Die Rolle von Wirtszellstress in der Infektion mit dem bakteriellen Krankheiserreger ShigellaShigella flexneriflexneri

The human-bacterial pathogen interaction is a complex process that results from a prolonged evolutionary arms race in the struggle for survival. The pathogen employs virulence strategies to achieve host colonization, and the latter counteracts using defense programs. The encounter of both organisms results in drastic physiological changes leading to stress, which is an ancient response accompanying infection. Recent evidence suggests that the stress response in the host converges with the innate immune pathways and influences the outcome of infection. However, the contribution of stress and the exact mechanism(s) of its involvement in host defense remain to be elucidated. Using the model bacterial pathogen Shigella flexneri, and comparing it with the closely related pathogen Salmonella Typhimurium, this study investigated the role of host stress in the outcome of infection. Shigella infection is characterized by a pronounced pro-inflammatory response that causes intense stress in host tissues, particularly the intestinal epithelium, which constitutes the first barrier against Shigella colonization. In this study, inflammatory stress was simulated in epithelial cells by inducing oxidative stress, hypoxia, and cytokine stimulation. Shigella infection of epithelial cells exposed to such stresses was strongly inhibited at the adhesion/binding stage. This resulted from the depletion of sphingolipidrafts in the plasma membrane by the stress-activated sphingomyelinases. Interestingly, Salmonella adhesion was not affected, by virtue of its flagellar motility, which allowed the gathering of bacteria at remaining membrane rafts. Moreover, the intracellular replication of Shigella lead to a similar sphingolipid-raft depletion in the membrane across adjacent cells inhibiting extracellular bacterial invasion. Additionally, this study shows that Shigella infection interferes with the host stress granule-formation in response to stress. Interestingly, infected cells exhibited a nuclear depletion of the global RNA-binding stress-granule associated proteins TIAR and TIA-1 and their accumulation in the cytoplasm. Overall, this work investigated different aspects of the host stress-response in the defense against bacterial infection. The findings shed light on the importance of the host stress-pathways during infection, and improve the understanding of different strategies in host-pathogen interaction.Die Interaktion von Mensch und bakteriellem Krankheitserreger ist ein komplexer Prozess, der aus dem anhaltenden evolutionären Wettrüsten im Kampf ums Überleben resultiert. Der Erreger setzt Virulenzstrategien zur Kolonisierung des Wirtes ein und dieser nutzt Verteidigungsprogramme um dem entgegenzuwirken. Die Begegnung der beiden Organismen resultiert in drastischen physiologischen Veränderungen, welche zu Stress führen, der eine klassische infektionsbegleitende Reaktion ist. Neuere Untersuchungen deuten darauf hin, dass die Stressantwort des Wirtes mit den Signalwegen der angeborenen Immunantwort konvergiert und im Ergebnis die Infektion beeinflusst. Jedoch bleiben die Bedeutung des Stresses und der exakte Mechanismus wie Stress an der Verteidigung des Wirtes beteiligt ist, noch zu klären. In dieser Studie dienten der bakterielle Krankheitserreger Shigella flexneri und vergleichend dazu der nah verwandte Erreger Salmonella Typhimurium als Modellorganismen, um die Rolle von Wirtszellstress für den Ausgang der Infektion zu untersuchen. Die Infektion mit Shigellen ist durch eine ausgeprägte pro-inflammatorische Reaktion gekennzeichnet. Diese versursacht in den Wirtsgeweben, insbesondere im Darmepithel, einen starken Stress, der die erste Barriere gegen die Besiedelung mit Shigellen darstellt. In der vorliegenden Arbeit wurde entzündlicher Stress in Epithelzellen durch die Induktion von oxidativem Stress, Hypoxie und Zytokinstimulation simuliert. Die Shigelleninfektion von Epithelzellen, die solchen Belastungen ausgesetzt waren, war stark im Adhäsions-/ Bindungsstadium gehemmt. Dies resultierte aus der Verarmung von Sphingolipidflößen in der Plasmamembran durch stressaktivierte Sphingomyelinasen. Interessanterweise wurde die Adhäsion von Salmonellen, aufgrund ihrer Flaggellenvermittelten Beweglichkeit, nicht beeinträchtigt und ermöglichte so die Ansammlung von Bakterien an den verbleibenden Membranflößen. Darüber hinaus führte die intrazelluläre Replikation von Shigellen zu einer ähnlichen Verminderung von Sphingolipidflößen in der Membran benachbarter Zellen, wodurch die extrazelluläre bakterielle Invasion gehemmt wurde. Zusätzlich zeigt diese Studie, dass eine Infektion mit Shigellen mit der Bildung von Stressgranula in der Wirtszelle interferiert. Interessanterweise zeigten infizierte Zellen eine nukleäre Depletion der globalen RNA-bindenden und Stressgranula assoziierten Proteine TIAR und TIA-1 sowie deren Akkumulation im Zytoplasma. Insgesamt untersuchte diese Arbeit verschiedene Aspekte der Stressreaktion der Wirtszelle bei der Verteidigung gegen bakterielle Infektionen. Die Ergebnisse beleuchten die Bedeutung der Stresssignalwege im Wirt während der Infektion und verbessern das Verständnis der verschiedenen Strategien in der Interaktion von Wirt und Krankheitserreger

