A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

Brucella, the agent causing brucellosis, is a major zoonotic pathogen with worldwide distribution. Brucella resides and replicates inside infected host cells in membrane-bound compartments called Brucella- containing vacuoles (BCVs). Following uptake, Brucella resides in endosomal BCVs (eBCVs) that gradually mature from early to late endosomal features. Through a poorly understood process that is key to the intracellular lifestyle of Brucella, the eBCV escapes fusion with lysosomes by transitioning to the replicative BCV (rBCV), a replicative niche directly connected to the endoplasmic reticulum (ER). Despite the notion that this complex intracellular lifestyle must depend on a multitude of host factors, a holistic view on which of these components control Brucella cell entry, trafficking, and replication is still missing. Here we used a systematic cell-based small interfering RNA (siRNA) knockdown screen in HeLa cells infected with Brucella abortus and identified 425 components of the human infectome for Brucella infection. These include multiple components of pathways involved in central processes such as the cell cycle, actin cytoskeleton dynamics, or vesicular trafficking. Using assays for pathogen entry, knockdown complementation, and colocalization at single-cell resolution, we identified the requirement of the VPS retromer for Brucella to escape the lysosomal degradative pathway and to establish its intracellular replicative niche. We thus validated the VPS retromer as a novel host factor critical for Brucella intracellular trafficking. Further, our genomewide data shed light on the interplay between central host processes and the biogenesis of the Brucella replicative niche.; IMPORTANCE; With >300,000 new cases of human brucellosis annually, Brucella is regarded as one of the most important zoonotic bacterial pathogens worldwide. The agent causing brucellosis resides inside host cells within vacuoles termed Brucella- containing vacuoles (BCVs). Although a few host components required to escape the degradative lysosomal pathway and to establish the ER-derived replicative BCV (rBCV) have already been identified, the global understanding of this highly coordinated process is still partial, and many factors remain unknown. To gain deeper insight into these fundamental questions, we performed a genomewide RNA interference (RNAi) screen aiming at discovering novel host factors involved in the Brucella intracellular cycle. We identified 425 host proteins that contribute to Brucella cellular entry, intracellular trafficking, and replication. Together, this study sheds light on previously unknown host pathways required for the Brucella infection cycle and highlights the VPS retromer components as critical factors for the establishment of the Brucella intracellular replicative niche

Elucidation of a molecular mechanism controlling inflammation during bacterial infection

Recently we described a mechanism of gap junction-mediated communication between infected and uninfected epithelial cells that potentiates innate immunity during infection by the enteroinvasive bacterium Shigella flexneri. We showed that although S. flexneri secretes multiple effector proteins that downregulate inflammation in infected epithelial cells, NF-κB and the MAP kinases p38, JNK and ERK are activated in uninfected cells surrounding the sites of infection. The propagation of these proinflammatory signals leads to massive secretion of proinflammatory cytokines such as interleukin-8 (IL-8) by uninfected bystander cells. A genome wide RNAi-screen on Shigella-induced bystander activation confirmed the roles of the proteins TAK1 and NF-κB. Besides this, new candidates for bystander activation were found, including Na+/K+-ATPase (ATP1A1), the TRAF-interacting protein with a FHA domain (TIFA) and the TNF receptor-associated factor 6 (TRAF6). These proteins together with NOD1 and RIPK2, members of the NOD1 signaling pathway, which is induced by invasive Shigella, were studied in more detail. To our surprise we found that signals underlying cell-cell communication are produced independently of the receptor NOD1 and the downstream signaling proteins RIPK2, TAK1 and NF-κB as well as independent of TIFA and TRAF6. Unexpectedly, in bystander cells NOD1 and RIPK2 contribute to the proinflammatory response, whereas TAK1, NF-κB, TIFA and TRAF6 are indispensable for the production of cytokines. Furthermore, TIFA and TRAF6 are upstream of TAK1 and are required for TAK1 activation. In addition, selective stimulation of TIFA or TRAF6 depleted cells with the NOD1 ligand iE-DAP unraveled that TIFA and TRAF6 contribute to NOD1 signaling in bystander cells of S. flexneri infection. And finally, we propose a link between intercellular calcium signaling triggered by invasive S. flexneri and bystander IL-8 expression, since inhibition of calcium signals via a calcium chelator or inhibition of the IP3-receptor or phospholipase C (PLC) lead to a decreased bystander IL-8 response

