369 results on '"Host cell cytosol"'
Search Results
2. Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry
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Z. Hong Zhou, Yanxiang Cui, Xian Xia, Polly Roy, and Weining Wu
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Models, Molecular ,Microbiology (medical) ,Endosome ,viruses ,Immunology ,Perforation (oil well) ,Sheep Diseases ,Endosomes ,Bluetongue ,Applied Microbiology and Biotechnology ,Microbiology ,Article ,Virus ,03 medical and health sciences ,0302 clinical medicine ,Models ,Viral entry ,Genetics ,2.1 Biological and endogenous factors ,2.2 Factors relating to the physical environment ,Animals ,Aetiology ,030304 developmental biology ,0303 health sciences ,Sheep ,Host cell cytosol ,Chemistry ,Cryoelectron Microscopy ,Molecular ,Cell Biology ,Hydrogen-Ion Concentration ,Virus Internalization ,Virus Release ,Cytosol ,Infectious Diseases ,Capsid ,Medical Microbiology ,Viruses ,Biophysics ,Capsid Proteins ,Generic health relevance ,Infection ,Bluetongue virus ,030217 neurology & neurosurgery - Abstract
Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4–3.9 A show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated β-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses. Cryo-electron microscopy and cryo-electron tomography reveal conformational changes of the bluetongue virus capsid protein VP5 that lead to membrane perforation and virus release into the cytosol.
- Published
- 2021
3. Bacterial Proteases and Virulence
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Frees, Dorte, Brøndsted, Lone, Ingmer, Hanne, and Dougan, David A., editor
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- 2013
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4. Pasteurella multocida Toxin Interaction with Host Cells: Entry and Cellular Effects
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Wilson, Brenda A., Ho, Mengfei, Aktories, Klaus, editor, Orth, Joachim H.C., editor, and Adler, Ben, editor
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- 2012
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5. Lipases as Pathogenicity Factors of Bacterial Pathogens of Humans
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Bender, J., Flieger, A., and Timmis, Kenneth N., editor
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- 2010
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6. Bacterial Toxins Activating Rho GTPases
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Munro, P., Lemichez, E., Compans, R.W., editor, Cooper, M.D., editor, Honjo, T., editor, Koprowski, H., editor, Melchers, F., editor, Oldstone, M.B.A., editor, Olsnes, S., editor, Potter, M., editor, Vogt, P.K., editor, Wagner, H., editor, Boquet, Patrice, editor, and Lemichez, Emmanuel, editor
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- 2005
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7. DNA Vaccine Delivery by Attenuated Intracellular Bacteria
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Dietrich, Guido, Goebel, Werner, Harris, J. Robin, editor, Hilderson, H. J., editor, Biswas, B. B., editor, Oelschlaeger, Tobias A., editor, and Hacker, Jörg, editor
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- 2000
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8. Physical and Gentic Organisation of pNGR234a
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Perret, X., Freiberg, C., Rosenthal, A., Broughton, W. J., Martĺnez, Esperanza, editor, and Hernández, Georgina, editor
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- 1999
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9. The Mycobacterium marinum ESX-1 system mediates phagosomal permeabilization and type I interferon production via separable mechanisms
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Victoria Lovins, Elin Movert, Esther Nobs, Christine Valfridsson, Fredric Carlsson, and Julia Lienard
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0303 health sciences ,Multidisciplinary ,Host cell cytosol ,Innate immune system ,biology ,030306 microbiology ,Chemistry ,Mutant ,Mitochondrion ,Type I interferon production ,biology.organism_classification ,Cell biology ,03 medical and health sciences ,Cytosol ,Secretion ,Mycobacterium marinum ,030304 developmental biology - Abstract
Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.
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- 2019
10. A predation assay using amoebae to screen for virulence factors unearthed the first W. chondrophila inclusion membrane protein
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Ludovic Pilloux, C. Kebbi-Beghdadi, Antony Croxatto, N. Tosetti, Trestan Pillonel, Gilbert Greub, Pilloux, L [0000-0002-2133-1891], Pillonel, T [0000-0002-5725-7929], Greub, G [0000-0001-9529-3317], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Phylum Chlamydiae ,Virulence Factors ,Chlamydiaceae ,030106 microbiology ,Virulence ,lcsh:Medicine ,Article ,Microbiology ,03 medical and health sciences ,Type III Secretion Systems ,Animals ,Secretion ,Amoeba ,Clinical microbiology ,lcsh:Science ,Multidisciplinary ,Host cell cytosol ,Waddlia chondrophila ,Chlamydiales ,biology ,Bacteria ,lcsh:R ,Computational Biology ,Membrane Proteins ,biology.organism_classification ,030104 developmental biology ,Membrane protein ,Cosmid ,lcsh:Q - Abstract
Waddlia chondrophila is an intracellular bacterium phylogenetically related to the well-studied human and animal pathogens of the Chlamydiaceae family. In the last decade, W. chondrophila was convincingly demonstrated to be associated with adverse pregnancy outcomes in humans and abortions in animals. All members of the phylum Chlamydiae possess a Type Three Secretion System that they use for delivering virulence proteins into the host cell cytosol to modulate their environment and create optimal conditions to complete their life cycle. To identify W. chondrophila virulence proteins, we used an original screening approach that combines a cosmid library with an assay monitoring resistance to predation by phagocytic amoebae. This technique combined with bioinformatic data allowed the identification of 28 candidate virulence proteins, including Wimp1, the first identified inclusion membrane protein of W. chondrophila.
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- 2019
11. T cell fate following Salmonella infection is determined by a STING-IRF1 signaling axis in mice
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Rachid Zagani, Yun Zhao, Sung-Moo Park, Hans Christian Reinecker, Naohiro Yoshida, Tatsushi Omatsu, and Pankaj Shah
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0301 basic medicine ,Host cell cytosol ,Innate immune system ,T cell ,Medicine (miscellaneous) ,Biology ,General Biochemistry, Genetics and Molecular Biology ,eye diseases ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,IRF1 ,Immune system ,Mucosal immunology ,lcsh:Biology (General) ,Stimulator of interferon genes ,medicine ,General Agricultural and Biological Sciences ,lcsh:QH301-705.5 ,030215 immunology ,Interferon regulatory factors - Abstract
The innate immune response following infection with entero-invasive bacterial species is triggered upon release of cyclic di-guanylate monophosphate (c-di-GMP) into the host cell cytosol. Bacterial c-di-GMP activates the intracellular Sensor Stimulator of Interferon Genes (STING), encoded by Tmem173 in mice. Here we identify Interferon Regulatory Factor (IRF) 1 as a critical effector of STING-mediated microbial DNA sensing that is responsible for TH17 cell generation in the mucosal immune system. We find that STING activation induces IRF1-dependent transcriptional programs in dendritic cells (DCs) that define T cell fate determination, including induction of Gasdermin D, IL-1 family member cytokines, and enzymes for eicosanoid synthesis. Our results show that IRF1-dependent transcriptional programs in DCs are a prerequisite for antigen-specific TH17 subspecification in response to microbial c-di-GMP and Salmonella typhimurium infection. Our identification of a STING-IRF1 signaling axis for adaptive host defense control will aid further understanding of infectious disease mechanisms.
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- 2019
12. Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence
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Matthew D. Welch, Patrik Engström, Michael Rape, Kevin G. Mark, Nadia Ingabire, Anthony T. Iavarone, Guillaume Golovkine, Gabriel Mitchell, Thomas P. Burke, and Jeffery S. Cox
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Applied Microbiology and Biotechnology ,Gene Knockout Techniques ,Mice ,Cytosol ,Ubiquitin ,Chlorocebus aethiops ,Rickettsia ,Polyubiquitin ,innate immunity ,autophagic recognition ,0303 health sciences ,Host cell cytosol ,Virulence ,biology ,OmpB ,OmpA ,Cell biology ,polyubiquitylation ,intracellular pathogens ,Female ,Bacterial outer membrane ,Microtubule-Associated Proteins ,Bacterial Outer Membrane Proteins ,Microbiology (medical) ,Immunology ,Microbiology ,Article ,Cell Line ,03 medical and health sciences ,Immune system ,Autophagy ,Genetics ,Animals ,Humans ,antimicrobial autophagy ,Vero Cells ,Immune Evasion ,030304 developmental biology ,030306 microbiology ,Macrophages ,Intracellular parasite ,Endothelial Cells ,Rickettsia Infections ,Cell Biology ,biology.organism_classification ,Mice, Inbred C57BL ,Disease Models, Animal ,A549 Cells ,biology.protein ,Transcriptome - Abstract
Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin-coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of the bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. OmpB is also required for the formation of a capsule-like layer. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice because of its ability to promote autophagy evasion in immune cells. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, thereby revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.
