183 results on '"Hilbi H"'
Search Results
2. AMP-PNP bound nanodisc reconstituted MsbA with nanobodies, spin-labeled at position A60C
- Author
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Parey, K., primary, Januliene, D., additional, Galazzo, L., additional, Meier, G., additional, Vecchis, D., additional, Striednig, B., additional, Hilbi, H., additional, Schaefer, L.V., additional, Kuprov, I., additional, Bordignon, E., additional, Seeger, M.A., additional, and Moeller, A., additional
- Published
- 2022
- Full Text
- View/download PDF
3. Nanodisc reconstituted MsbA in complex with nanobodies, spin-labeled at position T68C
- Author
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Parey, K., primary, Januliene, D., additional, Galazzo, L., additional, Meier, G., additional, Vecchis, D., additional, Striednig, B., additional, Hilbi, H., additional, Schaefer, L.V., additional, Kuprov, I., additional, Bordignon, E., additional, Seeger, M.A., additional, and Moeller, A., additional
- Published
- 2022
- Full Text
- View/download PDF
4. Nanodisc reconstituted MsbA in complex with nanobodies, spin-labeled at position A60C
- Author
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Januliene, D., primary, Parey, K., additional, Galazzo, L., additional, Meier, G., additional, Vecchis, D., additional, Striednig, B., additional, Hilbi, H., additional, Schaefer, L.V., additional, Kuprov, I., additional, Bordignon, E., additional, Seeger, M.A., additional, and Moeller, A., additional
- Published
- 2022
- Full Text
- View/download PDF
5. Migration of Acanthamoeba castellanii through Legionella Biofilms
- Author
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Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Hochstrasser, R, Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), and Hochstrasser, R
- Published
- 2019
6. Single cell analysis of Legionella and Legionella-infected Acanthamoeba by agarose embedment
- Author
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Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Personnic, Nicolas; https://orcid.org/0000-0001-8995-4617, Striednig, Bianca; https://orcid.org/0000-0001-7575-8965, Hilbi, Hubert; https://orcid.org/0000-0002-5462-9301, Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Personnic, Nicolas; https://orcid.org/0000-0001-8995-4617, Striednig, Bianca; https://orcid.org/0000-0001-7575-8965, and Hilbi, Hubert; https://orcid.org/0000-0002-5462-9301
- Published
- 2019
7. Perturbation of Legionella infection by RNA interference
- Author
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Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Steiner, B, Swart, L, Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Steiner, B, and Swart, L
- Published
- 2019
8. Preface
- Author
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Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), and Hilbi, H ( H )
- Published
- 2019
9. Quantitative imaging flow cytometry of Legionella-containing vacuoles in dually fluorescence-labelled Dictyostelium
- Author
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Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Welin, A, Weber, S, Buchrieser, C, Hilbi, H, Buchrieser, C ( C ), Hilbi, H ( H ), Welin, A, and Weber, S
- Published
- 2019
10. Perturbation of Legionella infection by RNA interference
- Author
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Steiner, B, Swart, L, Hilbi, H, University of Zurich, Buchrieser, C, and Hilbi, H
- Subjects
10179 Institute of Medical Microbiology ,570 Life sciences ,biology ,610 Medicine & health - Published
- 2019
11. Structure of the Legionella pneumophila response regulator LqsR
- Author
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Hochstrasser, R., primary, Hutter, C.A.J., additional, Arnold, F.M., additional, Baerlocher, K., additional, Seeger, M.A., additional, and Hilbi, H., additional
- Published
- 2020
- Full Text
- View/download PDF
12. PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection
- Author
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Buckley, C.M., Heath, V.L., Guého, A., Bosmani, C., Knobloch, P., Sikakana, P., Personnic, N., Dove, S.K., Michell, R.H., Meier, R., Hilbi, H., Soldati, T., Insall, R.H., and King, J.S.
- Abstract
By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.
- Published
- 2019
13. Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase
- Author
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Hilbi, H. and Dimroth, P.