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.Published versio

ALPK1 controls TIFA/TRAF6-dependent innate immunity against heptose-1,7-bisphosphate of gram-negative bacteria

<div><p>During infection by invasive bacteria, epithelial cells contribute to innate immunity via the local secretion of inflammatory cytokines. These are directly produced by infected cells or by uninfected bystanders via connexin-dependent cell-cell communication. However, the cellular pathways underlying this process remain largely unknown. Here we perform a genome-wide RNA interference screen and identify TIFA and TRAF6 as central players of <i>Shigella flexneri</i> and <i>Salmonella typhimurium</i>-induced interleukin-8 expression. We show that threonine 9 and the forkhead-associated domain of TIFA are necessary for the oligomerization of TIFA in both infected and bystander cells. Subsequently, this process triggers TRAF6 oligomerization and NF-κB activation. We demonstrate that TIFA/TRAF6-dependent cytokine expression is induced by the bacterial metabolite heptose-1,7-bisphosphate (HBP). In addition, we identify alpha-kinase 1 (ALPK1) as the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to infection with <i>S</i>. <i>flexneri</i> and <i>S</i>. <i>typhimurium</i> but also to <i>Neisseria meningitidis</i>. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.</p></div

TIFA and TRAF6 control <i>S</i>. <i>flexneri</i>-induced NF-κB activation.

<p><b>A)</b> TIFA and TRAF6 control <i>S</i>. <i>flexneri</i>-induced NF-κB activation in infected cells. HeLa cells were transfected with control, TIFA- or TRAF6-targeting siRNAs and infected with <i>S</i>. <i>flexneri</i> Δ<i>virG</i> (green) at MOI 20 for 60 minutes. After fixation, cells were stained for NF-κB p65 (red). <b>B</b>) Quantification of NF-κB translocation in infected cells after depletion of TIFA and TRAF6. NF-κB translocation was quantified by measuring the intensity ratio between the nucleus and the cytoplasm by automated image analysis, defining a threshold ratio and quantifying the fraction of NF-κB positive cells. Data correspond to the mean +/- SD of triplicate wells from a representative of 3 independent experiments, p***<0.0005. <b>C</b>) TIFA and TRAF6 control NF-κB activation both in infected and bystander cells. HeLa cells were treated as in A and infected at a MOI 0.5 (bacteria in green) for 60 minutes. After fixation, cells were stained for NF-κB p65 (red). <b>D</b>) Quantification of NF-κB translocation in bystander cells. The fluorescence intensity ratio between the cytoplasm and the nucleus was measured in bystander cells. Data correspond to the mean +/- SD of 3 independent experiments, p*<0.05. <b>E</b>) Impact of TIFA and TRAF6 depletion on PMA-induced NF-κB activation. After siRNA transfection, HeLa cells were stimulated with PMA (100 ng/ml) for 60 minutes. Data correspond to the mean +/- SD of 3 independent experiments, p**<0.005, ns: non-significant p>0.05. <b>F</b>) Impact of TIFA and TRAF6 depletion on TNFα-induced NF-κB activation. After siRNA transfection, HeLa cells were stimulated for 30 minutes with TNFα at the indicated concentrations. Data correspond to the mean +/- SD of 3 independent experiments, ns: non-significant p>0.05. <b>G</b>) Impact of TIFA and TRAF6 depletion on C12-iE-DAP-induced NF-κB activation. After siRNA transfection, HeLa cells were stimulated for 60 minutes with C12-iE-DAP at the indicated concentrations. Data correspond to the mean +/- SD of 3 independent experiments, non-significant p>0.05, p*<0.05, p***<0.0005.</p

Residue T9, the FHA domain and residue E178 of TIFA are necessary for IL-8 expression.

