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Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery:https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available athttps://phenaid.ardigen.com/jumpcpexplorer/.

Summary The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII., Graphical Abstract, Highlights • TMEM127 and WWP2 are required for MHCII depletion by Salmonella SteD • Tumor suppressor TMEM127 is an adaptor for oncoprotein E3 ligase WWP2 • SteD interacts with TMEM127 to induce ubiquitination and degradation of mature MHCII • SteD also undergoes TMEM127- and WWP2-dependent ubiquitination, Salmonella inhibits the adaptive immune response by reducing cell surface levels of mature MHC class II. Alix et al. reveal that the Salmonella effector SteD co-opts TMEM127, a tumor suppressor protein of previously unknown function. TMEM127 binds the E3 ubiquitin ligase WWP2 enabling ubiquitination and degradation of MHCII and SteD.

A convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1 A with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses.

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC-dependent activity. The crystal structure of the SidC N-terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.

Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.

The SPI-2 type III secretion system (T3SS) of intracellular Salmonella enterica translocates effector proteins into mammalian cells. Infection of antigen-presenting cells results in SPI-2 T3SS-dependent ubiquitination and reduction of surface-localized mature MHC class II (mMHCII). We identify the effector SteD as required and sufficient for this process. In Mel Juso cells, SteD localized to the Golgi network and vesicles containing the E3 ubiquitin ligase MARCH8 and mMHCII. SteD caused MARCH8-dependent ubiquitination and depletion of surface mMHCII. One of two transmembrane domains and the C-terminal cytoplasmic region of SteD mediated binding to MARCH8 and mMHCII, respectively. Infection of dendritic cells resulted in SteD-dependent depletion of surface MHCII, the co-stimulatory molecule B7.2, and suppression of T cell activation. SteD also accounted for suppression of T cell activation during Salmonella infection of mice. We propose that SteD is an adaptor, forcing inappropriate ubiquitination of mMHCII by MARCH8 and thereby suppressing T cell activation.

Mammalian phagocytes control bacterial infections effectively through phagocytosis, the process by which particles engulfed at the cell surface are transported to lysosomes for destruction. However, intracellular pathogens have evolved mechanisms to avoid this fate. Many bacterial pathogens use specialized secretion systems to deliver proteins into host cells that subvert signaling pathways controlling membrane transport. These bacterial effectors modulate the function of proteins that regulate membrane transport and alter the phospholipid content of membranes. Elucidating the biochemical function of these effectors has provided a greater understanding of how bacteria control membrane transport to create a replicative niche within the host and provided insight into the regulation of membrane transport in eukaryotic cells.

Identification of short coding sequences is challenging, both experimentally and in silico, and functional natural peptides (< 50 amino acids) have to a large extent been overlooked in Gram-negative bacteria. Recent results have converged to highlight the role of hydrophobic peptides that form a novel class of active molecules in Escherichia coli and Salmonella enterica serovar Typhimurium. These peptides can play a regulatory role by interacting with protein partners at the inner membrane and by modulating protein partner activity or stability. Genome-wide analyses in both bacterial species have identified several conserved short open reading frames encoding a single transmembrane segment. We discuss the known and predicted membrane-associated peptides and the tools for their identification. Besides the identification of novel regulatory networks, characterization of peptides with a single transmembrane helix segment and proteins that interact with them provides a powerful opportunity to study interactions between alpha helices within biological membranes. In addition, some bioactive membrane peptides could provide a basis for engineering membrane protein antagonists.

Aedes mosquitoes are the major vectors of (re)-emerging infections including arboviruses (dengue, Chikungunya, yellow fever) in developing countries. Moreover, the emergence of Aedes-borne diseases in the developed world is currently a source of concern. Evaluation of human immune responses to Aedes bites could be a useful immuno-epidemiological tool for evaluating exposure to Aedes-borne diseases and thus predicting the risk of such emerging diseases. Specific IgE and IgG4 antibody (Ab) responses to Aedes aegypti saliva were evaluated in young Senegalese children living in an area of exposure to the Aedes vector. Specific IgE and IgG4 responses increased during rainy season of high exposure to Aedes bites. In addition, the evolution of anti-saliva isotype levels during the rainy season presented spatial heterogeneity between the studied villages. These preliminaries results support the potential approach of using anti-saliva Ab responses for evaluating exposure to Aedes vectors and risks of emerging arbovirus infections.

MgtC is required for intramacrophage replication of intracellular pathogens and growth in low Mg(2+) medium. A link between these two phenotypes has been proposed due to putative Mg(2+) deprivation inside phagosome. MgtC is part of a family of proteins that share a conserved N-terminal transmembrane domain and a variable C-terminal domain. A combination of predictive and experimental approaches indicates that the Salmonella MgtC C-terminal domain is cytoplasmic, adopts a fold also found in metal transporters and RNA interacting domain, and does not bind Mg(2+). MgtC homologues from diverse gamma-proteobacteria, including the extracellular pathogens Yersinia pestis, Photorhabdus luminescens and Pseudomonas aeruginosa, have been expressed in a SalmonellaDeltamgtC strain. The Y. pestis MgtC fully replaced the Salmonella MgtC whereas P. luminescens or P. aeruginosa MgtC complemented only in low Mg(2+) medium, thus dissociating for the first time the two MgtC-related phenotypes. In addition, we identified single amino acids changes that prevent or promote MgtC role in macrophages without affecting MgtC role in low Mg(2+) culture. A SalmonellaDeltamgtC strain showed elongated and autoaggregated bacteria in low Mg(2+) medium but not in macrophages. Taken together our results suggest that MgtC has a dual role when bacteria localize in macrophages or low Mg(2+) environment.

