence of RNA synthesis, strongly supporting translational control. Evidence indicates that rpoH mRNA translation is normally repressed during steady-state growth and is transiently derepressed upon shift to higher temperature. Furthermore, such a thermal regulation probably involves an interplay of positive and negative cis-acting rpoH mRNA regions that correspond to the translational initiation region and an internal coding region, re...

Type IV secretion systems (T4SSs) play a central role in the pathogenicity of many important pathogens, including Agrobacterium tumefaciens, Helicobacter pylori, and Legionella pneumophila. The T4SSs are related to bacterial conjugation systems, and are classified into two subgroups, type IVA (T4ASS) and type IVB (T4BSS). The T4BSS, which is closely related to conjugation systems of IncI plasmids, was originally found in human pathogen L. pneumophila; pathogenesis by L. pneumophila infection requires functional Dot/Icm T4BSS. A zoonotic pathogen, Coxiella burnetii, and an arthropod pathogen, Rickettsiella grylli – both of which carry T4BSSs highly similar to the Legionella Dot/Icm system – are evolutionarily closely related and comprise a monophyletic group. A growing body of bacterial genomic information now suggests that T4BSSs are not limited to Legionella and related bacteria and IncI plasmids. Here, we review the current knowledge on T4BSS apparatus and component proteins, gained mainly from studies on L. pneumophila Dot/Icm T4BSS. Recent structural studies, along with previous findings, suggest that the Dot/Icm T4BSS contains components with primary or higher-order structures similar to those in other types of secretion systems – types II, III, IVA, and VI, thus highlighting the mosaic nature of T4BSS architecture.