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- 2019
13. Viewing Legionella pneumophila Pathogenesis through an Immunological Lens
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Sunny Shin and Xin Liu
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Virulence ,Context (language use) ,Biology ,Endoplasmic Reticulum ,Legionella pneumophila ,Article ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Structural Biology ,Macrophages, Alveolar ,Animals ,Humans ,Secretion ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Host cell cytosol ,Effector ,Intracellular parasite ,biology.organism_classification ,respiratory tract diseases ,Cytokines ,bacteria ,Legionnaires' Disease ,030217 neurology & neurosurgery - Abstract
Legionella pneumophila is the causative agent of the severe pneumonia Legionnaires' disease. L. pneumophila is ubiquitously found in freshwater environments, where it replicates within free-living protozoa. Aerosolization of contaminated water supplies allows the bacteria to be inhaled into the human lung, where L. pneumophila can be phagocytosed by alveolar macrophages and replicate intracellularly. The Dot/Icm type IV secretion system (T4SS) is one of the key virulence factors required for intracellular bacterial replication and subsequent disease. The Dot/Icm apparatus translocates more than 300 effector proteins into the host cell cytosol. These effectors interfere with a variety of cellular processes, thus enabling the bacterium to evade phagosome–lysosome fusion and establish an endoplasmic reticulum-derived Legionella-containing vacuole, which facilitates bacterial replication. In turn, the immune system has evolved numerous strategies to recognize intracellular bacteria such as L. pneumophila, leading to potent inflammatory responses that aid in eliminating infection. This review aims to provide an overview of L. pneumophila pathogenesis in the context of the host immune response.
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- 2019
14. Host inflammasome defense mechanisms and bacterial pathogen evasion strategies
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Sky W. Brubaker, Susan M. Brewer, and Denise M. Monack
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0301 basic medicine ,Inflammasomes ,Immunology ,Biology ,Bacterial Physiological Phenomena ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Immunity ,medicine ,Animals ,Humans ,Immunology and Allergy ,Disease Resistance ,Immune Evasion ,Innate immune system ,Host cell cytosol ,Pattern recognition receptor ,Inflammasome ,Bacterial Infections ,Evasion (ethics) ,Immunity, Innate ,030104 developmental biology ,Multiprotein Complexes ,Host-Pathogen Interactions ,Disease Susceptibility ,Biomarkers ,Protein Binding ,Signal Transduction ,030215 immunology ,medicine.drug - Abstract
Inflammasomes are a formidable armada of intracellular pattern recognition receptors. They recognize determinants of infection, such as foreign entities or danger signals within the host cell cytosol, rapidly executing innate immune defenses and initiating adaptive immune responses. Although inflammasomes are implicated in many diseases, they are especially critical in host protection against intracellular bacterial pathogens. Given this role, it is not surprising that many pathogens have evolved effective strategies to evade inflammasome activation. In this review, we will provide a brief summary of inflammasome activation during infection with the intent of highlighting recent advances in the field. Additionally, we will review known bacterial evasion strategies and countermeasures that impact pathogenesis.
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- 2019
15. Cytosolic detection of phagosomal bacteria-Mechanisms underlying PAMP exodus from the phagosome into the cytosol
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Stephanie A Ragland and Jonathan C. Kagan
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Host cell cytosol ,Innate immune system ,Bacteria ,animal diseases ,Phagocytosis ,Pathogen-associated molecular pattern ,Pathogen-Associated Molecular Pattern Molecules ,Pattern recognition receptor ,Caspase-11 ,Bacterial Infections ,Biology ,Microbiology ,Article ,Cell biology ,Cytosol ,Phagosomes ,Receptors, Pattern Recognition ,Macrophage ,Animals ,Humans ,Molecular Biology ,Phagosome - Abstract
The metazoan innate immune system senses bacterial infections by detecting highly conserved bacterial molecules, termed pathogen-associated molecular patterns (PAMPs). PAMPs are detected by a variety of host pattern recognition receptors (PRRs), whose function is to coordinate downstream immune responses. PRR activities are, in part, regulated by their subcellular localizations. Accordingly, professional phagocytes can detect extracellular bacteria and their PAMPs via plasma membrane-oriented PRRs. Conversely, phagocytosed bacteria and their PAMPs are detected by transmembrane PRRs oriented towards the phagosomal lumen. Even though PAMPs are unable to passively diffuse across membranes, phagocytosed bacteria are also detected by PRRs localized within the host cell cytosol. This phenomenon is explained upon phagocytosis of bacteria that specialize in phagosomal escape and cytosolic residence. Contrary to this cytosolic lifestyle, most bacteria studied to date spend their entire intracellular lifestyle contained within phagosomes, yet they also stimulate cytosolic PRRs. Herein, we will review our current understanding for how phagosomal PAMPs become accessible to cytosolic PRRs, as well as highlight knowledge gaps that should inspire future investigations.
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- 2021
16. Developing Cytokine Storm-Sensitive Therapeutic Strategy in COVID-19 Using 8P9R Chimeric Peptide and Soluble ACE2
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Yasaman Nazerian, Hassan Niknejad, Ali Ebrahimi, and Kimia Vakili
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QH301-705.5 ,exosomes ,Biology ,Virus ,NF-κB ,Proinflammatory cytokine ,Cell and Developmental Biology ,Viral entry ,Hypothesis and Theory ,medicine ,sACE2 ,Biology (General) ,Transcription factor ,mesenchymal stem cells ,Host cell cytosol ,COVID-19 ,Promoter ,Cell Biology ,medicine.disease ,Cell biology ,Viral replication ,cytokine storm ,8P9R chimeric peptide ,ARDS ,Cytokine storm ,Developmental Biology - Abstract
Currently, the COVID-19 pandemic is an international challenge, largely due to lack of effective therapies. Pharmacotherapy has not yet been able to find a definitive treatment for COVID-19. Since SARS-CoV-2 affects several organs, treatment strategies that target the virus in a wider range are expected to be ultimately more successful. To this end, a two-step treatment strategy has been presented. In the first phase of the disease, when the patient is newly infected with the virus and the cytokine storm has not yet been developed, a chimeric peptide is used to inhibit virus entry into the host cell cytosol (by inhibiting endosomal pH acidification) and viral replication. After the virus entry and decrease of angiotensin converting enzyme 2 (ACE2) level, some people are unable to properly compensate for the ACE2 pathway and progress toward the cytokine storm. In the beginning of the cytokine storm, sACE2 protein is very effective in regulating the immune system toward the anti-inflammatory pathway, including M2 macrophages. Hence, the genes of 8P9R chimeric peptide and sACE2 would be inserted in an episomal vector with a separate promoter for each gene: the chimeric peptide gene promoter is a CMV promoter, while the sACE2 gene promoter is a NF-κB-sensitive promoter. The NF-κB-sensitive promoter induces the expression of sACE2 gene soon after elevation of NF-κB which is the main transcription factor of inflammatory genes. Thus, as the expression of inflammatory cytokines increases, the expression of sACE2 increases simultaneously. In this condition, sACE2 can prevent the cytokine storm by inhibiting the pro-inflammatory pathways. To deliver the designed vector to the target cells, mesenchymal stem cell-derived (MSC-derived) exosome-liposome hybrids are used. Herein, the strategy can be considered as a personalized clinical therapy for COVID-19, that can prevent morbidity and mortality in the future.
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- 2021
17. Single-cell analyses reveal phosphate availability as critical factor for nutrition of Salmonella enterica within mammalian host cells
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Jennifer Röder, Michael Hensel, and Pascal Felgner
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Salmonella typhimurium ,Host cell cytosol ,biology ,Intracellular parasite ,Immunology ,Cell ,Salmonella enterica ,Vacuole ,biology.organism_classification ,Microbiology ,Type three secretion system ,Cell biology ,Phosphates ,Cytosol ,medicine.anatomical_structure ,Bacterial Proteins ,Virology ,Vacuoles ,medicine ,Animals ,Humans ,Single-Cell Analysis ,Intracellular ,HeLa Cells - Abstract
Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen and acquisition of nutrients from host cells is essential for survival and proliferation of intracellular STM. The nutritional environment of intracellular STM is only partially understood. We deploy bacteria harbouring reporter plasmids to interrogate the environmental cues acting on intracellular STM, and flow cytometry allows analyses on level of single STM. Phosphorus is a macro-element for cellular life, and in STM inorganic phosphate (Pi ), homeostasis is mediated by the two-component regulatory system PhoBR, resulting in expression of the high affinity phosphate transporter pstSCAB-phoU. Using fluorescent protein reporters, we investigated Pi availability for intracellular STM at single-cell level over time. We observed that Pi concentration in the Salmonella-containing vacuole (SCV) is limiting and activates the promoter of pstSCAB-phoU encoding a high affinity phosphate uptake system. Correlation between reporter activation by STM in defined media and in host cells indicates Pi concentration less 10 μM within the SCV. STM proliferating within the SCV experience increasing Pi limitations. Activity of the Salmonella pathogenicity island 2 (SPI2)-encoded type III secretion system (T3SS) is crucial for efficient intracellular proliferation, and SPI2-T3SS-mediated endosomal remodelling also reliefs Pi limitation. STM that are released from SCV to enter the cytosol of epithelial cells did not indicate Pi limitations. Addition of Pi to culture media of infected cells partially relieved Pi limitations in the SCV, as did inhibition of intracellular proliferation. We conclude that availability of Pi is critical for intracellular lifestyle of STM, and Pi acquisition is maintained by multiple mechanisms. Our work demonstrates the use of bacterial pathogens as sensitive single-cell reporters for their environment in host cell or host organisms. TAKE AWAY: Salmonella strains were engineered to report their intracellular niche and the availability of inorganic phosphate (Pi ) on level of single intracellular bacteria Within the Salmonella-containing vacuole (SCV), Pi is limited and limitation increases with bacterial proliferation Salmonella located in host cell cytosol are not limited in Pi availability Remodelling of the host cell endosomal system mediated by T3SS-2 reliefs Pi limitation in the SCV.
