Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Francisella tularensis enters a double membraned compartment following cell-cell transfer.

Previously, we found that phagocytic cells ingest bacteria directly from the cytosol of infected cells without killing the initially infected cell (Steele et al., 2016). Here, we explored the events immediately following bacterial transfer. Francisella tularensis bacteria acquired from infected cells were found within double-membrane vesicles partially composed from the donor cell plasma membrane. As with phagosomal escape, the F. tularensis Type VI Secretion System (T6SS) was required for vacuole escape. We constructed a T6SS inducible strain and established conditions where this strain is trapped in vacuoles of cells infected through bacterial transfer. Using this strain we identified bacterial transfer events in the lungs of infected mice, demonstrating that this process occurs in infected animals. These data and electron microscopy analysis of the transfer event revealed that macrophages acquire cytoplasm and membrane components of other cells through a process that is distinct from, but related to phagocytosis.

Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer.

Many bacterial pathogens can cause septicemia and spread from the bloodstream into internal organs. During leptospirosis, individuals are infected by contact with Leptospira-containing animal urine-contaminated water. The spirochetes invade internal organs after septicemia to cause disease aggravation, but the mechanism of leptospiral excretion and spreading remains unknown. Here, we demonstrated that Leptospira interrogans entered human/mouse endothelial and epithelial cells and fibroblasts by caveolae/integrin-ß1-PI3K/FAK-mediated microfilament-dependent endocytosis to form Leptospira (Lep)-vesicles that did not fuse with lysosomes. Lep-vesicles recruited Rab5/Rab11 and Sec/Exo-SNARE proteins in endocytic recycling and vesicular transport systems for intracellular transport and release by SNARE-complex/FAK-mediated microfilament/microtubule-dependent exocytosis. Both intracellular leptospires and infected cells maintained their viability. Leptospiral propagation was only observed in mouse fibroblasts. Our study revealed that L. interrogans utilizes endocytic recycling and vesicular transport systems for transcytosis across endothelial or epithelial barrier in blood vessels or renal tubules, which contributes to spreading in vivo and transmission of leptospirosis.

A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins.

Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. SIGNIFICANCE: Enterococcal infections, especially bacteremia and endocarditis, are challenging to treat because E. faecium have acquired resistance to multiple classes of antimicrobials, including ampicillin, aminoglycosides, and glycopeptides. Thus, research on different modes of enterococcal pathogenicity is warranted. This study utilized a proteomic approach to identify MV-associated proteins of different nosocomial E. faecium strains representing four clinically relevant sequence types (STs), namely ST17, ST18, ST78, and ST192. The presented data suggest that E. faecium MVs are involved in virulence and antimicrobial resistance.

Interaction of microbial pathogens with host exocytic pathways.

Many microbial pathogens co-opt or perturb host membrane trafficking pathways. This review covers recent examples in which microbes interact with host exocytosis, the fusion of intracellular vesicles with the plasma membrane. The bacterial pathogens Listeria monocytogenes and Staphylococcus aureus subvert recycling endosomal pathways of exocytosis in order to induce their entry into human cells. By contrast, entry of the protozoan pathogen Trypanosoma cruzi or the virus adenovirus into host cells involves exploitation of lysosomal exocytosis. Toxins produced by Bacillus anthracis or Vibrio cholerae interfere with exocytosis pathways mediated by the GTPase Rab11 and the exocyst complex. By doing so, anthrax or cholera toxins impair recycling of cadherins to cell-cell junctions and disrupt the barrier properties of endothelial cells or intestinal epithelial cells, respectively. Uropathogenic Escherichia coli (UPEC) is expelled from bladder epithelial cells through two different exocytic routes that involve sensing of bacteria in vacuoles by host Toll-like receptor 4 (TLR4) or monitoring of the pH of lysosomes harbouring UPEC. The TLR4 pathway is mediated by multiple Rab GTPases and the exocyst, whereas the other pathway involves exocytosis of lysosomes. Expulsion of UPEC through these pathways is thought to benefit the host.

Bacteria and endothelial cells: a toxic relationship.

Pathogenic bacteria use the bloodstream as a highway for getting around the body, and thus have to find ways to enter and exit through the endothelium. Many bacteria approach this problem by producing toxins that can breach the endothelial barrier through diverse creative mechanisms, including directly killing endothelial cells (ECs), weakening the cytoskeleton within ECs, and breaking the junctions between ECs. Toxins can also modulate the immune response by influencing endothelial biology, and can modulate endothelial function by influencing the response of leukocytes. Understanding these interactions, in both the in vitro and in vivo contexts, is of critical importance for designing new therapies for sepsis and other severe bacterial diseases.

Master manipulators: an update on Legionella pneumophila Icm/Dot translocated substrates and their host targets.