- Published
- 1994
- Full Text
- View/download PDF
14. Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress
- Author
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Hanna, N., primary, Burdet, F., additional, Melotti, A., additional, Bosmani, C., additional, Kicka, S., additional, Hilbi, H., additional, Cosson, P., additional, Pagni, M., additional, and Soldati, T., additional
- Published
- 2019
- Full Text
- View/download PDF
15. Amoebae-based screening reveals a novel family of compounds restricting intracellular Legionella
- Author
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Harrison, C F, Chiriano, G, Finsel, I, Manske, C, Hoffmann, C, Steiner, B, Kranjc, A, Patthey-Vuadens, O, Kicka, S, Trofimov, V, Ouertatani-Sakouhi, H, Soldati, T, Scapozza, L, Hilbi, H, University of Zurich, and Hilbi, H
- Subjects
10179 Institute of Medical Microbiology ,570 Life sciences ,biology ,610 Medicine & health ,2725 Infectious Diseases - Published
- 2015
16. Legionella pneumophila RidL N-terminal retromer binding domain
- Author
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Baerlocher, K., primary, Hutter, C.A.J., additional, Swart, A.L., additional, Steiner, B., additional, Welin, A., additional, Hohl, M., additional, Letourneur, F., additional, Seeger, M.A., additional, and Hilbi, H., additional
- Published
- 2017
- Full Text
- View/download PDF
17. Legionella pneumophila RidL N-terminal domain lacking beta hairpin
- Author
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Baerlocher, K., primary, Hutter, C.A.J., additional, Swart, A.L., additional, Steiner, B., additional, Welin, A., additional, Hohl, M., additional, Letourneur, F., additional, Seeger, M.A., additional, and Hilbi, H., additional
- Published
- 2017
- Full Text
- View/download PDF
18. IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid
- Author
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Zamboni, DS, Naujoks, J, Tabeling, C, Dill, BD, Hoffmann, C, Brown, AS, Kunze, M, Kempa, S, Peter, A, Mollenkopf, H-J, Dorhoi, A, Kershaw, O, Gruber, AD, Sander, LE, Witzenrath, M, Herold, S, Nerlich, A, Hocke, AC, van Driel, I, Suttorp, N, Bedoui, S, Hilbi, H, Trost, M, Opitz, B, Zamboni, DS, Naujoks, J, Tabeling, C, Dill, BD, Hoffmann, C, Brown, AS, Kunze, M, Kempa, S, Peter, A, Mollenkopf, H-J, Dorhoi, A, Kershaw, O, Gruber, AD, Sander, LE, Witzenrath, M, Herold, S, Nerlich, A, Hocke, AC, van Driel, I, Suttorp, N, Bedoui, S, Hilbi, H, Trost, M, and Opitz, B
- Abstract
Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.
- Published
- 2016
19. Identification of anti-infective compounds using amoebae
- Author
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Unden, Gottfried, Thines, Eckhard, Schuffler, Anja, Unden, G ( Gottfried ), Thines, E ( Eckhard ), Schuffler, A ( Anja ), Harrison, C F, Hilbi, H, Unden, Gottfried, Thines, Eckhard, Schuffler, Anja, Unden, G ( Gottfried ), Thines, E ( Eckhard ), Schuffler, A ( Anja ), Harrison, C F, and Hilbi, H
- Published
- 2016
20. Crystal structure of LppA from Legionella pneumophila
- Author
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Weber, S., primary, Stirnimann, C., additional, Wieser, M., additional, Meier, R., additional, Engelhardt, S., additional, Li, X., additional, Capitani, G., additional, Kammerer, R., additional, and Hilbi, H., additional
- Published
- 2014
- Full Text
- View/download PDF
21. Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions
- Author
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Tiaden, A, Spirig, T, Carranza, P, Brüggemann, H, Riedel, K, Eberl, L, Buchrieser, C, Hilbi, H, Tiaden, A, Spirig, T, Carranza, P, Brüggemann, H, Riedel, K, Eberl, L, Buchrieser, C, and Hilbi, H
- Abstract
The causative agent of Legionnaires' disease, Legionella pneumophila, is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase. L. pneumophila harbors the lqs (Legionella quorum sensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in Escherichia coli. LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterize L. pneumophila mutant strains lacking all four genes of the lqs cluster or only the hdeD gene. While an hdeD mutant strain did not have overt physiological or virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis, and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The deletion of the lqs cluster caused more-severe phenotypes than deletion of only lqsR, suggesting a synergistic effect of the other lqs genes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in the lqs mutant and wild-type L. pneumophila. Genes involved in protein production, metabolism, and bioenergetics were upregulated in the lqs mutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome analysis revealed that a set of Icm/Dot substrates is not produced in the absence of the lqs gene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of the lqs mutant strain.
- Published
- 2008
22. The interleukin 1beta-converting enzyme, caspase 1, is activated during Shigella flexneri-induced apoptosis in human monocyte-derived macrophages
- Author
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Hilbi, H, primary, Chen, Y, additional, Thirumalai, K, additional, and Zychlinsky, A, additional
- Published
- 1997
- Full Text
- View/download PDF
23. Macrophage apoptosis in microbial infections
- Author
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HILBI, H., primary, ZYCHLINSKY, A., additional, and SANSONETTI, P. J., additional
- Published
- 1997
- Full Text
- View/download PDF
24. Tripeptidyl peptidase II promotes maturation of caspase-1 in Shigella flexneri-induced macrophage apoptosis.
- Author
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Hilbi, H, Puro, R J, and Zychlinsky, A
- Abstract
The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages. Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) and clasto-lactacystin beta-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages from Shigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blocked Shigella-induced apoptosis by 95% at a concentration of 1 microM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect on Shigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1beta in Shigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability of Shigella to escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways.