- Published
- 2021
18. Parasite-Mediated Remodeling of the Host Microfilament Cytoskeleton Enables Rapid Egress of Trypanosoma cruzi following Membrane Rupture
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Alexis Bonfim-Melo, Jaime A. Costales, Éden Ramalho Ferreira, Renato A. Mortara, Barbara A. Burleigh, and Kevin M. Tyler
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Chagas’ disease ,microfilament ,Trypanosoma cruzi ,Biology ,Microfilament ,Filamentous actin ,Microbiology ,Host-Parasite Interactions ,host-parasite relationship ,Virology ,Chlorocebus aethiops ,parasitic diseases ,Animals ,Chagas Disease ,Cytoskeleton ,Vero Cells ,Host cell cytosol ,protozoan ,Cell Membrane ,lytic cycle ,biology.organism_classification ,Actin cytoskeleton ,cell invasion ,amastigote ,Actins ,QR1-502 ,Cell biology ,egress ,trypomastigote ,Actin Cytoskeleton ,Lytic cycle ,Host cell plasma membrane ,stage-regulated ,Research Article - Abstract
Chagas' disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this pathogen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for maintenance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell's actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages-a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to preserve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomastigotes may trigger sudden egress. IMPORTANCE Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite's lytic cycle: how parasites egress and thereby sustain round after round of infection. Our results show that once in the host cell cytosol and having undergone amastigogenesis, T. cruzi begins to alter the host cell cytoskeleton, remodeling normal F-actin meshworks into encapsulating spheroidal cages. Filamentous actin diminishes over the course of the lytic cycle, and just prior to egress, the filaments comprising the cages are severely degraded where adjacent to the parasites. We conclude that sudden egress follows breach of the containment afforded by the actin cytoskeleton and subsequent plasma membrane rupture-a process that when understood in molecular detail may serve as a target for future novel therapeutic interventions.
- Published
- 2021
19. The newly discovered role of endocytosis in artemisinin resistance
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Hannah Michaela Behrens, Tobias Spielmann, and Sabine Schmidt
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Combination therapy ,Plasmodium falciparum ,Drug Resistance ,Protozoan Proteins ,Endocytosis ,Antimalarials ,parasitic diseases ,Drug Discovery ,medicine ,Humans ,Artemisinin ,Malaria, Falciparum ,Pharmacology ,Genetics ,Host cell cytosol ,biology ,Artemisinin resistance ,biology.organism_classification ,medicine.disease ,Artemisinins ,Mutation ,Molecular Medicine ,Single point ,Malaria ,medicine.drug - Abstract
Artemisinin and its derivatives (ART) are the cornerstone of malaria treatment as part of artemisinin combination therapy (ACT). However, reduced susceptibility to artemisinin as well as its partner drugs threatens the usefulness of ACTs. Single point mutations in the parasite protein Kelch13 (K13) are necessary and sufficient for the reduced sensitivity of malaria parasites to ART but several alternative mechanisms for this resistance have been proposed. Recent work found that K13 is involved in the endocytosis of host cell cytosol and indicated that this is the process responsible for resistance in parasites with mutated K13. These studies also identified a series of further proteins that act together with K13 in the same pathway, including previously suspected resistance proteins such as UBP1 and AP-2μ. Here, we give a brief overview of artemisinin resistance, present the recent evidence of the role of endocytosis in ART resistance and discuss previous hypotheses in light of this new evidence. We also give an outlook on how the new insights might affect future research.
- Published
- 2021
20. Transport mechanisms at the malaria parasite-host cell interface
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Chi-Min Ho and Josh R. Beck
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Plasmodium ,Erythrocytes ,Cell Membranes ,Vacuole ,Review ,Medical Conditions ,Animal Cells ,Red Blood Cells ,Medicine and Health Sciences ,Malaria, Falciparum ,Biology (General) ,Integral membrane protein ,Protozoans ,0303 health sciences ,Host cell cytosol ,biology ,Effector ,Malarial Parasites ,Eukaryota ,Translocon ,Cell biology ,Cellular Structures and Organelles ,Cellular Types ,QH301-705.5 ,Immunology ,Plasmodium falciparum ,Virulence ,Microbiology ,Host-Parasite Interactions ,03 medical and health sciences ,Virology ,Parasite Groups ,parasitic diseases ,Genetics ,Parasitic Diseases ,Animals ,Humans ,Integral Membrane Proteins ,Molecular Biology ,030304 developmental biology ,Blood Cells ,030306 microbiology ,Host Cells ,Organisms ,Biology and Life Sciences ,Membrane Proteins ,Biological Transport ,Cell Biology ,RC581-607 ,biology.organism_classification ,Parasitic Protozoans ,Malaria ,Cytosol ,Parasitology ,Immunologic diseases. Allergy ,Apicomplexa ,Viral Transmission and Infection - Abstract
Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by thePlasmodiumTranslocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison toToxoplasma gondiiand other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.
- Published
- 2021
21. Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections
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Aaron S. Dhanda, Julian A. Guttman, and Connie Yu
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0301 basic medicine ,Histology ,Human pathogen ,Cypa ,Microbiology ,Enteropathogenic Escherichia coli ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Escherichia coli Infections ,Ecology, Evolution, Behavior and Systematics ,Host cell cytosol ,biology ,Effector ,Cell Membrane ,Salmonella enterica ,biology.organism_classification ,Actin cytoskeleton ,Actin Cytoskeleton ,030104 developmental biology ,Salmonella Infections ,Basigin ,bacteria ,Anatomy ,030217 neurology & neurosurgery ,Bacteria ,HeLa Cells ,Biotechnology - Abstract
Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are highly infectious gastrointestinal human pathogens. These microbes inject bacterial-derived effector proteins directly into the host cell cytosol as part of their disease processes. A common host subcellular target of these pathogens is the actin cytoskeleton, which is commandeered by the bacteria and is used during their attachment onto (EPEC) or invasion into (S. Typhimurium) the host cells. We previously demonstrated that the host enzyme cyclophilin A (CypA) is recruited to the actin-rich regions of EPEC pedestals and S. Typhimurium membrane ruffles. To further expand the growing catalogue of host proteins usurped by actin-hijacking bacteria, we examined the host plasma membrane protein and cognate receptor of CypA, CD147, during EPEC and S. Typhimurium infections. Here, we show that CD147 is enriched at the basolateral regions of pedestals but, unlike CypA, it is absent from their actin-rich core. We show that the CD147 recruitment to these areas requires EPEC pedestal formation and not solely bacteria-host cell contact. Additionally, we demonstrate that the depletion of CD147 by siRNA does not alter the formation of pedestals. Finally, we show that CD147 is also a component of actin-rich membrane ruffles generated during S. Typhimurium invasion of host cells. Collectively, our findings establish CD147 as another host component present at dynamic actin-rich structures formed during bacterial infections. Anat Rec, 302:2224-2232, 2019. © 2019 American Association for Anatomy.
