immunofluorescence-analysis-stress-granule-formation-after-bacterial

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Université Paris Descartes-Sorbonne Paris Cité

Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila, two Pax6 genes function in a pathway in which twin of eyeless (toy) directly regulates eyeless (ey), which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, we used different truncated forms of toy and small eyes (sey), and tested their capacity to induce ectopic eye development in an ey-independent manner. Even though the Paired domains of TOY and SEY have DNA-binding properties that differ from those of the Paired domain of EY, they all are capable of inducing ectopic eye development in an ey mutant background. We also show that one of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, we show that only the homeodomain (HD) of EY is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of TOY and EY, and significant evolutionary divergence between the two Drosophila Pax6 genes.

Functional divergence between eyeless and twin of eyeless in Drosophila melanogaster.

Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.

Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis.

Listeria monocytogenes is a bacterial pathogen that grows within the cytosol of infected host cells. Entry into the cytosol is largely mediated by a secreted bacterial cytolysin, listeriolysin O (LLO). In order to prevent host cell damage, the pore-forming activity of LLO is restricted to the phagosome. Compartmentalization of LLO requires a PEST-like sequence; PEST sequences can direct eukaryotic proteins for proteasomal degradation. Here we test the hypothesis that LLO's PEST-like sequence compartmentalizes pore-forming activity by targeting this bacterial protein for degradation in the host cytosol. We show that intracellular LLO was degraded in a proteasome-dependent manner, and that, prior to degradation, LLO was ubiquitinated and was phosphorylated within the PEST-like sequence. However, wild-type LLO and PEST region mutants had similarly short intracellular half-lives and both the wild-type and mutant proteins were stabilized by inhibitors of host proteasomes. Additionally, blocking host proteasomes did not cause toxicity in a wild-type infection, but enhanced the cytotoxicity of PEST region mutants. Together with the observation that PEST region mutants exhibit higher intracellular LLO levels than wild-type bacteria, these data suggest that LLO's PEST-like region does not mediate proteasomal degradation by the host, but controls LLO production in the cytosol.

Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence.

Listeria monocytogenes is an intracytosolic bacterial pathogen that escapes from the phagosome using a secreted cytolysin, listeriolysin O (LLO). In the host cytosol, LLO activity is minimized to prevent pore formation in the host plasma membrane; premature lysis of the infected host cell exposes the bacteria to extracellular immune defences of the host and is detrimental to infection. Here we identified nucleotide substitutions in the coding sequence of the LLO gene (hly) that did not alter the protein sequence, yet caused over-production of LLO, cytotoxicity and loss of virulence. These phenotypes were independent of the promoter and, under conditions in which the mutants produced more LLO protein than wild type, levels of hly mRNA were similar. Finally, negative regulation of LLO was maintained even when bacteria were engineered to produce elevated levels of the wild-type hly transcript. Together, our data demonstrate that translational regulation of LLO is critical for L. monocytogenes pathogenesis.

Regulated translation of listeriolysin O controls virulence of Listeria monocytogenes.

The intracellular pathogen Listeria monocytogenes escapes from a phagosomal compartment into the cytosol by secreting the pore-forming cytolysin listeriolysin O (LLO). During the proliferation of L. monocytogenes bacteria in the mammalian cell cytosol, the secreted LLO is targeted for degradation by the ubiquitin system. We report here that LLO is a substrate of the ubiquitin-dependent N-end rule pathway, which recognizes LLO through its N-terminal Lys residue. Specifically, we demonstrated by reverse-genetic and pharmacological methods that LLO was targeted for degradation by the N-end rule pathway in reticulocyte extracts and mouse NIH 3T3 cells and after its secretion by intracellular bacteria into the mouse cell cytosol. Replacing the N-terminal Lys of LLO with a stabilizing residue such as Val increased the in vivo half-life of LLO but did not strongly affect the intracellular growth or virulence of L. monocytogenes. Nevertheless, this replacement decreased the virulence of L. monocytogenes by nearly twofold, suggesting that a destabilizing N-terminal residue of LLO may stem from positive selection during the evolution of this and related bacteria. A double mutant strain of L. monocytogenes in which upregulated secretion of LLO was combined with a stabilizing N-terminal residue was severely toxic to infected mammalian cells, resulting in reduced intracellular growth of bacteria and an approximately 100-fold-lower level of virulence. In summary, we showed that LLO is degraded by the N-end rule pathway and that the degradation of LLO can reduce the toxicity of L. monocytogenes during infection, a property of LLO that may have been selected for its positive effects on fitness during the evolution of L. monocytogenes.

Listeriolysin O secreted by Listeria monocytogenes into the host cell cytosol is degraded by the N-end rule pathway.

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin family and a primary virulence factor of the gram-positive, facultative intracellular pathogen Listeria monocytogenes. During the intracellular life cycle of L. monocytogenes, LLO is largely responsible for mediating rupture of the phagosomal membrane, thereby allowing the bacterium access to the host cytosol, its replicative niche. In the host cytosol, LLO activity is controlled at numerous levels to prevent perforation of the plasma membrane and loss of the intracellular environment. In this review, we focus primarily on the role of LLO in phagosomal escape and the multiple regulatory mechanisms that control LLO activity in the host cytosol.

Listeriolysin O: a phagosome-specific lysin.

Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions.

Quantitative RT-PCR profiling of the rabbit immune response: assessment of acute Shigella flexneri infection.