A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins

Abstract Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins

RNA landscape of the emerging cancer-associated microbe Fusobacterium nucleatum.

Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered renewed attention for its association with several different human cancers. The growing interest in this emerging cancer-associated bacterium contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. As fusobacteria lack all established small RNA-associated proteins, post-transcriptional networks in these bacteria are also unknown. In the present study, using differential RNA-sequencing, we generate high-resolution global RNA maps for five clinically relevant fusobacterial strains-F. nucleatum subspecies nucleatum, animalis, polymorphum and vincentii, as well as F. periodonticum-for early, mid-exponential growth and early stationary phase. These data are made available in an online browser, and we use these to uncover fundamental aspects of fusobacterial gene expression architecture and a suite of non-coding RNAs. Developing a vector for functional analysis of fusobacterial genes, we discover a conserved fusobacterial oxygen-induced small RNA, FoxI, which serves as a post-transcriptional repressor of the major outer membrane porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum adaptation to different environments, which may elucidate how these bacteria colonize different compartments of the human body

Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens.

While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in <i>Shigella</i> capture by filopodia

<div><p>MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by <i>Shigella flexneri</i>, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors <i>Shigella</i> infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of <i>Shigella</i> binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing <i>Shigella</i> capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon <i>Shigella</i> replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient <i>Shigella</i> capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract <i>Shigella</i> infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of <i>Shigella</i> with host cells.</p></div

Increase of <i>Shigella</i> infection induced by miR-29b-2-5p is dependent on multiple targets.

<p>A. Schematic of the workflow for identification of miR-29b-2-5p targets relevant during <i>Shigella</i> infection. Transcriptomic analysis revealed fifty-two genes down-regulated by miR-29b-2-5p overexpression (≥1.5-fold) and up-regulated upon <i>Shigella</i> infection (≥2-fold, <i>Shigella</i> + cell population; 6 hpi). A RNAi screening targeting 46 of these genes was performed to identify siRNAs able to recapitulate the phenotype of miR-29b-2-5p. B. Percentage of HeLa cells infected with <i>Shigella</i> WT upon treatment with control siRNA or siRNAs targeting 33 genes, selected as described in Fig 3A. SiRNAs that decreased cell viability to less than 65% of control were excluded. MiR-29b-2-5p is shown for comparison. Results are shown normalized to control siRNA. C. Representative images of HeLa cells infected with <i>Shigella</i> WT, upon treatment with the 6 siRNAs that increase percentage of infected cells by at least 2.5-fold, compared with control siRNA. Cells treated with control siRNA and miR-29b-2-5p mimic are shown for comparison. Scale bar, 100 μm. D. UNC5C expression in HeLa cells infected with <i>Shigella</i> WT (MOI 10 and 100), at 0.5, 3 and 6 hpi. Results are shown normalized to mock-treated cells. E. UNC5C expression in HeLa cells treated with miR-29b-2-5p mimic. Results are shown normalized to cells transfected with control miRNA mimic. F. Schematic representation of the UNC5C 3'UTR constructs used for the miRNA binding site reporter assays and of the two identified regions of miR-29b-2-5p complementarity to the UNC5C 3’UTR. Shaded regions denote the UNC5C 3’UTR regions deleted in the reporter constructs. G. Results of the luciferase reporter assays. Luciferase activity in cells treated with miR-29b-2-5 is shown compared to control miRNA mimic. For panels B and C, <i>Shigella</i> infection was performed at MOI 10 and analyzed at 6 hpi. Results are shown as mean ± s.e.m. from 3 (panel B and E), 4 (panel D), >5 (panel G) independent experiments; *P<0.05, ***P<0.001.</p

MiR-29b-2-5p is decreased upon <i>Shigella</i> infection.