- Published
- 2019
22. Anthrax toxin channel: What we know based on over 30 years of research
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Wenxing Liu and Ekaterina M. Nestorovich
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Pore-forming toxin ,Antigens, Bacterial ,Host cell cytosol ,biology ,Chemistry ,Toxin ,Protein Conformation ,Anthrax toxin ,Bacterial Toxins ,Lipid Bilayers ,Biophysics ,Cell Biology ,Hydrogen-Ion Concentration ,medicine.disease_cause ,Biochemistry ,Cell biology ,Cytosol ,Protein Transport ,medicine ,biology.protein ,Translocase ,Lipid bilayer ,Exotoxin - Abstract
Protective antigen channel is the central component of the deadly anthrax exotoxin responsible for binding and delivery of the toxin's enzymatic lethal and edema factor components into the cytosol. The channel, which is more than three times longer than the lipid bilayer membrane thickness and has a 6-A limiting diameter, is believed to provide a sophisticated unfoldase and translocase machinery for the foreign protein transport into the host cell cytosol. The tripartite toxin can be reengineered, one component at a time or collectively, to adapt it for the targeted cancer therapeutic treatments. In this review, we focus on the biophysical studies of the protective antigen channel-forming activity, small ion transport properties, enzymatic factor translocation, and blockage comparing it with the related clostridial binary toxin channels. We address issues linked to the anthrax toxin channel structural dynamics and lipid dependence, which are yet to become generally recognized as parts of the toxin translocation machinery.
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- 2021
23. The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry
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Cody J. Lauritsen, Christopher R. Gourley, Geremy Clair, Joshua N. Adkins, Nicholas M. Negretti, Prabhat K. Talukdar, Michael E. Konkel, and Courtney M. Klappenbach
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0301 basic medicine ,Science ,General Physics and Astronomy ,RAC1 ,Flagellum ,medicine.disease_cause ,Campylobacter jejuni ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,IQGAP1 ,Bacterial Proteins ,Campylobacter Infections ,medicine ,Humans ,Small GTPase ,Cellular microbiology ,Cytoskeleton ,Multidisciplinary ,Host cell cytosol ,Bacteria ,030102 biochemistry & molecular biology ,biology ,Effector ,Chemistry ,Campylobacter ,Epithelial Cells ,General Chemistry ,biology.organism_classification ,Cell biology ,030104 developmental biology ,Flagella ,ras GTPase-Activating Proteins ,Gene Knockdown Techniques ,Host-Pathogen Interactions ,Pathogens ,Transcriptome ,Signal Transduction - Abstract
Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1., The pathogen Campylobacter jejuni invades intestinal cells after secreting protein effectors into the host cell cytosol via the flagellum. Here, Negretti et al. show that one of these effectors, CiaD, binds to host protein IQGAP1, thus leading to unconstrained activity of small GTPase Rac1, which modulates actin reorganization and bacterial internalization.
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- 2021
24. Site-Specific Labeling and (19)F NMR Provide Direct Evidence for Dynamic Behavior of the Anthrax Toxin Pore ϕ-Clamp Structure
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James G. Bann, William M. Westler, Masaru Miyagi, and Srinivas Gonti
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Models, Molecular ,Antigens, Bacterial ,Protein Folding ,0303 health sciences ,Host cell cytosol ,Protein Conformation ,Direct evidence ,Chemistry ,Anthrax toxin ,Bacterial Toxins ,030302 biochemistry & molecular biology ,Cell ,Fluorine-19 NMR ,Biochemistry ,Article ,Fluorine-19 Magnetic Resonance Imaging ,03 medical and health sciences ,Residue (chemistry) ,Cytosol ,medicine.anatomical_structure ,medicine ,Biophysics ,Nuclear Magnetic Resonance, Biomolecular ,Binding domain - Abstract
The anthrax toxin protective antigen (PA), the membrane binding and pore-forming component of the anthrax toxin, was studied using (19)F NMR. We site-specifically labeled PA with p-fluorophenylalanine (pF-Phe) at Phe427, a critically important residue that comprises the ϕ-clamp that is required for translocation of edema factor (EF) and lethal factor (LF) into the host cell cytosol. We utilized (19)F NMR to follow low-pH-induced structural changes in the prepore, alone and bound to the N-terminal PA binding domain of LF, LF(N). Our studies indicate that pF-Phe427 is dynamic in the prepore state and then becomes more dynamic in the transition to the pore. An increase in dynamic behavior at the ϕ-clamp may provide the necessary room for movement needed in translocating EF and LF into the cell cytosol.
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- 2021
25. Evolution and Adaptation of Legionella pneumophila to Manipulate the Ubiquitination Machinery of Its Amoebae and Mammalian Hosts
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Yousef Abu Kwaik and Christopher T. D. Price
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amoebae ,F-box ,lcsh:QR1-502 ,Biochemistry ,Legionella pneumophila ,lcsh:Microbiology ,Deubiquitinating enzyme ,03 medical and health sciences ,Ubiquitin ,ubiquitin ,Ligase activity ,U-box ,Molecular Biology ,E3-ubiquitin ligase ,030304 developmental biology ,0303 health sciences ,Host cell cytosol ,biology ,030306 microbiology ,Effector ,legionella pneumophila ,biology.organism_classification ,Ubiquitin ligase ,Cell biology ,biology.protein ,Function (biology) - Abstract
The ubiquitin pathway is highly conserved across the eukaryotic domain of life and plays an essential role in a plethora of cellular processes. It is not surprising that many intracellular bacterial pathogens often target the essential host ubiquitin pathway. The intracellular bacterial pathogen Legionella pneumophila injects into the host cell cytosol multiple classes of classical and novel ubiquitin-modifying enzymes that modulate diverse ubiquitin-related processes in the host cell. Most of these pathogen-injected proteins, designated as effectors, mimic known E3-ubiquitin ligases through harboring F-box or U-box domains. The classical F-box effector, AnkB targets host proteins for K48-linked polyubiquitination, which leads to excessive proteasomal degradation that is required to generate adequate supplies of amino acids for metabolism of the pathogen. In contrast, the SidC and SdcA effectors share no structural similarity to known eukaryotic ligases despite having E3-ubiquitin ligase activity, suggesting that the number of E3-ligases in eukaryotes is under-represented. L. pneumophila also injects into the host many novel ubiquitin-modifying enzymes, which are the SidE family of effectors that catalyze phosphoribosyl-ubiquitination of serine residue of target proteins, independently of the canonical E1-2-3 enzymatic cascade. Interestingly, the environmental bacterium, L. pneumophila, has evolved within a diverse range of amoebal species, which serve as the natural hosts, while accidental transmission through contaminated aerosols can cause pneumonia in humans. Therefore, it is likely that the novel ubiquitin-modifying enzymes of L. pneumophila were acquired by the pathogen through interkingdom gene transfer from the diverse natural amoebal hosts. Furthermore, conservation of the ubiquitin pathway across eukaryotes has enabled these novel ubiquitin-modifying enzymes to function similarly in mammalian cells. Studies on the biological functions of these effectors are likely to reveal further novel ubiquitin biology and shed further lights on the evolution of ubiquitin.
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- 2021
26. The bacterial effector HopZ1a acetylates MKK7 to suppress plant immunity
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Javier Ruiz-Albert, Javier Rueda-Blanco, Diego López-Márquez, Carmen R. Beuzón, Alberto P. Macho, and José S. Rufián
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0106 biological sciences ,0301 basic medicine ,Physiology ,Arabidopsis ,Plant Immunity ,Pseudomonas syringae ,Plant Science ,Mitogen-activated protein kinase kinase ,01 natural sciences ,Type three secretion system ,03 medical and health sciences ,Immune system ,Bacterial Proteins ,Arabidopsis thaliana ,Kinase activity ,Protein kinase A ,Plant Diseases ,Host cell cytosol ,biology ,Chemistry ,Effector ,Arabidopsis Proteins ,fungi ,biology.organism_classification ,Cell biology ,030104 developmental biology ,010606 plant biology & botany - Abstract
ThePseudomonas syringaetype III secretion system translocates effector proteins into the host cell cytosol, suppressing plant basal immunity triggered upon recognition of pathogen-associated molecular patterns (PAMPs), and effector-triggered immunity. Effector HopZ1a suppresses local and systemic immunity triggered by PAMPs and effectors, through target acetylation. HopZ1a has been shown to target several plant proteins, but none fully substantiates HopZ1a-associated immune suppression. Here, we investigateArabidopsis thalianamitogen-activated protein kinase kinases (MKKs) as potential targets, focusing on AtMKK7, a positive regulator of local and systemic immunity. We analyse HopZ1a interference with AtMKK7 by translocation of HopZ1a from bacteria inoculated into Arabidopsis expressing MKK7 from an inducible promoter. Reciprocal phenotypes are analysed on plants expressing a construct quenching MKK7 native expression. We analyse HopZ1a-MKK7 interaction by three independent methods, and the relevance of acetylation byin vitrokinase andin plantafunctional assays. We demonstrate AtMKK7 contribution to immune signalling showing MKK7-dependent flg22-induced ROS burst, MAPK activation, and callose accumulation, plus AvrRpt2-triggered MKK7-dependent signalling. Further, we demonstrate HopZ1a suppression of all MKK7-dependent responses, HopZ1a-MKK7 interaction in planta, and HopZ1a acetylation of MKK7 in a lysine required for full kinase activity. We demonstrate that HopZ1a targets AtMKK7 to suppress local and systemic plant immunity.