- Published
- 2000
25. Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB.
- Author
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Hilbi, H, Moss, J E, Hersh, D, Chen, Y, Arondel, J, Banerjee, S, Flavell, R A, Yuan, J, Sansonetti, P J, and Zychlinsky, A
- Abstract
We report here that the Shigella invasion plasmid antigen (Ipa)B, which is sufficient to induce apoptosis in macrophages, binds to caspase (Casp)-1, but not to Casp-2 or Casp-3. Casp-1 is activated and its specific substrate interleukin-1beta is cleaved shortly after Shigella infection. Macrophages isolated from Casp-1 knock-out mice are not susceptible to Shigella-induced apoptosis, although they respond normally to other apoptotic stimuli. Shigella kills macrophages from casp-3, casp-11, and p53 knock-out mice as well as macrophages overexpressing Bcl-2. We propose that Shigella induces apoptosis by directly activating Casp-1 through IpaB, bypassing signal transduction events and caspases upstream of Casp-1. Taken together these data indicate that Shigella-induced apoptosis is distinct from other forms of apoptosis and seems uniquely dependent on Casp-1.
- Published
- 1998
26. Legionella: A versatile model to study hostpathogen interactions
- Author
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Hilbi, H. and Carmen Buchrieser
27. Quantitative imaging flow cytometry of Legionella-containing vacuoles in dually fluorescence-labelled Dictyostelium
- Author
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Stephen Weber, Amanda Welin, Hubert Hilbi, University of Zurich, Buchrieser, C, Hilbi, H, and Welin, A
- Subjects
0303 health sciences ,biology ,medicine.diagnostic_test ,030306 microbiology ,Chemistry ,10179 Institute of Medical Microbiology ,Endoplasmic reticulum ,Cellular Vesicle ,610 Medicine & health ,Vacuole ,biology.organism_classification ,Legionella pneumophila ,Dictyostelium discoideum ,Cell biology ,Flow cytometry ,03 medical and health sciences ,1311 Genetics ,Fluorescence microscope ,medicine ,1312 Molecular Biology ,570 Life sciences ,030304 developmental biology ,Phagosome - Abstract
Legionella pneumophila enters and replicates within protozoan and mammalian phagocytes by forming through a conserved mechanism a specialized intracellular compartment termed the Legionella-containing vacuole (LCV). This compartment avoids fusion with bactericidal lysosomes but communicates extensively with different cellular vesicle trafficking pathways and ultimately interacts closely with the endoplasmic reticulum. In order to delineate the process of pathogen vacuole formation and to better understand L. pneumophila virulence, an analysis of markers of the different trafficking pathways on the pathogen vacuole is crucial. Here, we describe a method for rapid, objective and quantitative analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we employ an imaging flow cytometry approach and use the D. discoideum -L. pneumophila infection model. Imaging flow cytometry enables quantification of many different parameters by fluorescence microscopy of cells in flow, rapidly producing statistically robust data from thousands of cells. We also describe the generation of D. discoideum strains simultaneously producing two different fluorescently tagged probes that enable visualization of compartments and processes in parallel. The quantitative imaging flow technique can be corroborated and enhanced by laser scanning confocal microscopy.
- Published
- 2019
28. Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment
- Author
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Personnic, Nicolas, Striednig, Bianca, Hilbi, Hubert, University of Zurich, Buchrieser, C, Hilbi, H, and Personnic, Nicolas
- Subjects
Fastidious organism ,0303 health sciences ,biology ,10179 Institute of Medical Microbiology ,030306 microbiology ,Legionella ,610 Medicine & health ,biology.organism_classification ,Legionella pneumophila ,Microbiology ,Acanthamoeba ,03 medical and health sciences ,1311 Genetics ,Single-cell analysis ,parasitic diseases ,1312 Molecular Biology ,570 Life sciences ,Protozoa ,Acanthamoeba castellanii ,Bacteria ,030304 developmental biology - Abstract
Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and its natural host A. castellanii at single cell level by using an agarose embedment assay. Agarose-embedded bacteria and infected cells can be monitored over several hours up to several days. Using properly adapted flow chambers, agarose-embedded specimens can be subjected to a wide range of fluctuating conditions.