Membrane traffic, which is the “cellular postal service” that shuttles biomolecules around the cell and organizes the structure of organelles, is among the primary targets of effectors injected by ...

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover mono-ubiquitination of Rab1 as a new SidC-dependent activity. The crystal structure of the SidC N-terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.

Identification of short coding sequences is challenging, both experimentally and in silico, and functional natural peptides (50 amino acids) have to a large extent been overlooked in Gram-negative bacteria. Recent results have converged to highlight the role of hydrophobic peptides that form a novel class of active molecules in Escherichia coli and Salmonella enterica serovar Typhimurium. These peptides can play a regulatory role by interacting with protein partners at the inner membrane and by modulating protein partner activity or stability. Genome-wide analyses in both bacterial species have identified several conserved short open reading frames encoding a single transmembrane segment. We discuss the known and predicted membrane-associated peptides and the tools for their identification. Besides the identification of novel regulatory networks, characterization of peptides with a single transmembrane helix segment and proteins that interact with them provides a powerful opportunity to study interactions between alpha helices within biological membranes. In addition, some bioactive membrane peptides could provide a basis for engineering membrane protein antagonists.

In Salmonella enterica serovar Typhimurium, MgtC and PagC are positively regulated by the PhoP–PhoQ two-component system, which is activated under magnesium deprivation. Both MgtC and PagC are of unknown function but have been involved in intramacrophage survival. We have found that the amount of PagC is lowered in a ΔmgtC mutant strain grown in magnesium depleted medium. However, the effect of MgtC on PagC does not account for the growth defect of a ΔmgtC mutant in macrophages since, in contrast to previous reports, our results indicate that PagC does not contribute to intramacrophage survival. In addition, a pagC null mutant is only poorly attenuated in Nramp1-negative or Nramp1-positive mice. On the other hand, a mgtC null mutant is significantly more attenuated with Nramp1-positive than Nramp1-negative mice, suggesting that a functional Nramp1 (Slc11a1) further limits the multiplication of this mutant within the host.

International audience; MgtC is a virulence factor common to several intracellular pathogens that is required for intramacrophage survival and growth in magnesium-depleted medium. In Salmonella enterica, MgtC is coexpressed with the MgtB magnesium transporter and transcription of the mgtCB operon is induced by magnesium deprivation. Despite the high level of mgtCB transcriptional induction in magnesium-depleted medium, the MgtC protein is hardly detected in a wild-type Salmonella strain. Here, we show that downregulation of MgtC expression is dependent on a hydrophobic peptide, MgtR, which is encoded by the mgtCB operon. Our results suggest that MgtR promotes MgtC degradation by the FtsH protease, providing a negative regulatory feedback. Bacterial two-hybrid assays demonstrate that MgtR interacts with the inner-membrane MgtC protein. We identified mutant derivatives of MgtR and MgtC that prevent both regulation and interaction between the two partners. In macrophages, overexpression of the MgtR peptide led to a decrease of the replication rate of Salmonella. This study highlights the role of peptides in bacterial regulatory mechanisms and provides a natural antagonist of the MgtC virulence factor.

The Legionella pneumophila effector protein RalF functions as a guanine nucleotide exchange factor (GEF) that activates the host small GTPase protein ADP-ribosylation factor (Arf), and recruits this host protein to the vacuoles in which this pathogen resides. GEF activity is conferred by the Sec7 domain located in the N-terminal region of RalF. Structural studies indicate that the C-terminal region of RalF makes contacts with residues in the Sec7 domain important for Arf interactions. Theoretically, the C-terminal region of RalF could prevent nucleotide exchange activity by blocking the ability of Arf to interact with the Sec7 domain. For this reason, the C-terminal region of RalF has been termed a capping domain. Here, the role of the RalF capping domain was investigated by comparing biochemical and effector activities mediated by this domain in both the Legionella RalF protein (LpRalF) and in a RalF ortholog isolated from the unrelated intracellular pathogen Rickettsia prowazekii (RpRalF). These data indicate that both RalF proteins contain a functional Sec7 domain and that the capping domain regulates RalF GEF activity. The capping domain has intrinsic determinants that mediate localization of the RalF protein inside of host cells and confer distinct effector activities. Localization mediated by the capping domain of LpRalF enables the GEF to modulate membrane transport in the secretory pathway, whereas, the capping domain of RpRalF enables this bacterial GEF to modulate actin dynamics occurring near the plasma membrane. Thus, these data reveal that divergence in the function of the C-terminal capping domain alters the in vivo functions of the RalF proteins., Author Summary Legionella pneumophila and Rickettsia prowazekii are two pathogenic intracellular bacteria, phylogenetically distant and presenting different intracellular lifestyles. Interestingly, both organisms encode a protein called RalF, which in Legionella has been shown to be an effector protein that functions as a guanine nucleotide exchange factor (GEF) for the Arf family of eukaryotic GTPases. We show that the Sec7 domains in both proteins have a similar enzymatic ability to activate Arf1, however, the two proteins differ in their effector functions inside mammalian cells. The Legionella RalF protein targets secretory transport functions, whereas, the Rickettsia RalF protein modulates actin dynamics at the plasma membrane. These differences in RalF effector activities are mediated by a C-terminal domain that directs localization and interaction with host cell determinants. These data reveal important properties of the RalF protein that provide insight into differential roles for Arf during infection by Legionella and Rickettsia, and also provide a model for investigating how intracellular localization of structurally similar Arf GEFs enables a protein to regulate functionally distinct host cell processes.