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- 2020
27. Host Cell Targets of Released Lipid and Secreted Protein Effectors of Mycobacterium tuberculosis
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Jacques Augenstreich and Volker Briken
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0301 basic medicine ,Microbiology (medical) ,030106 microbiology ,Immunology ,lcsh:QR1-502 ,Virulence ,Microbiology ,lcsh:Microbiology ,lipids ,03 medical and health sciences ,MHC class I ,Phagosome maturation ,Xenophagy ,Secretion ,Host cell cytosol ,biology ,Chemistry ,Effector ,phagosome maturation ,Mycobacterium tuberculosis ,cytokines ,Cell biology ,effector ,cell death ,030104 developmental biology ,Infectious Diseases ,biology.protein ,Bacterial outer membrane - Abstract
Mycobacterium tuberculosis (Mtb) is a very successful pathogen, strictly adapted to humans and the cause of tuberculosis. Its success is associated with its ability to inhibit host cell intrinsic immune responses by using an arsenal of virulence factors of different nature. It has evolved to synthesize a series of complex lipids which form an outer membrane and may also be released to enter host cell membranes. In addition, secreted protein effectors of Mtb are entering the host cell cytosol to interact with host cell proteins. We briefly discuss the current model, involving the ESX-1 type seven secretion system and the Mtb lipid phthiocerol dimycoserosate (PDIM), of how Mtb creates pores in the phagosomal membrane to allow Mtb proteins to access to the host cell cytosol. We provide an exhaustive list of Mtb secreted proteins that have effector functions. They modify (mostly inhibit but sometimes activate) host cell pathways such as: phagosome maturation, cell death, cytokine response, xenophagy, reactive oxygen species (ROS) response via NADPH oxidase 2 (NOX2), nitric oxide (NO) response via NO Synthase 2 (NOS2) and antigen presentation via MHC class I and class II molecules. We discuss the host cell targets for each lipid and protein effector and the importance of the Mtb effector for virulence of the bacterium.
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- 2020
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28. Atg5-Deficient Mice Infected with Francisella tularensis LVS Demonstrate Increased Survival and Less Severe Pathology in Internal Organs
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Marija Ćurlin, Marina Šantić, Ina Kelava, Valentina Marečić, Sanja Štifter, Maša Knežević, Mirna Mihelčić, Anders Sjöstedt, and Mateja Ožanič
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Microbiology (medical) ,autophagy ,mice ,ATG5 ,Biology ,Microbiology ,Article ,Tularemia ,03 medical and health sciences ,BIOMEDICINE AND HEALTHCARE. Clinical Medical Sciences. Medical Microbiology ,Virology ,medicine ,Francisella ,lcsh:QH301-705.5 ,Francisella tularensis ,030304 developmental biology ,Phagosome ,0303 health sciences ,Host cell cytosol ,Attenuated vaccine ,030306 microbiology ,Autophagy ,medicine.disease ,biology.organism_classification ,bacterial infections and mycoses ,3. Good health ,lcsh:Biology (General) ,Atg5 ,BIOMEDICINA I ZDRAVSTVO. Kliničke medicinske znanosti. Medicinska mikrobiologija - Abstract
Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.
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- 2020
29. Cyclophilin 19 secreted in the host cell cytosol by Trypanosoma cruzi promotes ROS production required for parasite growth
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Carolina B. Moraes, Bradford S. McGwire, Paula Bittencourt-Cunha, Sergio Schenkman, Bijay Kumar Jha, Normanda Souza-Melo, Fernanda Midori Abukawa, Gregory Pedroso Dos Santos, Laura M. Alcântara, and Nilmar Silvio Moretti
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Trypanosoma cruzi ,Immunology ,Protozoan Proteins ,Microbiology ,Article ,Host-Parasite Interactions ,Myoblasts ,03 medical and health sciences ,Cyclophilin A ,Cyclophilins ,Cytosol ,Virology ,Cyclosporin a ,Animals ,Cyclophilin ,030304 developmental biology ,0303 health sciences ,Host cell cytosol ,NADPH oxidase ,biology ,030306 microbiology ,Wild type ,biology.organism_classification ,Cell biology ,Rats ,biology.protein ,Reactive Oxygen Species ,Intracellular - Abstract
Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47(phox), as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47(phox) in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.
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- 2020
30. Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite <named-content content-type='genus-species'>Toxoplasma gondii</named-content>
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Yuan Xue, Suchita Rastogi, John C. Boothroyd, and Stephen R. Quake
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Virulence Factors ,parasitology ,Cell ,Population ,Protozoan Proteins ,Microbiology ,effector functions ,single-cell RNA sequencing ,Host-Microbe Biology ,host-parasite relationship ,03 medical and health sciences ,Immune system ,Virology ,parasitic diseases ,medicine ,Humans ,education ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,education.field_of_study ,Host cell cytosol ,Rhoptry ,biology ,Effector ,Gene Expression Profiling ,Intracellular parasite ,030302 biochemistry & molecular biology ,Toxoplasma gondii ,Fibroblasts ,biology.organism_classification ,QR1-502 ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,Host-Pathogen Interactions ,Single-Cell Analysis ,Dense granule ,Toxoplasma ,Research Article - Abstract
This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology., The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.
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- 2020
31. Human GBP1 promotes pathogen vacuole rupture and inflammasome activation during Legionella pneumophila infection
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Antonia Ronalda Bass and Sunny Shin
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Host cell cytosol ,biology ,Chemistry ,Pyroptosis ,Inflammasome ,Vacuole ,biology.organism_classification ,Legionella pneumophila ,Cell biology ,Interferon ,medicine ,biology.protein ,Caspase ,Intracellular ,medicine.drug - Abstract
The inflammasome is an essential component of host defense against intracellular bacterial pathogens, such as Legionella pneumophila, the causative agent of the severe pneumonia Legionnaires’ disease. Inflammasome activation leads to recruitment and activation of caspases, which promote IL-1 family cytokine release and pyroptosis. In mice, interferon (IFN) signaling promotes inflammasome responses against L. pneumophila, in part through the functions of a family of IFN-inducible GTPases known as guanylate binding proteins (GBPs) (1). Within murine macrophages, IFN signaling promotes rupture of the L. pneumophila-containing vacuole (LCV), whereas GBPs are dispensable for vacuole rupture. Instead, GBPs facilitate the lysis of cytosol-exposed L. pneumophila. In contrast to mouse GBPs, the functions of human GBPs in inflammasome responses to L. pneumophila are poorly understood. Here, we show that IFN-γ promotes caspase-1, caspase-4, and caspase-5 inflammasome activation during L. pneumophila infection and upregulates GBP expression in primary human macrophages. We find that human GBP1 is important for maximal IFN-γ-driven inflammasome responses to L. pneumophila. Furthermore, IFN-γ signaling promotes the rupture of LCVs. Intriguingly, in contrast to murine GBPs, human GBP1 targets the LCV in a T4SS-dependent manner and promotes vacuolar lysis, resulting in increased bacterial access to the host cell cytosol. Our findings show a key role for human GBP1 in targeting and disrupting pathogen-containing vacuoles and reveal mechanistic differences in how mouse and human GBPs promote inflammasome responses to L. pneumophila.