- Published
- 2019
29. Migration of Acanthamoeba castellanii through Legionella Biofilms
- Author
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Hubert Hilbi, Ramon Hochstrasser, University of Zurich, Buchrieser, C, and Hilbi, H
- Subjects
food.ingredient ,Legionella ,610 Medicine & health ,Legionella pneumophila ,Microbiology ,Amoeba (genus) ,03 medical and health sciences ,Molecular level ,food ,1311 Genetics ,parasitic diseases ,1312 Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,10179 Institute of Medical Microbiology ,Biofilm ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,biology.organism_classification ,respiratory tract diseases ,bacteria ,Protozoa ,Acanthamoeba castellanii ,570 Life sciences ,Bacteria - Abstract
The amoeba-resistant bacterium Legionella pneumophila infects humans through aerosols and thereby can cause a life-threatening pneumonia termed Legionnaires' disease. In the environment L. pneumophila forms and colonizes biofilms, which usually comprise complex multispecies communities. In these biofilms L. pneumophila persists and replicates intracellularly in protozoa, such as the amoeba Acanthamoeba castellanii. The interactions between sessile L. pneumophila in biofilms and their natural protozoan hosts are not understood on a molecular level. Here, we describe a method to visualize by confocal microscopy the formation and architecture of mono-species L. pneumophila biofilms. Furthermore, we describe and quantify the migration or "grazing" of A. castellanii in the biofilm. This allows investigating on a molecular and cellular level L. pneumophila biofilm formation and Legionella-amoeba interactions within biofilms.
- Published
- 2019
30. Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions
- Author
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André N. Tiaden, Thomas Spirig, Hubert Hilbi, Carmen Buchrieser, Kathrin Riedel, Leo Eberl, Holger Brüggemann, Paula Carranza, University of Zurich, and Hilbi, H
- Subjects
Proteome ,Mutant ,Virulence ,580 Plants (Botany) ,Microbiology ,Legionella pneumophila ,Cell Line ,Bacterial Proteins ,Microscopy, Electron, Transmission ,10126 Department of Plant and Microbial Biology ,Gene cluster ,1312 Molecular Biology ,Animals ,Molecular Biology ,Gene ,Oligonucleotide Array Sequence Analysis ,Sequence Deletion ,Molecular Biology of Pathogens ,Genetics ,Acanthamoeba castellanii ,Microscopy, Confocal ,biology ,Macrophages ,2404 Microbiology ,biology.organism_classification ,Phenotype ,Quorum sensing ,Multigene Family ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Host-Pathogen Interactions ,Electrophoresis, Polyacrylamide Gel ,Autoinducer ,Genome, Bacterial - Abstract
The causative agent of Legionnaires' disease, Legionella pneumophila , is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase. L. pneumophila harbors the lqs ( Legionella q uorum s ensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in Escherichia coli . LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterize L. pneumophila mutant strains lacking all four genes of the lqs cluster or only the hdeD gene. While an hdeD mutant strain did not have overt physiological or virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis, and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The deletion of the lqs cluster caused more-severe phenotypes than deletion of only lqsR , suggesting a synergistic effect of the other lqs genes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in the lqs mutant and wild-type L. pneumophila . Genes involved in protein production, metabolism, and bioenergetics were upregulated in the lqs mutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome analysis revealed that a set of Icm/Dot substrates is not produced in the absence of the lqs gene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of the lqs mutant strain.
- Published
- 2008
- Full Text
- View/download PDF
31. Small molecule communication of Legionella : the ins and outs of autoinducer and nitric oxide signaling.
- Author
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Michaelis S, Gomez-Valero L, Chen T, Schmid C, Buchrieser C, and Hilbi H
- Subjects
- Humans, Lactones metabolism, Quorum Sensing, Homoserine analogs & derivatives, Homoserine metabolism, Legionnaires' Disease microbiology, Legionnaires' Disease metabolism, Bacterial Proteins metabolism, Legionella metabolism, Animals, Nitric Oxide metabolism, Signal Transduction, Legionella pneumophila metabolism
- Abstract
SUMMARY Legionella pneumophila is a Gram-negative environmental bacterium, which survives in planktonic form, colonizes biofilms, and infects protozoa. Upon inhalation of Legionella -contaminated aerosols, the opportunistic pathogen replicates within and destroys alveolar macrophages, thereby causing a severe pneumonia termed Legionnaires' disease. Gram-negative bacteria employ low molecular weight organic compounds as well as the inorganic gas nitric oxide (NO) for cell-cell communication. L. pneumophila produces, secretes, and detects the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1, 3-hydroxypentadecane-4-one). LAI-1 is secreted by L. pneumophila in outer membrane vesicles and not only promotes communication among bacteria but also triggers responses from eukaryotic cells. L. pneumophila detects NO through three different receptors, and signaling through the volatile molecule translates into fluctuations of the intracellular second messenger cyclic-di-guanylate monophosphate. The LAI-1 and NO signaling pathways are linked via the pleiotropic transcription factor LvbR. In this review, we summarize current knowledge about inter-bacterial and inter-kingdom signaling through LAI-1 and NO by Legionella species., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
- Full Text
- View/download PDF
32. Nitric oxide signaling through three receptors regulates virulence, biofilm formation, and phenotypic heterogeneity of Legionella pneumophila .
