Legionella pneumophila is an intracellular pathogen that multiplies in a specialized vacuole within host cells. Biogenesis of this vacuole requires the Dot/Icm type IV protein translocation system. By using a Cre/ loxP -based protein translocation assay, we found that proteins translocated by the Dot/Icm complex across the host phagosomal membrane can also be transferred from one bacterial cell to another. The flexibility of this system allowed the identification of several families of proteins translocated by the Dot/Icm complex. When analyzed by immunofluorescence microscopy, a protein identified by this procedure, SidC, was shown to translocate across the phagosomal membranes to the cytoplasmic face of the L. pneumophila phagosome. The identification of large numbers of these substrates, and the fact that the absence of any one substrate rarely results in strong defects in intracellular growth, indicate that there is significant functional redundancy among the Dot/Icm translocation targets.

An unprecedented mechanism of ubiquitination that is independent of E1 and E2 enzymes, instead relying on activation of ubiquitin by ADP-ribosylation, and which is mediated by members of the SidE effector family encoded by the bacterial pathogen Legionella pneumophila, establishes that ubiquitination can be carried out by a single enzyme. Ubiquitination is an important regulatory mechanism for many cellular processes, and several bacterial pathogens have evolved to exploit and target this protein degradation pathway to facilitate their own proliferation. Ubiquitination is catalysed by a sequential multi-enzyme cascade that involves three enzymes: E1, E2 and E3. In this study, Zhao-Qing Luo and colleagues describe an unprecedented mechanism of ubiquitination that is independent of E1 and E2 enzymes, and which is mediated by members of the SidE effector family encoded by the bacterial pathogen Legionella pneumophila. They show that SidE effectors ubiquitinate multiple Rab small GTPases in a mechanism that relies on activation of ubiquitin by ADP-ribosylation, thereby circumventing the requirement for activation by the E1 and E2 enzymes. This work establishes that ubiquitination can be carried out by a single enzyme, but future work is required to resolve the functional implications of this unique mechanism of post-translational modification. Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade1, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents2. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases3 or as enzymes that directly attack ubiquitin4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes5. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

Ubiquitination regulates many essential cellular processes in eukaryotes. This post-translational modification (PTM) is typically achieved by E1, E2, and E3 enzymes that sequentially catalyze activation, conjugation, and ligation reactions, respectively, leading to covalent attachment of ubiquitin, usually to lysine residues of substrate proteins. Ubiquitin can also be successively linked to one of the seven lysine residues on ubiquitin to form distinctive forms of polyubiquitin chains, which, depending upon the lysine used and the length of the chains, dictate the fate of substrate proteins. Recent discoveries revealed that this ubiquitin code is further expanded by PTMs such as phosphorylation, acetylation, deamidation, and ADP-ribosylation, on ubiquitin, components of the ubiquitination machinery, or both. These PTMs provide additional regulatory nodes to integrate development or insulting signals with cellular homeostasis. Understanding the precise roles of these PTMs in the regulation of ubiquitin signaling will provide new insights into the mechanisms and treatment of various human diseases linked to ubiquitination, including neurodegenerative diseases, cancer, infection, and immune disorders.

Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell–cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell–cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa.

Significance: Many effectors of the Coxiella burnetii Dot/Icm transporter are important for its virulence, but the lack of understanding of their biochemical activity prevents the use of them as potential therapeutic targets. Here, we found that the effector Cbu0513 (CinF) is a protein phosphatase that attacks IκBα, a key regulatory protein in NF-κB signaling. Unlike its homologs such as ST0318 from the archaeon Sulfolobus tokodaii , CinF has lost the enzymatic activity as a fructose-1,6-bisphosphate aldolase/phosphatase. Instead, it specifically targets IκBα to make it resistant to proteasome-mediated degradation in cells stimulated with NF-κB agonists. Our finding has expanded the strategy used by bacterial pathogens to inhibit host immunity, which may provide leads for the development of immune modulators for disease treatment.

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.