<p><b>A)</b> Schematic representation of wild-type TIFA and the T9, RKN and E178A TIFA mutants. <b>B</b>) Only wild-type TIFA rescues IL-8 expression after siRNA-mediated depletion of TIFA. HeLa cells were transfected for 72 hours with TIFA-targeting siRNA. 24 hours prior infection, cells were transfected with empty vector, wild-type or mutated TIFA cDNA constructs. All TIFA cDNA constructs are TIFA siRNA-resistant. Cells were infected with <i>S</i>. <i>flexneri</i> Δ<i>virG</i> (green) for 3.5 hours. After fixation, cells were stained for F-actin (grey), DNA (blue) and IL-8 (red). Scale bars, 20 μm. <b>C</b>) Quantification of IL-8 as shown in B. Data correspond to the mean +/- SD of 3 independent experiments, p***<0.0005, ns: non-significant p>0.05.</p

Sensing of HBP triggers TIFA/TRAF6-dependent innate immunity.

<p><b>A</b>) TIFA and TRAF6 are not involved in <i>L</i>. <i>monocytogenes</i>-induced IL-8 production. Cells were transfected with control, TIFA- or TRAF6-targeting siRNAs, infected with <i>L</i>. <i>monocytogenes</i> for 3.5 hours and stained for IL-8. Data show the mean +/- SD of 3 independent experiments, ns: p>0.05. <b>B</b>) <i>S</i>. <i>typhimurium</i> infection induces TIFA oligomers. Hela cells were transfected with wild-type TIFA cDNA, infected with <i>L</i>. <i>monocytogenes</i> or <i>S</i>. <i>typhimurium</i> for 45 minutes and stained for TIFA (green) and DNA (blue). Arrows indicate bacteria (red). <b>C</b>) TIFA and TRAF6 are involved in IL-8 expression after <i>S</i>. <i>typhimurium</i> infection. Cells were transfected as in A, infected with <i>S</i>. <i>typhimurium</i> for 3.5 hours and stained for IL-8. Data show the mean +/- SD of 3 independent experiments, p***<0.0005. <b>D</b>) HBP is required for IL-8 induction after <i>S</i>. <i>typhimurium</i> infection. Cells were infected with wt, Δ<i>hldE</i> or Δ<i>gmhB S</i>. <i>typhimurium</i> (green) and stained for IL-8 (red), F-actin (grey) and DNA (blue). <b>E</b>) Quantification of IL-8 after infection with wt, Δ<i>hldE</i> or Δ<i>gmhB S</i>. <i>typhimurium</i>. Data show the mean +/- SD of 3 independent experiments, p*<0.05. <b>F</b>) HBP is required for IL-8 expression after <i>S</i>. <i>flexneri</i> infection. Cells were infected with wt, Δ<i>hldE</i> or Δ<i>gmhB S</i>. <i>flexneri</i> (green) and stained as in D. Scale bars, 20 μm. <b>G</b>) Comparison of the infection rates after infection with wt, Δ<i>hldE</i> or Δ<i>gmhB S</i>. <i>flexneri</i> at multiple MOIs. Data show the mean +/- SD of triplicate wells, graph representative of 3 independent experiments. <b>H</b>) Quantification of IL-8 after infection with wt, Δ<i>hldE</i> or Δ<i>gmhB S</i>. <i>flexneri</i>. Data show the mean +/- SD of triplicate wells, graph representative of 3 independent experiments. <b>I</b>) TIFA oligomerization is HBP-dependent. Cells were transfected with TIFA cDNA and infected with wt, Δ<i>hldE</i> or Δ<i>gmhB S</i>. <i>flexneri</i> (red). Cells were stained for TIFA (green) and DNA (blue). <b>J</b>) IL-8 secretion of <i>S</i>. <i>flexneri</i>-infected Caco-2 cells is largely HBP-dependent. ELISA assay measuring the secretion of IL-8 after infection of Caco-2 cells. Cells were infected for 6 hours with wt (MOI 400), Δ<i>hldE</i> (MOI 4) or Δ<i>waaC</i> (MOI 4) <i>S</i>. <i>flexneri</i>. Data correspond to the mean +/- SD of 3 independent experiments, p*<0.05.</p

TIFA and TRAF6 form co-localizing oligomers in infected and bystander cells.