- Author
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Michaelis S, Chen T, Schmid C, and Hilbi H
- Subjects
- Virulence, Phenotype, Macrophages microbiology, Quorum Sensing, Biofilms growth & development, Legionella pneumophila genetics, Legionella pneumophila pathogenicity, Legionella pneumophila physiology, Legionella pneumophila metabolism, Nitric Oxide metabolism, Signal Transduction, Gene Expression Regulation, Bacterial, Bacterial Proteins genetics, Bacterial Proteins metabolism
- Abstract
The causative agent of Legionnaires' disease, Legionella pneumophila , is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. L. pneumophila employs the Legionella quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of L. pneumophila to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (P
flaA ) and the 6S small regulatory RNA (P6SRNA ). Marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of PflaA expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the Δ nosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that L. pneumophila regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP., Importance: The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation., Competing Interests: The authors declare no conflict of interest.- Published
- 2024
- Full Text
- View/download PDF
33. The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling.
- Author
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Fan M, Kiefer P, Charki P, Hedberg C, Seibel J, Vorholt JA, and Hilbi H
- Subjects
- Humans, Bacterial Proteins genetics, Dictyostelium, Escherichia coli, Legionella, Legionnaires' Disease microbiology, Legionella pneumophila physiology, Quorum Sensing
- Abstract
Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (P
flaA or P6SRNA ), but not under control of its endogenous promoter (PlqsA ), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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34. ER-dependent membrane repair of mycobacteria-induced vacuole damage.
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Anand A, Mazur A-C, Rosell-Arevalo P, Franzkoch R, Breitsprecher L, Listian SA, Hüttel SV, Müller D, Schäfer DG, Vormittag S, Hilbi H, Maniak M, Gutierrez MG, and Barisch C
- Subjects
- Humans, Vacuoles metabolism, Endoplasmic Reticulum, Dictyostelium microbiology, Mycobacterium marinum metabolism, Mycobacterium tuberculosis metabolism, Tuberculosis metabolism
- Abstract
Importance: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis , the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum / Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis ., Competing Interests: The authors declare no conflict of interest.
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- 2023
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35. Tapping lipid droplets: A rich fat diet of intracellular bacterial pathogens.
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Hüsler D, Stauffer P, and Hilbi H
- Subjects
- Lipid Metabolism, Lipid Droplets metabolism, Diet
- Abstract
Lipid droplets (LDs) are dynamic and versatile organelles present in most eukaryotic cells. LDs consist of a hydrophobic core of neutral lipids, a phospholipid monolayer coat, and a variety of associated proteins. LDs are formed at the endoplasmic reticulum and have diverse roles in lipid storage, energy metabolism, membrane trafficking, and cellular signaling. In addition to their physiological cellular functions, LDs have been implicated in the pathogenesis of several diseases, including metabolic disorders, cancer, and infections. A number of intracellular bacterial pathogens modulate and/or interact with LDs during host cell infection. Members of the genera Mycobacterium, Legionella, Coxiella, Chlamydia, and Salmonella exploit LDs as a source of intracellular nutrients and membrane components to establish their distinct intracellular replicative niches. In this review, we focus on the biogenesis, interactions, and functions of LDs, as well as on their role in lipid metabolism of intracellular bacterial pathogens., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)
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- 2023
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36. The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila .
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Hüsler D, Stauffer P, Keller B, Böck D, Steiner T, Ostrzinski A, Vormittag S, Striednig B, Swart AL, Letourneur F, Maaß S, Becher D, Eisenreich W, Pilhofer M, and Hilbi H
- Subjects
- Humans, GTP Phosphohydrolases metabolism, Macrophages metabolism, Lipid Droplets metabolism, Vacuoles metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Legionella pneumophila metabolism, Dictyostelium metabolism, Legionella metabolism, Legionnaires' Disease microbiology
- Abstract
The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella -containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum . Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δ sey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila ., Competing Interests: DH, PS, BK, DB, TS, AO, SV, BS, AS, FL, SM, DB, WE, MP, HH No competing interests declared, (© 2023, Hüsler et al.)
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- 2023
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37. Pathogen vacuole membrane contact sites - close encounters of the fifth kind.
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Vormittag S, Ende RJ, Derré I, and Hilbi H
- Abstract
Vesicular trafficking and membrane fusion are well-characterized, versatile, and sophisticated means of 'long range' intracellular protein and lipid delivery. Membrane contact sites (MCS) have been studied in far less detail, but are crucial for 'short range' (10-30 nm) communication between organelles, as well as between pathogen vacuoles and organelles. MCS are specialized in the non-vesicular trafficking of small molecules such as calcium and lipids. Pivotal MCS components important for lipid transfer are the VAP receptor/tether protein, oxysterol binding proteins (OSBPs), the ceramide transport protein CERT, the phosphoinositide phosphatase Sac1, and the lipid phosphatidylinositol 4-phosphate (PtdIns(4) P ). In this review, we discuss how these MCS components are subverted by bacterial pathogens and their secreted effector proteins to promote intracellular survival and replication., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)
- Published
- 2023
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38. Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.