<p>A. Schematic representation of the pri-miR-29b-1/a and pri-miR-29b-2/c genomic loci, located respectively on Chr.7q32.3 and Chr.1q32.2. Precursor miRNAs are represented as hairpins in the diagram and exons as black boxes. The sequences of the mature miRNAs derived from the pri-miR-29b-1/a and pri-miR-29b-2/c precursors are shown below the diagram. The unique seed sequence of hsa-miR-29b-2-5p is highlighted in red and the seed sequence common to hsa-miR-29a/b/c-3p is highlighted in gray. B. Levels of mature miR-29b-2-5p in HeLa cells infected with <i>Shigella</i> WT (MOI 10 and 100), determined at 0.5, 3 and 6 hpi. C. Mature miR-29b-2-5p levels in HeLa cells infected with <i>Shigella</i> WT, ΔIcsA (defective in intercellular spreading), ΔIpaB (able to bind, but invasion deficient) and BS176 (unable to bind and invade) strains, or incubated with heat-killed <i>Shigella</i>. Infection was performed at MOI 10 and 100 for <i>Shigella</i> WT and MOI 100 and 250 for all other strains, and analyzed at 6 hpi. D. Levels of mature miR-29b-2-5p, miR-29b-3p, miR-29c-5p and miR-29c-3p in the total cell population, <i>Shigella</i> + and <i>Shigella</i> - fractions, at 6 hpi. HeLa cells were infected with <i>Shigella</i> WT expressing GFP at MOI 10 and subjected to cell sorting to separate the population of cells with internalized bacteria (<i>Shigella</i> +) and bystander cells (<i>Shigella</i> -). E. Expression levels of pri-miR-29b-2/c in the total cell population, <i>Shigella</i> + and <i>Shigella</i> - fractions, at 6 hpi. F. Levels of miR-29b-2-5p in HeLa cells infected with <i>Shigella</i> WT (MOI 100), determined at 6 hpi, in cells transfected with control siRNA or with siRNAs targeting RRP41, PNPT1 or XRN1, which have been involved in mature miRNA degradation. Expression levels of pri-miR-29b-2/c and mature miRNAs was determined by qRT-PCR. Results are normalized to mock-treated cells and shown as mean ± s.e.m. from 3 (panels B, D and E) or 5 (panel C and F) independent experiments; *P<0.05, **P<0.01, ***P<0.001.</p

MiR-29b-2-5p favors <i>Shigella</i> binding and intracellular replication.

<p>A. Effect of human miRNA mimics (genome-wide library of miRNA mimics corresponding to miRBase 19) on the percentage of <i>Shigella</i> infected cells (expressed as log2 fold change compared to control miRNA). MiRNAs highlighted in blue and red significantly increase or decrease infection by at least 2-fold, respectively (P<0.05). B and C. Representative images (B) and cfu quantification (C) of intracellular bacteria in HeLa cells infected with <i>Shigella</i> WT, upon treatment with miR-29b-2-5p or control miRNA mimics, and analyzed at three times post-infection (0.5, 3 and 6 hpi). Scale bar, 20 μm. D and E. Representative images (D) and cfu quantification (E) of bacteria bound to HeLa cells transfected with miR-29b-2-5p or control miRNA mimics and incubated with <i>Shigella</i> WT or ΔIpaB mutant strain for 10 min. Scale bar, 20 μm. F. Distribution of the number of <i>Shigella</i> per infected cell at different times post-infection (binding, 0.5, 3 and 6 hpi), in HeLa cells transfected with miR-29b-2-5p or control miRNA mimics. Results are shown for at least 50 infected cells per condition and independent experiment. Values in the X-axis correspond to the extremities of the defined bins. G. Percentage of 7-AAD positive cells following treatment with miR-29b-2-5p or control miRNA mimics for the cell population with internalized <i>Shigella</i> (<i>Shigella</i> +), analyzed at 3 and 6 hpi. <i>Shigella</i> infection was performed at MOI 10, except for the binding experiments (panels D and E) in which MOI 50 was used. Results are shown as mean ± s.e.m. from 4 (panel F), 5 (panels C and E) or 15 (panel G) independent experiments; *P<0.05, ***P<0.001.</p