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in β-defensin, IL-10, interferon-β, transforming growth factor-β, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.

Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers.

Segmented Filamentous Bacteria (SFB) are present in the gut microbiota of a large number of vertebrate species where they are found intimately attached to the intestinal epithelium. SFB has recently attracted considerable attention due to its outstanding capacity to stimulate innate and adaptive host immune responses without causing pathology. Recent genomic analysis placed SFB between obligate and facultative symbionts, unraveled its highly auxotrophic needs, and provided a rationale for the complex SFB life-style in close contact with the epithelium. Herein, we examine how the SFB life-style may underlie its potent immunostimulatory properties and discuss how the trade-off set up between SFB and its hosts can simultaneously help to establish and maintain the ecological niche of SFB in the intestine and drive the post-natal maturation of the host gut immune barrier.

Host interactions with Segmented Filamentous Bacteria: an unusual trade-off that drives the post-natal maturation of the gut immune system.

Numerous pathogenic Gram-negative bacteria use a type three secretion apparatus (T3SA) to translocate effector proteins into host cells. Detecting and monitoring the T3SA of intracellular bacteria within intact host cells has been challenging. Taking advantage of the tight coupling between T3S effector-gene transcription and T3SA activity in Shigella flexneri, together with a fast-maturing green fluorescent protein, we developed reporters to monitor T3SA activity in real time. These reporters reveal a dynamic temporal regulation of the T3SA during the course of infection. T3SA is activated initially during bacterial entry and downregulated subsequently when bacteria gain access to the host cell cytoplasm, allowing replenishment of the bacterial stores of T3S substrates necessary for invading neighboring cells. Reactivation of the T3SA was strictly dependent on actin-based motility and formation of plasma membrane protrusions during cell-to-cell spread. Thus, the T3SA is subject to a tight on/off regulation within the bacterial intracellular niche.

A fluorescent reporter reveals on/off regulation of the Shigella type III secretion apparatus during entry and cell-to-cell spread.

"Candidatus Arthromitus" sp. strain SFB-mouse-NL (SFB, segmented filamentous bacteria) is a commensal bacterium necessary for inducing the postnatal maturation of homeostatic innate and adaptive immune responses in the mouse gut. Here, we report the genome sequence of this bacterium, which sets it apart from earlier sequenced mouse SFB isolates.

Genome Sequence of "Candidatus Arthromitus" sp. Strain SFB-Mouse-NL, a Commensal Bacterium with a Key Role in Postnatal Maturation of Gut Immune Functions.

The gut microbiota plays a crucial role in the maturation of the intestinal mucosal immune system of its host. Within the thousand bacterial species present in the intestine, the symbiont segmented filamentous bacterium (SFB) is unique in its ability to potently stimulate the post-natal maturation of the B- and T-cell compartments and induce a striking increase in the small-intestinal Th17 responses. Unlike other commensals, SFB intimately attaches to absorptive epithelial cells in the ileum and cells overlying Peyer's patches. This colonization does not result in pathology; rather, it protects the host from pathogens. Yet, little is known about the SFB-host interaction that underlies the important immunostimulatory properties of SFB, because SFB have resisted in vitro culturing for more than 50 years. Here we grow mouse SFB outside their host in an SFB-host cell co-culturing system. Single-celled SFB isolated from monocolonized mice undergo filamentation, segmentation, and differentiation to release viable infectious particles, the intracellular offspring, which can colonize mice to induce signature immune responses. In vitro, intracellular offspring can attach to mouse and human host cells and recruit actin. In addition, SFB can potently stimulate the upregulation of host innate defence genes, inflammatory cytokines, and chemokines. In vitro culturing thereby mimics the in vivo niche, provides new insights into SFB growth requirements and their immunostimulatory potential, and makes possible the investigation of the complex developmental stages of SFB and the detailed dissection of the unique SFB-host interaction at the cellular and molecular levels.

Growth and host interaction of mouse segmented filamentous bacteria in vitro.

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.

Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation.

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 Shigella flexneri and Salmonella typhimurium-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 S. flexneri and S. typhimurium but also to Neisseria meningitidis. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.

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

The Th17 cell composition in the murine gut is strikingly dependent on the presence of the commensal segmented filamentous bacteria (SFB). SFB potently stimulates innate and adaptive immune responses and protects the host from pathogens both in and outside of the gut, partly due to its unique ability to promote a Th17-fostering environment. Recent work has highlighted the role of the tight adherence of SFB to the intestinal surface in mediating the potent immunostimulatory potential of SFB. Progress has also been made in our understanding of how SFB fosters this protective immune environment on the cellular and molecular level. This review focuses on the ability of SFB to specifically stimulate Th17 immunity.

Pamela Schnupf

Laboratory of Intestinal Immunity,
Institut Imagine-INSERM UMR 1163,
Laboratory of Intestinal Immunity, Institut Imagine-INSERM UMR 1163

Université Paris Descartes-Sorbonne Paris Cité

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Pamela Schnupf

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Intestinal Immunity,Institut Imagine-INSERM UMR 1163,Laboratory of Intestinal Immunity, Institut Imagine-INSERM UMR 1163

Université Paris Descartes-Sorbonne Paris Cité

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Laboratory of Intestinal Immunity,
Institut Imagine-INSERM UMR 1163,
Laboratory of Intestinal Immunity, Institut Imagine-INSERM UMR 1163