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Vormittag S, Hüsler D, Haneburger I, Kroniger T, Anand A, Prantl M, Barisch C, Maaß S, Becher D, Letourneur F, and Hilbi H
- Subjects
- Vacuoles metabolism, Phosphatidylinositols metabolism, Membrane Proteins metabolism, Bacterial Proteins metabolism, Legionella metabolism, Dictyostelium microbiology, Legionella pneumophila
- Abstract
Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation., (© 2022 The Authors.)
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- 2023
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39. Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyostelium.
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Welin A, Hüsler D, and Hilbi H
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- Animals, Vacuoles metabolism, Flow Cytometry, Kinetics, Bacterial Proteins metabolism, Mammals, Legionella, Dictyostelium genetics, Dictyostelium metabolism, Legionella pneumophila genetics, Legionnaires' Disease metabolism
- Abstract
The causative agent of a severe pneumonia termed "Legionnaires' disease", Legionella pneumophila, replicates within protozoan and mammalian phagocytes in a specialized intracellular compartment called the Legionella-containing vacuole (LCV). This compartment does not fuse with bactericidal lysosomes but communicates extensively with several cellular vesicle trafficking pathways and eventually associates tightly with the endoplasmic reticulum. In order to comprehend in detail the complex process of LCV formation, the identification and kinetic analysis of cellular trafficking pathway markers on the pathogen vacuole are crucial. This chapter describes imaging flow cytometry (IFC)-based methods for the objective, quantitative and high-throughput analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we use the haploid amoeba Dictyostelium discoideum as an infection model for L. pneumophila, to analyze either fixed intact infected host cells or LCVs from homogenized amoebae. Parental strains and isogenic mutant amoebae are compared in order to determine the contribution of a specific host factor to LCV formation. The amoebae simultaneously produce two different fluorescently tagged probes enabling tandem quantification of two LCV markers in intact amoebae or the identification of LCVs using one probe and quantification of the other probe in host cell homogenates. The IFC approach allows rapid generation of statistically robust data from thousands of pathogen vacuoles and can be applied to other infection models., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2023
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40. 5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells.
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Ifrid E, Ouertatani-Sakouhi H, Jauslin T, Kicka S, Chiriano G, Harrison CF, Hilbi H, Scapozza L, Soldati T, and Cosson P
- Subjects
- Humans, Klebsiella pneumoniae genetics, Phagocytes, Anti-Bacterial Agents, Dictyostelium microbiology, Klebsiella Infections microbiology
- Abstract
Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 μM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
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41. The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells.
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Galazzo L, Meier G, Januliene D, Parey K, De Vecchis D, Striednig B, Hilbi H, Schäfer LV, Kuprov I, Moeller A, Bordignon E, and Seeger MA
- Subjects
- Adenosine Triphosphate metabolism, Bacterial Proteins metabolism, Detergents metabolism, Lipid A, Liposomes metabolism, Membrane Proteins metabolism, Protein Conformation, ATP-Binding Cassette Transporters chemistry, Escherichia coli metabolism, Escherichia coli Proteins chemistry
- Abstract
Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.
- Published
- 2022
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42. Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs-LvbR network, effector proteins and the flagellum.
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Hochstrasser R, Michaelis S, Brülisauer S, Sura T, Fan M, Maaß S, Becher D, and Hilbi H
- Subjects
- Bacterial Proteins metabolism, Biofilms, Flagella genetics, Flagella metabolism, Humans, Quorum Sensing, Acanthamoeba castellanii, Legionella metabolism, Legionella pneumophila genetics, Legionnaires' Disease
- Abstract
The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic 'fitness island', metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆lqsR or ∆flaA mutants form 'patchy' biofilms like the parental strain JR32, while ∆lvbR forms a 'mat-like' biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms but not in ∆lqsR or ∆flaA biofilms. The amoeba-adherent bacteria induced promoters implicated in motility (P
flaA ) or virulence (PsidC , PralF ). Taken together, the Lqs-LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and - apart from the T4SS - govern bacterial cluster formation on the amoebae., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)- Published
- 2022
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43. Bacterial quorum sensing and phenotypic heterogeneity: how the collective shapes the individual.