<p><b>A)</b> TIFA forms large oligomers in infected and bystander cells. HeLa cells were transfected with wild-type TIFA cDNA and infected or not with <i>S</i>. <i>flexneri</i> Δ<i>virG</i> (red) at MOI 0.5. Cells were stained for TIFA (green) and DNA (blue). <b>B</b>) Quantification of cells showing TIFA oligomers post infection. Cells were treated as in A. Cells showing TIFA punctuates were manually quantified for infected and bystander cells. Graph shows the mean of triplicate wells with a total of n = 130 cells per condition, data representative of 3 experiments. <b>C</b>) TIFA oligomerization occurs within minutes of infection in infected and bystander cells. HeLa cells were transfected with wt TIFA cDNA, infected or not for 15 minutes and co-stained for TIFA (green) and NF-κB p65 (red). Arrows indicate bacteria. <b>D</b>) Localization of wt, T9A, RKN and E178A TIFA mutants. Cells were transfected with wt TIFA or the different mutants, infected for 1 hour and stained as in A. Images are representative of three independent experiments. <b>E</b>) TRAF6 oligomerization is TIFA-dependent. HeLa cells were co-transfected with wild-type TIFA or E178A TIFA and Flag-TRAF6. After infection, cells were stained for TIFA (green) and Flag (red). Arrows indicate <i>S</i>. <i>flexneri</i>. <b>F</b>) Co-localizing TIFA and TRAF6 oligomers after <i>S</i>. <i>flexneri</i> infection in Caco-2 cells. Arrows indicate <i>S</i>. <i>flexneri</i>. Scale bars, 20 μm <b>G</b>) Co-immunoprecipitation of TIFA and TRAF6 after <i>S</i>. <i>flexneri</i> infection. HeLa cells were co-transfected with wt or E178A myc-TIFA and Flag-TRAF6 and infected for 1 hour at MOI 10. Myc IP was blotted with an anti-Flag antibody and the input lysate with anti-Flag and anti-myc antibodies. Data representative of two independent experiments.</p

RNAi screen reveals the roles of TIFA and TRAF6 in <i>S</i>. <i>flexneri</i> infection-induced IL-8 expression.

<p><b>A</b>) Schematic representation of the assay used to monitor IL-8 expression in the <i>S</i>. <i>flexneri</i> infection assay. <b>B</b>) Illustration of the image-based assay developed for the screen. HeLa cells were infected for 3.5 hours with <i>S</i>. <i>flexneri</i> Δ<i>virG</i> expressing dsRed under the control of the <i>uhpT</i> promoter (green). Cells were stained for F-actin (grey), DNA (blue) and IL-8 (red). Scale bars, 20 μm. <b>C</b>) Genome-wide RNAi screening data of IL-8 expression after <i>S</i>. <i>flexneri</i> infection in HeLa cells. IL-8 measurements were extracted with CellProfiler, Z-scored and ranked. <b>D</b>) Validation of the role of TIFA and TRAF6 in <i>S</i>. <i>flexneri</i> infection-induced IL-8. HeLa cells were transfected with control, TIFA- or TRAF6-targeting siRNAs and infected with <i>S</i>. <i>flexneri</i> Δ<i>virG</i> for 3.5 hours. Cells were stained as in B. <b>E</b>) Impact of TIFA and TRAF6 depletion on IL-8 expression. Quantification of cells producing IL-8 as shown in D by automated image analysis (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006224#sec011" target="_blank">Methods</a>). Data show the mean +/- SD of 3 independent experiments, p**<0.005, p***<0.0005. <b>F)</b> TIFA and TRAF6 control inflammation after wild-type <i>S</i>. <i>flexneri</i> infection of HeLa cells. Cells were treated as in D and infected with wild-type <i>S</i>. <i>flexneri</i> for 3.5 hours. <b>G)</b> Impact of TIFA and TRAF6 depletion on IL-8 expression. Quantification of cells producing IL-8 as shown in F. Data show the mean +/- SD of 3 independent experiments, p*<0.05. <b>H)</b> TIFA and TRAF6 regulate IL-8 expression in HEK293 cells. HEK293 cells were transfected and infected as in D. IL-8 was measured by image analysis. Data correspond to the mean +/- SD of 3 independent experiments, p***<0.0005. <b>I)</b> Images showing the implication of TIFA and TRAF6 in HEK293 cells after infection as quantified in H.</p