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- 2020
32. Inflammasome-mediated antagonism of type I interferon enhances Rickettsia pathogenesis
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Russell E. Vance, Thomas P. Burke, Patrik Engström, Matthew D. Welch, Joshua A. Fonbuena, and Roberto A. Chavez
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Lipopolysaccharides ,Male ,Inflammasomes ,medicine.medical_treatment ,Inbred C57BL ,Applied Microbiology and Biotechnology ,Mice ,0302 clinical medicine ,Cytosol ,Interferon ,Innate ,Rickettsia ,Receptor ,Mice, Knockout ,0303 health sciences ,Host cell cytosol ,Pyroptosis ,Intracellular Signaling Peptides and Proteins ,Initiator ,Inflammasome ,Caspases, Initiator ,Cytokine ,Infectious Diseases ,Medical Microbiology ,Caspases ,Interferon Type I ,Interferon Regulatory Factors ,Host-Pathogen Interactions ,Female ,Infection ,medicine.drug ,Microbiology (medical) ,Knockout ,Immunology ,Biology ,Microbiology ,Article ,Cell Line ,Vaccine Related ,03 medical and health sciences ,Immunity ,Biodefense ,Genetics ,medicine ,Animals ,Humans ,Transcription factor ,030304 developmental biology ,Innate immune system ,030306 microbiology ,Animal ,Prevention ,Inflammatory and immune system ,Rickettsia Infections ,Cell Biology ,Phosphate-Binding Proteins ,biology.organism_classification ,Immunity, Innate ,Mice, Inbred C57BL ,Disease Models, Animal ,Emerging Infectious Diseases ,Disease Models ,Carrier Proteins ,IRF5 ,030215 immunology ,Interferon regulatory factors - Abstract
SummaryInflammasomes and interferons constitute two critical arms of innate immunity. Most facultative bacterial pathogens that inhabit the host cell cytosol avoid activating inflammasomes and are often resistant to killing by type I interferon (IFN-I). We report that the human pathogenRickettsia parkeri,an obligate intracellular pathogen that resides in the cytosol, is sensitive to IFN-I. The mechanism of IFN-I-dependent restriction requires the transcription factor IRF5, which upregulates anti-rickettsial factors including guanylate-binding proteins and iNOS. However,R. parkericurtails cGAS-dependent IFN-I production by causing caspase-11-dependent pyroptosis.In vivo, inflammasome activation antagonizes IFN-I production, enhancingR. parkeriabundance in the spleen. Mice lacking either IFN-I or IFN-γ signaling are resistant to infection, but mice lacking both rapidly succumb, revealing that both interferons are required to controlR. parkeri. This study illuminates how an obligate cytosolic pathogen exploits the intrinsic trade-off between cell death and cytokine production to escape killing by innate immunity.HighlightsRickettsiakilled by GBPs activates caspase-11 and GSDMD, promoting pyroptosisRickettsiaexploits pyroptosis to avoid cGAS-dependent type I interferonIRF5, GBPs, and iNOS contribute to controllingR. parkeriinfectionIfnar-/-Ifngr-/-mice succumb to infection, uncovering a mouse model to studyR. parkeri
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- 2020
33. Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes
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Hendrik Koliwer-Brandl, Thierry Soldati, Paulina Knobloch, Nabil Hanna, I. Gonda, Nicolas Personnic, Fabian M. Arnold, Markus A. Seeger, Hubert Hilbi, Caroline Barisch, Sophia Adenau, University of Zurich, and Hilbi, Hubert
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Siderophore ,animal structures ,Phagocyte ,Iron ,Immunology ,Mutant ,Siderophores ,Mycobactin ,610 Medicine & health ,Biology ,Microbiology ,Mass Spectrometry ,Mycobacterium tuberculosis ,Mice ,03 medical and health sciences ,Bacterial Proteins ,Virology ,medicine ,Animals ,Peptide Synthases ,Oxazoles ,Mycobacterium marinum ,030304 developmental biology ,Acanthamoeba castellanii ,Phagocytes ,0303 health sciences ,2403 Immunology ,Host cell cytosol ,030306 microbiology ,10179 Institute of Medical Microbiology ,2404 Microbiology ,bacterial infections and mycoses ,biology.organism_classification ,3. Good health ,RAW 264.7 Cells ,medicine.anatomical_structure ,Vacuoles ,ddc:540 ,2406 Virology ,570 Life sciences ,biology ,Transcriptome ,Bacteria - Abstract
Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen.
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- 2020
34. Why is<scp>Listeria monocytogenes</scp>such a potent inducer of CD8+ T‐cells?
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Daniel A. Portnoy and Alfredo Chávez-Arroyo
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Cytotoxic ,T-Lymphocytes ,CD8-Positive T-Lymphocytes ,medicine.disease_cause ,Mice ,Cytosol ,vaccine ,Phagosomes ,Cytotoxic T cell ,Listeriosis ,bacteria ,Immunity, Cellular ,0303 health sciences ,Host cell cytosol ,biology ,Antigen processing ,intracellular pathogen ,Foodborne Illness ,Infectious Diseases ,Medical Microbiology ,Host-Pathogen Interactions ,Cytokines ,Infection ,dendritic cell ,Immunology ,chemical and pharmacologic phenomena ,macrophage ,Major histocompatibility complex ,Microbiology ,Article ,Vaccine Related ,03 medical and health sciences ,Listeria monocytogenes ,Antigen ,inflammasome ,Immunity ,Biodefense ,Virology ,medicine ,cancer ,Animals ,Humans ,030304 developmental biology ,tumour ,030306 microbiology ,Prevention ,Inflammatory and immune system ,Intracellular parasite ,Emerging Infectious Diseases ,Good Health and Well Being ,biology.protein ,Immunization ,Cellular ,Digestive Diseases ,T-Lymphocytes, Cytotoxic - Abstract
Listeria monocytogenes is a rapidly growing, Gram-positive, facultative intracellular pathogen that has been used for over 5 decades as a model to study basic aspects of infection and immunity. In a murine intravenous infection model, immunisation with a sublethal infection of L. monocytogenes initially leads to rapid intracellular multiplication followed by clearance of the bacteria and ultimately culminates in the development of long-lived cell-mediated immunity (CMI) mediated by antigen-specific CD8+ cytotoxic T-cells. Importantly, effective immunisation requires live, replicating bacteria. In this review, we summarise the cell and immunobiology of L. monocytogenes infection and discuss aspects of its pathogenesis that we suspect lead to robust CMI. We suggest five specific features of L. monocytogenes infection that positively impact the development of CMI: (a) the bacteria have a predilection for professional antigen-presenting cells; (b) the bacteria escape from phagosomes, grow, and secrete antigens into the host cell cytosol; (c) bacterial-secreted proteins enter the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation; (d) the bacteria do not induce rapid host cell death; and (e) cytosolic bacteria induce a cytokine response that favours CMI. Collectively, these features make L. monocytogenes an attractive vaccine vector for both infectious disease applications and cancer immunotherapy.
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- 2020
35. The Sinorhizobium fredii HH103 type III secretion system effector NopC blocks nodulation with Lotus japonicus Gifu
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Francisco Javier López-Baena, Francisco Pérez-Montaño, Francisco Javier Ollero, Cynthia Alias-Villegas, Irene Jiménez-Guerrero, Carlos Medina, Sebastián Acosta-Jurado, and José María Vinardell
- Subjects
0106 biological sciences ,0301 basic medicine ,Physiology ,Lotus japonicus ,Mutant ,Sinorhizobium ,Plant Science ,Sinorhizobium fredii ,01 natural sciences ,Plant Root Nodulation ,Type three secretion system ,Rhizobia ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,Type III Secretion Systems ,Secretion ,Symbiosis ,Host cell cytosol ,biology ,Effector ,fungi ,food and beverages ,biology.organism_classification ,030104 developmental biology ,Lotus ,010606 plant biology & botany - Abstract
The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.
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- 2020
36. Irgm2 and Gate-16 cooperatively dampen targeting of caspase-11 to Gram-negative bacterial products
- Author
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Pierre-Jean Bordignon, Audrey Hessel, Karima Chaoui, Rémi Planès, Jonathan C. Howard, Elif Eren, Salimata Bagayoko, Karin Santoni, Miriam Pinilla, Masahiro Yamamoto, Etienne Meunier, and Odile Burlet-Schiltz
- Subjects
0303 health sciences ,Host cell cytosol ,Chemistry ,Intracellular parasite ,medicine.medical_treatment ,Pyroptosis ,Inflammasome ,Caspase-11 ,Cell biology ,03 medical and health sciences ,Classical complement pathway ,0302 clinical medicine ,Immune system ,Cytokine ,medicine ,030217 neurology & neurosurgery ,030304 developmental biology ,medicine.drug - Abstract
Inflammatory caspase-11 (rodent) and caspases-4 and -5 (human) detect gram-negative bacterial component LPS in the host cell cytosol, which promotes activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1 related cytokine release is of importance to mount an efficient immune response against various bacteria, its unrestrained activation drives sepsis. This suggests that cellular components might tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but not deleterious inflammatory response. Here we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively slow down non-canonical inflammasome activation both in macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lower caspase-11-mediated pyroptosis and cytokine release. Specifically, deficiency in Irgm2 or Gate16 opens an alternative road for caspase-11 targeting to intracellular bacteria, independently of the classical pathway driven by the Guanylate Binding Proteins (GBPs). Thus, our findings provide new molecular effectors involved at fine-tuning the optimal non-canonical inflammasome response and add novel insights in the understanding of the immune pathways they control.