- Author
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Striednig B and Hilbi H
- Subjects
- Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Phenotype, Bacteria genetics, Bacteria metabolism, Quorum Sensing
- Abstract
Bacteria communicate with each other through a plethora of small, diffusible organic molecules called autoinducers. This cell-density-dependent regulatory principle is termed quorum sensing, and in many cases the process indeed coordinates group behavior of bacterial populations. Yet, even clonal bacterial populations are not uniform entities; rather, they adopt phenotypic heterogeneity to cope with consecutive, rapid, and frequent environmental fluctuations (bet-hedging) or to concurrently interact with each other by exerting different, often complementary, functions (division of labor). Quorum sensing is mainly regarded as a coordinator of bacterial collective behavior. However, it can also be a driver or a target of individual phenotypic heterogeneity. Hence, quorum sensing increases the overall fitness of a bacterial community by orchestrating group behavior as well as individual traits., Competing Interests: Declaration of interests There are no interests to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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44. The Legionella Lqs-LvbR Regulatory Network Controls Temperature-Dependent Growth Onset and Bacterial Cell Density.
- Author
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Hochstrasser R and Hilbi H
- Subjects
- Bacterial Proteins genetics, Cell Count, Humans, Quorum Sensing, Temperature, Legionella genetics, Legionella pneumophila, Legionnaires' Disease microbiology
- Abstract
Legionella species are facultative intracellular pathogens that cause a life-threatening pneumonia termed Legionnaires' disease. Legionella pneumophila employs the Lqs-LvbR ( Legionella quorum sensing- Legionella virulence and biofilm regulator) network to regulate virulence and motility, but its role for growth in media is ill-defined. Here, we report that compared to the L. pneumophila reference strain JR32, a Δ lqsR mutant showed a reduced lag phase at 30°C and reached a higher cell density at 45°C, while the Δ lqsA , Δ lqsS , and Δ lqsT mutants showed a longer lag phase and reached a lower cell density. A Δ lvbR mutant resumed growth like the parental strain at 30°C but exhibited a substantially reduced cell density at 45°C. Thus, LvbR is an important cell density regulator at elevated temperatures. Environmental and clinical L. pneumophila strains grew in N -(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) medium after distinct lag phases with similar rates at 30°C, reached different cell densities at the optimal growth temperature of 40°C, and no longer grew at 50°C. Legionella longbeachae reached a rather low cell density at 40°C and did not grow at and beyond 45°C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and upon growth at 30°C or 45°C, the P
lqsR , PlqsA , PlqsS , and PlvbR promoters from strain JR32 were expressed in these strains with distinct patterns. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32 and possibly also of environmental and clinical L. pneumophila isolates. IMPORTANCE Environmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires' disease, the incidence of which is on the rise worldwide. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a mechanism similar to that in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical, or biological means will reduce the incidence of Legionnaires' disease. Here, we show that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is determined not only by the temperature and nutrient availability but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.- Published
- 2022
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45. Microbe Profile: Legionella pneumophila - a copycat eukaryote.
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Hilbi H and Buchrieser C
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Eukaryota, Gene Transfer, Horizontal, Humans, Legionella pneumophila genetics, Legionella pneumophila metabolism
- Abstract
Legionella pneumophila is an environmental bacterium that parasitizes aquatic protozoa and uses the same processes to infect humans. The facultative intracellular pathogen causes a life-threatening pneumonia with possible systemic complications. The co-evolution with protozoa is reflected in an armoury of bacterial effectors, and many of these type IV-secreted proteins have likely been acquired by interdomain horizontal gene transfer (HGT) from hosts. The unique features of L. pneumophila are the largest bacterial effector repertoire known to date, subversion of virtually all eukaryotic signalling pathways and acquisition of eukaryotic enzyme activities used to manipulate the host cell to the pathogen's advantage.
- Published
- 2022
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46. Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection.
- Author
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Maffei E, Shaidullina A, Burkolter M, Heyer Y, Estermann F, Druelle V, Sauer P, Willi L, Michaelis S, Hilbi H, Thaler DS, and Harms A
- Subjects
- Escherichia coli immunology, Host Specificity, Immunity, Phenotype, Phylogeny, Polysaccharides metabolism, Receptors, Cell Surface metabolism, Salmonella virology, Viral Proteins metabolism, Coliphages physiology, Escherichia coli virology, Host-Pathogen Interactions physiology
- Abstract
Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage-host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage-host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages' host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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47. The Polar Legionella Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane.
