Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. 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 agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalyzed by these enzymes is energized by NAD which activates ubiquitin by the formation of ADP-ribosylated ubiquitin (ADPR-Ub). These results establish that ubiquitination can be catalyzed by a single enzyme whose activity does not require ATP.

Highlights d Deep profiling of proteome and phosphoproteome in AD progression d Validation of protein alterations in two independent AD cohorts d Identification of Ab-induced protein changes in AD and the 5xFAD mouse model d Prioritization of proteins and pathways in AD by multi-omics

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Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ϳ3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways. While infectious diseases continue to be a major threat to human health, there is an urgent need in rational design and development of new treatment strategies. As a model for studying bacterial pathogenesis, Salmonella spp. cause millions of infections per year ranging from food poisoning to life-threatening systemic typhoid fever (1). Central to its pathogenesis is a syringelike structure known as the type III secretion system (T3SS), which delivers bacterial proteins called effectors directly into eukaryotic host cells (2, 3). The injected proteins are able to modulate various host cellular functions, and over the years extensive studies have been carried out on these effector proteins and/or their interacting host targets. While these studies have contributed substantially to our initial understanding of infection biology, daunting challenges are encountered because conventional reductionism-based studies certainly cannot explain the complex multifactorial nature of host-pathogen interactions (4). Systems-level analyses can provide a panoramic view of the functional host-pathogen interplay and thus are promising in this regard.During infection, the intracellular environment is likely to be very different from what bacteria encounter in rich culture media. It has long been known that various nutritional and environmental differences within host cells force bacterial pathogens to reprogram their gene expression. In fact, transcriptomic studies of intracellular Salmonella within infected macrophages and epithelial cells contributed to our initial understanding of bacterial adaptations in the host environment (5, 6). Because proteins are the final gene products and their changes may not correlate with those of mRNA, direct readout of the bacterial proteome is highly desired. Such measurements, however, have been technically challenging due to the presence of vast amounts of host proteins (7). As a ...

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Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies have revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attack several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. In this case, ubiquitin is first activated by ADP-ribosylation at Arg42 by a mono-ADP-ribosyltransferase activity; the intermediate is then cleaved by a phosphodiesterase activity also residing within SdeA, concomitant with the attachment of ubiquitin to serine residues of substrate proteins via a phosphoribosyl linker. Here we demonstrate that the effect of SidEs is antagonized by SidJ, an effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ reverses ubiquitination of SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. SidJ also displays classical deubiquitinase activity but does not require catalytic cysteine residues. Further, these deubiquitinase activities of SidJ are essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a ubiquitin-deconjugating enzyme that functions to impose temporal regulation on the activity of SidE effectors. SidJ may be important in future studies of signaling cascades mediated by this unique ubiquitination, one that also potentially regulates cellular processes in eukaryotic cells.

Multiplexed isobaric labeling methods, such as tandem mass tags (TMT), remarkably improve the throughput of quantitative mass spectrometry. Here, we present a 27-plex TMT method coupled with two-dimensional liquid chromatography (LC/LC) for extensive peptide fractionation and high-resolution tandem mass spectrometry (MS/MS) for peptide quantification and then apply the method to profile the complex human brain proteome of Alzheimer’s disease (AD). The 27-plex method combines multiplexed capacities of the 11-plex and the 16-plex TMT, as the peptides labeled by the two TMT sets display different mass and hydrophobicity, which can be well separated in LC-MS/MS. We first systematically optimized the protocol for the newly developed 16-plex TMT, including labeling reaction, desalting, and MS conditions, and then directly compared the 11-plex and 16-plex methods by analyzing the same human AD samples. Both methods yielded similar proteome coverage, analyzing >100 000 peptides in >10 000 human proteins. Furthermore, the 11-plex and 16-plex samples were mixed for a 27-plex assay, resulting in more than 8000 protein measurements within the same MS time. The 27-plex results are highly consistent with those of the individual 11-plex and 16-plex TMT analyses. We also used these proteomics data sets to compare the AD brain with the nondementia controls, discovering major AD-related proteins and revealing numerous novel protein alterations enriched in the pathways of amyloidosis, immunity, mitochondrial, and synaptic functions. Overall, our data strongly demonstrate that this new 27-plex strategy is highly feasible for routine large-scale proteomic analysis.

Novel Staphylococcus aureus clones continue to emerge that cause infections in otherwise healthy people. One example is the sequence type (ST) 398 lineage, which we show here is increasing in importance as a significant cause of community-associated (CA) human infections in China. We have a profound lack of understanding about what determines the considerable virulence potential of such newly emerging clones. Information about the contribution to virulence of the more recently discovered ESAT-6 secretion system (ESS) has remained particularly scarce. The Chinese ST398 isolates exhibited significantly increased expression of ESS genes as compared to predominant hospital-associated clones, which we found is likely due to increased expression of the accessory gene regulator (Agr) system and control of ESS by Agr. Importantly, deletion of essB in ST398 resulted in significantly reduced resistance to neutrophil killing and decreased virulence in murine skin and blood infection models. Our results demonstrate a key function of ESS in promoting virulence and mechanisms of resistance to innate host defense in an important emerging CA-S. aureus lineage. They suggest that ESS has a so far underestimated role in promoting aggressive virulence and epidemiological success of S. aureus.