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- 2020
37. Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm
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Gilbert Greub, Virginie Martin, Ludovic Pilloux, Carole Kebbi-Beghdadi, Pilloux, Ludovic [0000-0002-2133-1891], Greub, Gilbert [0000-0001-9529-3317], and Apollo - University of Cambridge Repository
- Subjects
Microbiology (medical) ,nuclear effector ,selective permeabilisation ,virulence factors ,Chlamydia trachomatis ,Biology ,medicine.disease_cause ,type iii secretion system ,Microbiology ,Article ,Type three secretion system ,03 medical and health sciences ,waddlia chondrophila ,Virology ,medicine ,host-pathogen interactions ,Secretion ,lcsh:QH301-705.5 ,030304 developmental biology ,0303 health sciences ,Host cell cytosol ,030306 microbiology ,Effector ,Intracellular parasite ,Cell biology ,lcsh:Biology (General) ,Host cell cytoplasm ,chlamydia trachomatis ,perfringolysin o ,Heterologous expression ,effectors - Abstract
Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.
- Published
- 2020
38. Endocytosis in Plasmodium and Toxoplasma Parasites
- Author
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Ricarda Sabitzki, Tobias Spielmann, Markus Meissner, and Simon Gras
- Subjects
0301 basic medicine ,Plasmodium ,030231 tropical medicine ,Endocytic cycle ,Drug Resistance ,Endocytosis ,03 medical and health sciences ,0302 clinical medicine ,parasitic diseases ,Extracellular ,Animals ,Humans ,Host cell cytosol ,biology ,Antiparasitic Agents ,Toxoplasma gondii ,Plasmodium falciparum ,biology.organism_classification ,Artemisinins ,Cell biology ,030104 developmental biology ,Infectious Diseases ,Parasitology ,Toxoplasma ,Intracellular - Abstract
Endocytosis is critical for many functions in eukaryotic cells. Uptake of host cell cytosol, an indispensable endocytic process in malaria blood-stage parasites, has been known for a long time. However, it is only recently that the proteins involved in this process have started to emerge. Unexpectedly, some of these proteins revealed a critical role for endocytosis in artemisinin resistance. More recently, endocytosis was discovered in both intracellular and extracellular Toxoplasma gondii parasites. Here, we review these findings, compare the endolysosomal systems of Toxoplasma and Plasmodium parasites, and present current knowledge about endocytic mechanisms in apicomplexans.
- Published
- 2020
39. Role of the ESCRT‐III complex in controlling integrity of the <scp> Salmonella </scp> ‐containing vacuole
- Author
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Vera Göser, Alexander Kehl, Michael Hensel, and Jennifer Röder
- Subjects
Salmonella typhimurium ,ESCRT III complex ,Endosome ,Immunology ,Endosomes ,Vacuole ,Microbiology ,ESCRT ,03 medical and health sciences ,Bacterial Proteins ,Salmonella ,Virology ,Type III Secretion Systems ,Humans ,Adaptor Proteins, Signal Transducing ,030304 developmental biology ,0303 health sciences ,Host cell cytosol ,Endosomal Sorting Complexes Required for Transport ,biology ,030306 microbiology ,Intracellular parasite ,Salmonella enterica ,Epithelial Cells ,biology.organism_classification ,Cell biology ,Vacuoles ,CRISPR-Cas Systems ,Lysosomes ,Intracellular ,HeLa Cells - Abstract
Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2-encoded type III secretion system (SPI2-T3SS). Fusion events of endosomal compartments with the Salmonella-containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella-modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT-III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT-III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2-T3SS-deficient mutant strain was partially abrogated. ESCRT-dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT-III recruitment in Salmonella-infected host cells.
- Published
- 2020
40. Chlamydia trachomatis Oligopeptide Transporter Performs Dual Functions of Oligopeptide Transport and Peptidoglycan Recycling
- Author
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Anthony T. Maurelli, George W. Liechti, Jessica A. Slade, and Raghuveer Singh
- Subjects
Immunology ,Chlamydia trachomatis ,Peptidoglycan ,Oligopeptide transport ,Biology ,medicine.disease_cause ,Microbiology ,Bacterial cell structure ,chemistry.chemical_compound ,Cytosol ,Bacterial Proteins ,Cell Wall ,Cell Line, Tumor ,Operon ,medicine ,Escherichia coli ,Humans ,Amino Acids ,Oligopeptide ,Host cell cytosol ,Intracellular parasite ,Membrane Transport Proteins ,Biological Transport ,Chlamydia Infections ,Molecular Pathogenesis ,Immunity, Innate ,Infectious Diseases ,Biochemistry ,chemistry ,Genes, Bacterial ,Parasitology ,Carrier Proteins ,Oligopeptides ,HeLa Cells - Abstract
Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF(Ct)) encodes multiple SBPs (OppA1(Ct), OppA2(Ct), and OppA3(Ct)). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coli opp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1(Ct), oppA2(Ct), oppA3(Ct)) along with oppBCDF(Ct) in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis. Importantly, we found that one chlamydial SBP, OppA3(Ct), possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis. These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.
- Published
- 2020
41. The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion
- Author
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Thomas Rudel, Daniela Auer, Annette Fischer, Sophie Dorothea Hügelschäffer, and HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
- Subjects
autophagy ,Immunology ,Golgi Apparatus ,Chlamydia trachomatis ,Vacuole ,DUB ,medicine.disease_cause ,Microbiology ,Deubiquitinating enzyme ,Mice ,03 medical and health sciences ,symbols.namesake ,Ubiquitin ,xenophagy ,ddc:570 ,Virology ,Cdu1 ,Golgi ,Autophagy ,medicine ,Animals ,Humans ,RNA, Small Interfering ,030304 developmental biology ,Inclusion Bodies ,0303 health sciences ,Host cell cytosol ,Bacteria ,Deubiquitinating Enzymes ,biology ,030306 microbiology ,Intracellular parasite ,ChlaDUB1 ,Biological Transport ,Golgi apparatus ,Cell biology ,Protein Transport ,Gene Knockdown Techniques ,Host-Pathogen Interactions ,Vacuoles ,symbols ,biology.protein ,HeLa Cells - Abstract
Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1‐deficient Chlamydia . Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface.
- Published
- 2020
42. Irgm2 and Gate‐16 cooperatively dampen Gram‐negative bacteria‐induced caspase‐11 response
- Author
-
Thomas Henry, Rémi Planès, Karima Chaoui, Karin Santoni, Jonathan C. Howard, Odile Burlet-Schiltz, Masahiro Yamamoto, Elif Eren, Audrey Hessel, Salimata Bagayoko, Brice Lagrange, Etienne Meunier, Miriam Pinilla, Pierre-Jean Bordignon, Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Inflammasome, Infections bactériennes et autoinflammation, Inflammasome, Bacterial Infections and Autoinflammation (I2BA), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Gram-negative bacteria ,infections/Interferons ,medicine.medical_treatment ,Immunology ,Caspase-11 ,Biochemistry ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Irgm2 ,Genetics ,medicine ,Molecular Biology ,Gate-16 ,030304 developmental biology ,0303 health sciences ,Host cell cytosol ,biology ,Chemistry ,Intracellular parasite ,[SDV.BA]Life Sciences [q-bio]/Animal biology ,Pyroptosis ,Inflammasome ,Articles ,biology.organism_classification ,non-canonical inflammasome Subject Categories Autophagy & Cell Death ,Cell biology ,Virology & Host Pathogen Interaction ,Cytokine ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Inflammatory caspase-11 (rodent) and caspases-4/5 (humans) detect the Gram-negative bacterial component LPS within the host cell cytosol, promoting activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1-related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but non-deleterious inflammatory responses. Here, we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively counteract Gram-negative bacteria-induced non-canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lowers caspase-11-mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)-dependent and GBP-independent routes for caspase-11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine-tune bacteria-activated non-canonical inflammasome responses and shed light on the understanding of the immune pathways they control. info:eu-repo/semantics/publishedVersion
- Published
- 2020
43. An Inducible Cre-lox System to Analyze the Role of LLO in Listeria monocytogenes Pathogenesis
- Author
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Brittney N. Nguyen and Daniel A. Portnoy
- Subjects
Health, Toxicology and Mutagenesis ,lcsh:Medicine ,Toxicology ,medicine.disease_cause ,cholesterol-dependent cytolysin ,Hemolysin Proteins ,Mice ,Cytosol ,vaccine ,2.1 Biological and endogenous factors ,Aetiology ,pore-forming toxin ,Heat-Shock Proteins ,0303 health sciences ,Host cell cytosol ,Virulence ,Cytotoxins ,pathogenesis ,Bacterial ,Listeriolysin O ,Pharmacology and Pharmaceutical Sciences ,Foodborne Illness ,3. Good health ,Infectious Diseases ,cytotoxicity ,Bacterial Toxins ,Cre recombinase ,Biology ,Cholesterol-dependent cytolysin ,Article ,Microbiology ,Cell Line ,Vaccine Related ,03 medical and health sciences ,Listeria monocytogenes ,Bacterial Proteins ,Biodefense ,Genetics ,medicine ,Animals ,030304 developmental biology ,Integrases ,030306 microbiology ,Prevention ,Macrophages ,lcsh:R ,Gene Expression Regulation, Bacterial ,Emerging Infectious Diseases ,Gene Expression Regulation ,Vacuoles ,Biochemistry and Cell Biology ,Cytolysin ,Cre-Lox recombination ,Digestive Diseases - Abstract
Listeriolysin O (LLO) is a pore-forming cytolysin that allows Listeria monocytogenes to escape from phagocytic vacuoles and enter the host cell cytosol. LLO is expressed continuously during infection, but it has been a challenge to evaluate the importance of LLO secreted in the host cell cytosol because deletion of the gene encoding LLO (hly) prevents localization of L. monocytogenes to the cytosol. Here, we describe a L. monocytogenes strain (hlyfl) in which hly is flanked by loxP sites and Cre recombinase is under the transcriptional control of the L. monocytogenes actA promoter, which is highly induced in the host cell cytosol. In less than 2 h after infection of bone marrow-derived macrophages (BMMs), bacteria were 100% non-hemolytic. hlyfl grew intracellularly to levels 10-fold greater than wildtype L. monocytogenes and was less cytotoxic. In an intravenous mouse model, 90% of bacteria were non-hemolytic within three hours in the spleen and eight hours in the liver. The loss of LLO led to a 2-log virulence defect in the spleen and a 4-log virulence defect in the liver compared to WT L. monocytogenes. Thus, the production of LLO in the cytosol has significant impact on the pathogenicity of L. monocytogenes.