Macrophages are the front line of immune defense against invading microbes. Microbes, however, have evolved numerous and diverse mechanisms to thwart these host immune defenses and thrive intracellularly. Legionella pneumophila, a Gram-negative pathogen of amoebal and mammalian phagocytes, is one such microbe. In humans, it causes a potentially fatal pneumonia referred to as Legionnaires' disease. Armed with the Icm/Dot type IV secretion system, which is required for virulence, and approximately 300 translocated proteins, Legionella is able to enter host cells, direct the biogenesis of its own vacuolar compartment, and establish a replicative niche, where it grows to high levels before lysing the host cell. Efforts to understand the pathogenesis of this bacterium have focused on characterizing the molecular activities of its many effectors. In this article, we highlight recent strides that have been made in understanding how Legionella effectors mediate host-pathogen interactions.

Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis.

Incorporation of locally produced signaling molecules into cell-derived vesicles may serve as an endogenous mediator delivery system. We recently reported that levels alpha-2-macroglobulin (A2MG)-containing microparticles are elevated in plasma from patients with sepsis. Herein, we investigated the immunomodulatory actions of A2MG containing microparticles during sepsis. Administration of A2MG-enriched (A2MG-E)-microparticles to mice with microbial sepsis protected against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates and reduced systemic inflammation. A2MG-E microparticles also enhanced survival in murine sepsis, an action lost in mice transfected with siRNA for LRP1, a putative A2MG receptor. In vitro, A2MG was functionally transferred onto endothelial cell plasma membranes from microparticles, augmenting neutrophil-endothelial adhesion. A2MG also modulated human leukocyte responses: enhanced bacterial phagocytosis, reactive oxygen species production, cathelicidin release, prevented endotoxin induced CXCR2 downregulation and preserved neutrophil chemotaxis in the presence of LPS. A significant association was also found between elevated plasma levels of A2MG-containing microparticles and survival in human sepsis patients. Taken together, these results identify A2MG enrichment in microparticles as an important host protective mechanism in sepsis.

The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.

The Gram-positive bacterium Listeria monocytogenes can enter the human central nervous system and cause life-threatening meningitis. During this process the pathogen has to invade and cross diverse cellular barriers involving the functions of the surface proteins Internalin (InlA) and InlB. Whereas the internalin-dependent crossing of the intestinal epithelium and the fetoplacental barrier have been subject to intensive investigation, limited research elucidating the crossing of the human blood-cerebrospinal fluid barrier (BCSFB) has been reported. We have recently established a functional in vitro model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells. We show polarized expression of receptors involved in listerial invasion (i.e. E-Cadherin, Met) in HIBCPP cells. Infecting HIBCPP cells with the L. monocytogenes strain EGD, we demonstrate polar invasion exclusively from the in vivo relevant basolateral cell side. Intracellular listeria were found in vacuoles and the cytoplasm, where they were often associated with "actin tail"-like structures. Furthermore, the L. monocytogenes wild type strain shows significantly higher internalization rates than isogenic mutants lacking either InlA, InlB or both surface proteins. Deletion of either one or both proteins leads to a similarly decreased invasion, suggesting an interdependent function of InlA and InlB during invasion of choroid plexus epithelial cells.

Enterocyte microvillus-derived vesicles detoxify bacterial products and regulate epithelial-microbial interactions.

The continuous monolayer of intestinal epithelial cells (IECs) lining the gut lumen functions as the site of nutrient absorption and as a physical barrier to prevent the translocation of microbes and associated toxic compounds into the peripheral vasculature. IECs also express host defense proteins such as intestinal alkaline phosphatase (IAP), which detoxify bacterial products and prevent intestinal inflammation. Our laboratory recently showed that IAP is enriched on vesicles that are released from the tips of IEC microvilli and accumulate in the intestinal lumen. Here, we show that these native "lumenal vesicles" (LVs) (1) contain catalytically active IAP that can dephosphorylate lipopolysaccharide (LPS), (2) cluster on the surface of native lumenal bacteria, (3) prevent the adherence of enteropathogenic E. coli (EPEC) to epithelial monolayers, and (4) limit bacterial population growth. We also find that IECs upregulate LV production in response to EPEC and other Gram-negative pathogens. Together, these results suggest that microvillar vesicle shedding represents a novel mechanism for distributing host defense machinery into the intestinal lumen and that microvillus-derived LVs modulate epithelial-microbial interactions.

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway.

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion.

Autophagy defends the mammalian cytosol against bacterial infection. Efficient pathogen engulfment is mediated by cargo-selecting autophagy adaptors that rely on unidentified pattern-recognition or danger receptors to label invading pathogens as autophagy cargo, typically by polyubiquitin coating. Here we show in human cells that galectin 8 (also known as LGALS8), a cytosolic lectin, is a danger receptor that restricts Salmonella proliferation. Galectin 8 monitors endosomal and lysosomal integrity and detects bacterial invasion by binding host glycans exposed on damaged Salmonella-containing vacuoles. By recruiting NDP52 (also known as CALCOCO2), galectin 8 activates antibacterial autophagy. Galectin-8-dependent recruitment of NDP52 to Salmonella-containing vesicles is transient and followed by ubiquitin-dependent NDP52 recruitment. Because galectin 8 also detects sterile damage to endosomes or lysosomes, as well as invasion by Listeria or Shigella, we suggest that galectin 8 serves as a versatile receptor for vesicle-damaging pathogens. Our results illustrate how cells deploy the danger receptor galectin 8 to combat infection by monitoring endosomal and lysosomal integrity on the basis of the specific lack of complex carbohydrates in the cytosol.

Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles.

The mechanism underlying bacterial conjugation through protozoa was investigated. Kanamycin-resistant Escherichia coli SM10λ+ carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. coli clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Page's amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10⁻6, but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10⁻8. Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.

Salmonella enterica replication in hemophagocytic macrophages requires two type three secretion systems.

Salmonella enterica serotype Typhimurium is a natural pathogen of mice, which acquire the bacteria orally and develop systemic acute infections that can become subacute to chronic infections. S. Typhimurium can reside within hemophagocytic macrophages (HMs) in SV129S6 mice, an Slc11a1/Nramp1(+/+) inbred strain. HMs are activated macrophages which have ingested viable hematopoietic cells and are a key characteristic of infectious and inflammatory diseases. Here we show that modest S. Typhimurium replication in HMs begins at 18 h postinfection, while activated macrophages kill the bacteria. For bacterial replication to occur, the phagocytosed viable cells must be grown to a low cell density and the multiplicity of infection must be low. HMs are able to kill phagocytosed Escherichia coli, produce reactive nitrogen species, and retain S. Typhimurium within membrane-bound vesicles. S. Typhimurium does not rescue E. coli upon coinfection of HMs. This indicates that S. Typhimurium does not cause HMs to become permissive for other microbes; rather, S. Typhimurium is especially equipped to survive within HMs. Two type three secretion systems (T3SS) encoded by S. Typhimurium are required for replication within HMs. While the T3SS within Salmonella pathogenicity island 2 (SPI-2) has been previously shown to be important for bacterial survival in cells, a role for SPI-1 in replication in macrophages has not been reported. The requirement for SPI-1 in HMs may help explain the role of SPI-1 during long-term colonization of mice.

Legionella pneumophila multiplication is enhanced by chronic AMPK signalling in mitochondrially diseased Dictyostelium cells.

Human patients with mitochondrial diseases are more susceptible to bacterial infections, particularly of the respiratory tract. To investigate the susceptibility of mitochondrially diseased cells to an intracellular bacterial respiratory pathogen, we exploited the advantages of Dictyostelium discoideum as an established model for mitochondrial disease and for Legionella pneumophila pathogenesis. Legionella infection of macrophages involves recruitment of mitochondria to the Legionella-containing phagosome. We confirm here that this also occurs in Dictyostelium and investigate the effect of mitochondrial dysfunction on host cell susceptibility to Legionella. In mitochondrially diseased Dictyostelium strains, the pathogen was taken up at normal rates, but it grew faster and reached counts that were twofold higher than in the wild-type host. We reported previously that other mitochondrial disease phenotypes for Dictyostelium are the result of the activity of an energy-sensing cellular alarm protein, AMP-activated protein kinase (AMPK). Here, we show that the increased ability of mitochondrially diseased cells to support Legionella proliferation is suppressed by antisense-inhibiting expression of the catalytic AMPKalpha subunit. Conversely, mitochondrial dysfunction is phenocopied, and intracellular Legionella growth is enhanced, by overexpressing an active form of AMPKalpha in otherwise normal cells. These results indicate that AMPK signalling in response to mitochondrial dysfunction enhances Legionella proliferation in host cells.

Neuritogenic actions of botulinum neurotoxin A on cultured motor neurons.

Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.

Bacterial particle endocytosis by epithelial cells is selective and enhanced by tumor necrosis factor receptor ligands.

Bacterial pathogens use virulence strategies to invade epithelial barriers, but active processes of epithelial cells may also contribute to the endocytosis of microbial particles. To focus on the latter, we studied the uptake of fixed and fluorescently labeled bacterial particles in intestinal and bronchoepithelial cell cultures and found it to be enhanced in Caco-2BBe and NCI-H292 cells after treatment with tumor necrosis factor alpha and an agonist antibody against the lymphotoxin beta receptor. Confocal fluorescence microscopy, flow cytometry, and transmission electron microscopy revealed that Staphylococcus aureus and Yersinia enterocolitica were readily endocytosed, although there was scant uptake of Shigella sonnei, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae particles. Endocytosed Staphylococcus was often associated with cytoplasmic claudin-4 vesicles; this was not found for Yersinia, suggesting that cytokine treatment upregulated two distinct endocytosis pathways. Interestingly, when Staphylococcus and Yersinia were coincubated with epithelial monolayers, the cells were unlikely to take up Yersinia unless they had also endocytosed large numbers of Staphylococcus particles, although the two bacteria were apparently processed in distinct compartments. Cytokine treatment induced an upregulation and redistribution of beta1 integrin to the apical surface of NCI-H292 cells; consistent with this effect, treatment with anti-beta1 integrin antibody blocked uptake of both Yersinia and Staphylococcus in NCI-H292 and Caco-2BBe cells. Our results suggest that capture of bacterial particles by mucosal epithelial cells is selective and that different endocytic mechanisms are enhanced by proinflammatory cytokines.

The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole.

Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.