Search

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.

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.

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.

The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations., Author Summary The intracellular pathogens Legionella pneumophila (Lp) and Rickettsia prowazekii (Rp) inject an effector (RalF) that diverts the host trafficking small GTPase Arf1. In the case of Lp, LpRalF recruits Arf1 to the Legionella-containing vacuole (LCV), where the pathogen replicates. RalF proteins are related to eukaryotic ArfGEFs, from which they depart by a unique auto-inhibitory capping domain that contains localization and functional determinants. In this work, we combined the reconstitution of RalF ArfGEF activity on artificial membranes with cellular and Lp infection assays to uncover how auto-inhibition is released for RalF proteins to function in vivo. We find that LpRalF and RpRalF are activated by membranes by about 1000-fold and map the membrane sensor to a unique motif in the capping domain. This motif is identical to the auto-inhibitory motif, thus establishing a binary switch that controls the ArfGEF activity of RalF in accordance with specific lipid environments. Finally, we show that the membrane sensor drives LpRalF binding to the LCV and timing of Arf activation during Lp infection. These results establish how RalF proteins are derepressed when they encounter a favorable lipid environment, and provide an evolutionary explanation to the presence of RalF in pathogens with diverging lifestyles.

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.

Several bacterial pathogens have evolved strategies to survive in macrophages and create a replicative niche within phagosomes. The bacterial factor MgtC is a key player in intramacrophage survival, being important for virulence in diverse intracellular pathogens. MgtC is also required for growth under magnesium limitation. Recent studies provide new clues on the role of MgtC in macrophages, which seems to be unlinked to adaptation to a low Mg(2+) microenvironment. In addition, we discuss the unexpected finding that MgtC modulates host P-type ATPase activity.

MgtC is a virulence factor common to several intracellular pathogens, including Mycobacterium tuberculosis , that might have been acquired through horizontal gene transfer. In the present study, we investigated the polymorphism of mgtC in clinical isolates representative of the main epidemic groups of M. tuberculosis . MgtC appears to have a low polymorphism rate in M. tuberculosis that consists exclusively of nonsynonymous mutations. We identified a single nucleotide polymorphism (SNP) at mgtC codon 182 ( mgtC 182 ) specifically associated with the Haarlem genotype. A simple PCR assay, called the “on/off switch assay,” using phosphorothioate-modified primers and Pfu polymerase allowed us to distinguish Haarlem from non-Haarlem strains based on the mgtC 182 SNP. The amino acid change (H182R) associated with the mgtC 182 SNP in Haarlem strains does not appear to procure a selective advantage. Our results offer a simple and rapid tool to distinguish between Haarlem and non-Haarlem strains. In addition, the on/off switch assay, which allows the detection of SNPs on chromosomal DNA and M. tuberculosis cultures, provides a novel approach for the screening of known SNPs in M. tuberculosis .

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.