- Author
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Böck D, Hüsler D, Steiner B, Medeiros JM, Welin A, Radomska KA, Hardt WD, Pilhofer M, and Hilbi H
- Subjects
- Bacterial Proteins genetics, Cell Polarity, Humans, Legionella pneumophila cytology, Legionella pneumophila genetics, Protein Transport, Type IV Secretion Systems genetics, Bacterial Proteins metabolism, Legionella pneumophila metabolism, Legionnaires' Disease microbiology, Type IV Secretion Systems metabolism, Vacuoles microbiology
- Abstract
Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, termed the " Legionella -containing vacuole" (LCV). To form an LCV and subvert pivotal host pathways, L. pneumophila employs a type IV secretion system (T4SS), which translocates more than 300 different effector proteins into the host cell. The L. pneumophila T4SS complex has been shown to span the bacterial cell envelope at the bacterial poles. However, the interactions between the T4SS and the LCV membrane are not understood. Using cryo-focused ion beam milling, cryo-electron tomography, and confocal laser scanning fluorescence microscopy, we show that up to half of the intravacuolar L. pneumophila bacteria tether their cell pole to the LCV membrane. Tethering coincides with the presence and function of T4SSs and likely promotes the establishment of distinct contact sites between T4SSs and the LCV membrane. Contact sites are characterized by indentations in the limiting LCV membrane and localize juxtaposed to T4SS machineries. The data are in agreement with the notion that effector translocation occurs by close membrane contact rather than by an extended pilus. Our findings provide novel insights into the interactions of the L. pneumophila T4SS with the LCV membrane in situ . IMPORTANCE Legionnaires' disease is a life-threatening pneumonia, which is characterized by high fever, coughing, shortness of breath, muscle pain, and headache. The disease is caused by the amoeba-resistant bacterium L. pneumophila found in various soil and aquatic environments and is transmitted to humans via the inhalation of small bacteria-containing droplets. An essential virulence factor of L. pneumophila is a so-called "type IV secretion system" (T4SS), which, by injecting a plethora of "effector proteins" into the host cell, determines pathogen-host interactions and the formation of a distinct intracellular compartment, the " Legionella -containing vacuole" (LCV). It is unknown how the T4SS makes contact to the LCV membrane to deliver the effectors. In this study, we identify indentations in the host cell membrane in close proximity to functional T4SSs localizing at the bacterial poles. Our work reveals first insights into the architecture of Legionella -LCV contact sites.
- Published
- 2021
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48. Dictyostelium Dynamin Superfamily GTPases Implicated in Vesicle Trafficking and Host-Pathogen Interactions.
- Author
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Katic A, Hüsler D, Letourneur F, and Hilbi H
- Abstract
The haploid social amoeba Dictyostelium discoideum is a powerful model organism to study vesicle trafficking, motility and migration, cell division, developmental processes, and host cell-pathogen interactions. Dynamin superfamily proteins (DSPs) are large GTPases, which promote membrane fission and fusion, as well as membrane-independent cellular processes. Accordingly, DSPs play crucial roles for vesicle biogenesis and transport, organelle homeostasis, cytokinesis and cell-autonomous immunity. Major progress has been made over the last years in elucidating the function and structure of mammalian DSPs. D. discoideum produces at least eight DSPs, which are involved in membrane dynamics and other processes. The function and structure of these large GTPases has not been fully explored, despite the elaborate genetic and cell biological tools available for D. discoideum . In this review, we focus on the current knowledge about mammalian and D. discoideum DSPs, and we advocate the use of the genetically tractable amoeba to further study the role of DSPs in cell and infection biology. Particular emphasis is put on the virulence mechanisms of the facultative intracellular bacterium Legionella pneumophila ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Katic, Hüsler, Letourneur and Hilbi.)
- Published
- 2021
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49. Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery.
- Author
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Striednig B, Lanner U, Niggli S, Katic A, Vormittag S, Brülisauer S, Hochstrasser R, Kaech A, Welin A, Flieger A, Ziegler U, Schmidt A, Hilbi H, and Personnic N
- Subjects
- Bacterial Proteins genetics, Humans, Quorum Sensing, Vacuoles, Legionella genetics, Legionella pneumophila genetics, Legionnaires' Disease
- Abstract
The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non-virulent and a non-replicating, virulent/transmissive phase. Here, we show on a single-cell level that at late stages of infection, individual motile (P
flaA -GFP-positive) and virulent (PralF - and PsidC -GFP-positive) L. pneumophila emerge in the cluster of non-growing bacteria within an LCV. Comparative proteomics of PflaA -GFP-positive and PflaA -GFP-negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA -GFP-positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi-phasic life cycle of L. pneumophila., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)- Published
- 2021
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50. Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.
- Author
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Hüsler D, Steiner B, Welin A, Striednig B, Swart AL, Molle V, Hilbi H, and Letourneur F
- Subjects
- Dictyostelium growth & development, Dictyostelium microbiology, Dictyostelium ultrastructure, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum Stress, Endoplasmic Reticulum, Rough microbiology, Endoplasmic Reticulum, Rough physiology, GTP Phosphohydrolases genetics, Homeostasis, Host-Pathogen Interactions, Legionella pneumophila growth & development, Movement, Muramidase metabolism, Phosphatidylinositol Phosphates metabolism, Protozoan Proteins genetics, Vacuoles physiology, Dictyostelium physiology, Endoplasmic Reticulum ultrastructure, GTP Phosphohydrolases metabolism, Legionella pneumophila physiology, Protozoan Proteins metabolism, Vacuoles microbiology
- Abstract
Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila., (© 2021 John Wiley & Sons Ltd.)
- Published
- 2021
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