Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.Legionella pneumophila | sphingosine-1-phosphate lyase | autophagy | sphingolipids | virulence T he Gram-negative intracellular bacterium Legionella pneumophila is an opportunistic human pathogen responsible for Legionnaires' disease. The bacteria are naturally found in freshwater systems where they replicate within protozoan hosts (1). It is thought that the adaptation to replication within amoebas has equipped L. pneumophila with the factors required to replicate successfully within human macrophages following opportunistic infection (2). Through genome sequencing, we have discovered that L. pneumophila encodes a high number and variety of proteins similar in sequence to eukaryotic proteins that are never or rarely found in other prokaryotic genomes (3). Subsequent phylogenetic analyses have suggested that many of these proteins were acquired by horizontal gene transfer (3, 4). One of these proteins exhibits a high degree of similarity to eukaryotic sphingosine-1 phosphate lyase (SPL). The L. pneumophila SPL homolog (LpSpl encoded by gene lpp2128, lpg2176, or legS2) is conserved in all L. pneumophila strains sequenced to date, but absent from Legionella longbeachae (SI Appendix, Table S1). Phylogenetic analysis of SPL sequences showed that the L. pneumophila spl gene was most likely acquired early during evolution by horizontal gene transfer from a protozoan organism (4, 5). With the increase in genome sequences available...

Coxiella burnetiiis an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of theC. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical forC. burnetiivirulence. Indeed, theC. burnetiiPmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate theC. burnetiiPmrA/B two-component system. This study has further enhanced our understanding ofC. burnetiipathogenesis, the host–pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.

Enteropathogenic Escherichia coli (EPEC) interferes with host cell signaling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a single Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrheagenic pathogen of children that utilizes a type III secretion system (T3SS) to inject virulence effector proteins directly into host intestinal cells. These effectors subvert host cell function and are encoded by genes either within the locus of enterocyte effacement (LEE) pathogenicity island (1) or outside the LEE (non-Lee-encoded [Nle] effectors) (2). A number of non-LEEencoded effectors that disrupt host cell signaling have been characterized in recent years (3, 4). For example, both NleC and NleE inhibit NF-B signaling and, consequently, block the production of inflammatory cytokines, such as interleukin-8 (5-7). NleB1 was recently characterized to be a glycosyltransferase effector that modifies a conserved arginine residue in the Fas-associated death domain protein (FADD) as well as the death domain-containing proteins TRADD and RIPK1, thereby blocking host death receptor signaling and apoptosis (8, 9). Modification of FADD prevents its recruitment to the Fas receptor and the subsequent recruitment and activation of caspase-8 (8, 9).Glycosyltransferases are enzymes that catalyze the transfer of a sugar moiety from an activated sugar donor substrate containing a phosphate-leaving group, such as nucleoside diphosphate sugars, to a specific acceptor substrate (10, 11). Cytosolic glycosylation is a vital molecular mechanism by which various bacterial toxins and effector proteins subvert eukaryotic host cell function (12). For example, Clostridium difficile toxins A and B modify small Rho GTPases by mono-O-glucosylation at specific threonine residues (13). Glycosylated Rho GTPases are locked in an inactive state, inhibiting downstream signaling pathways, resulting in the disruption of the cell cytoskeleton, the induction of apoptotic cell death, and fluid accumulation in the large intestine (14).The transfer of carbohydrate components to proteins usually occurs at the oxygen nucleophile of the hydroxyl group of a serine or threonine side chain, resulting in the formation of an O-linked glycosidic linkage, or at the nitrogen nucleophile of the amide group of an asparagine side chain, forming an N-linked glycosidic linkage (15)(16)(17)(18)(19). O-GlcNAc modification, unlike classic O-or Nlinked protein glycosylation, involves the transfer of a single GlcNAc moiety to the hydroxyl group of a serine or threonine residue, and this modification is not elongated (17,20). A nonclassical N-linked modification involves the addition of single glycosyl moieties from activated nucleotide sugars to asparagine residues of the target protein (21-23). NleB1 catalyzes an unusual glycosylation reaction that results in the addition of GlcNAc in an N-glycosidic linkage to arginine 117 (R117) of FADD (8,9). Th...