- Published
- 2020
44. Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS
- Author
-
Charles L. Larson, Robert A. Heinzen, and Paul A. Beare
- Subjects
0303 health sciences ,Host cell cytosol ,biology ,030306 microbiology ,Effector ,Mutant ,Vacuole ,bacterial infections and mycoses ,Coxiella burnetii ,biology.organism_classification ,Microbiology ,Legionella pneumophila ,Cell biology ,03 medical and health sciences ,Chaperone (protein) ,embryonic structures ,biology.protein ,bacteria ,Secretion ,Molecular Biology ,030304 developmental biology - Abstract
Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetiiicmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation. IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella. Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella. Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.
- Published
- 2019
45. Generation and screening of efficient neutralizing single domain antibodies (VHHs) against the critical functional domain of anthrax protective antigen (PA)
- Author
-
Abbas Shali, Fatemeh Gashtasbi, Sadegh Hasannia, Shirin Jalili, S. Shirin Shahangian, and Masoud Abdous
- Subjects
0301 basic medicine ,Camelus ,Phage display ,medicine.drug_class ,Anthrax toxin ,Bacterial Toxins ,030106 microbiology ,Biopanning ,Monoclonal antibody ,Biochemistry ,03 medical and health sciences ,Antigen ,Structural Biology ,medicine ,Animals ,Molecular Biology ,Antigens, Bacterial ,Host cell cytosol ,biology ,Chemistry ,General Medicine ,biology.organism_classification ,Antibodies, Bacterial ,Virology ,Bacillus anthracis ,Molecular Docking Simulation ,030104 developmental biology ,biology.protein ,Antibody ,Single-Chain Antibodies - Abstract
Since anthrax is an acute infectious disease, detection and neutralization of the toxins of pathogenic Bacillus anthracis are of great importance. The critical role of protective antigen (PA) component of tripartite anthrax toxin in toxin entry into the host cell cytosol provided a great deal of effort to generate monoclonal antibodies against this constitute. Regarding the importance of anthrax detection/neutralization and unique physicochemical and pharmacological features of VHHs as single domain antibodies, the present study aimed to generate VHHs against the receptor binding domain of PA, termed PAD4. After camel immunization, a gene repertoire of VHH fragments with a diversity of 4.7 × 108 clones was produced, followed by constructing a VHH phage display library. A stringent successive biopanning was then carried out to isolate the phages displaying high affinity VHHs against PAD4.Polyclonal and monoclonal Enzyme-linked immunosorbent assay (ELISA) verified binding specificity of phages to the target protein. Modeling of VHHs together with the docking simulation studies, illustrated the binding site of antibodies on antigen. Docking analysis revealed that all selected VHHs potently cover the key functional residues of PAD4. Since the selected VHHs could cover and block the receptor binding loops of PA, they could be proposed as hopeful anti-Anthrax candidates.
- Published
- 2018
46. Identification of interactions among host and bacterial proteins and evaluation of their role early during Shigella flexneri infection
- Author
-
Yiuka Leung, Marcia B. Goldberg, Anna Cristina Garza-Mayers, and Kelly Miller
- Subjects
0301 basic medicine ,Cytoplasm ,Phagocytosis ,030106 microbiology ,Biology ,medicine.disease_cause ,Microbiology ,Cell Line ,Shigella flexneri ,03 medical and health sciences ,Bacterial Proteins ,Type III Secretion Systems ,medicine ,Humans ,Secretion ,Shigella ,Dysentery, Bacillary ,Host cell cytosol ,Innate immune system ,Effector ,Macrophages ,Interleukin-18 ,Epithelial Cells ,biology.organism_classification ,030104 developmental biology ,Secretory protein ,Gene Knockdown Techniques ,Host-Pathogen Interactions ,Carrier Proteins ,Gene Deletion ,Research Article ,Protein Binding - Abstract
Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.
- Published
- 2018
47. Delivery of Chicken Egg Ovalbumin to Dendritic Cells by Listeriolysin O-Secreting Vegetative Bacillus subtilis
- Author
-
Michał Kamiński, Radosław Stachowiak, Jacek Bielecki, Tomasz Jagielski, and Katarzyna Roeske
- Subjects
Host cell cytosol ,biology ,Chemistry ,Listeriolysin O ,02 engineering and technology ,General Medicine ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Applied Microbiology and Biotechnology ,Epitope ,0104 chemical sciences ,Cell biology ,Ovalbumin ,Antigen ,MHC class I ,biology.protein ,Cytotoxic T cell ,0210 nano-technology ,Antigen-presenting cell ,Biotechnology - Abstract
Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of CD8⁺ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.
- Published
- 2018
48. Some molecular mechanisms of bacteria parasitization in host cell cytosol
- Author
-
E.V. E.V.Volchkova, O.F. O.F.Belaya, N.N. N.N.Kanshina, N.V. N.V.Kolaeva, E.O. E.O.Kravtsova, and M.N. M.N.Boychenko
- Subjects
Infectious Diseases ,Host cell cytosol ,biology ,Epidemiology ,Chemistry ,Virology ,biology.organism_classification ,Bacteria ,Cell biology - Published
- 2018
49. Cleansing the cytosol
- Author
-
Stella M. Hurtley
- Subjects
Multidisciplinary ,Host cell cytosol ,Chemistry ,medicine.medical_treatment ,Intracellular parasite ,Epithelium ,Microbiology ,Cytosol ,Cytokine ,Immune system ,medicine.anatomical_structure ,Immunity ,medicine ,CRISPR - Abstract
Microbiology Most human cells, not just those belonging to the immune system, mount protective responses to infection when activated by the immune cytokine interferon-gamma (IFN-γ). How IFN-γ confers this function in nonimmune cells and tissues is poorly understood. Gaudet et al. used genome-scale CRISPR/Cas9 gene editing to identify apolipoprotein L-3 (APOL3) as an IFN-γ–induced bactericidal protein that protects human epithelium, endothelium, and fibroblasts against infection (see the Perspective by Nathan). APOL3 directly targets bacteria in the host cell cytosol and kills them by dissolving their anionic membranes into lipoprotein complexes. This work reveals a detergent-like mechanism enlisted during human cell-autonomous immunity to combat intracellular pathogens. Science , abf8113, this issue p. [eabf8113][1]; see also abj5637, p. [276][2] [1]: /lookup/doi/10.1126/science.abf8113 [2]: /lookup/doi/10.1126/science.abj5637
- Published
- 2021
50. The Edwardsiella piscicida thioredoxin-like protein inhibits ASK1-MAPKs signaling cascades to promote pathogenesis during infection
- Author
-
Yuanxing Zhang, Shu Quan, Qin Liu, Dahai Yang, Zhuang Wang, Lingzhi Zhang, Wenting Xu, Xin Zheng, Jinchao Tan, Xiaohong Liu, Zhaoyan Gu, and Cuiting Yang
- Subjects
Models, Molecular ,Life Cycles ,Cell signaling ,Cultured tumor cells ,Signal transduction ,Crystallography, X-Ray ,Biochemistry ,Redox Signaling ,Thioredoxins ,Larvae ,Post-Translational Modification ,Phosphorylation ,Biology (General) ,Zebrafish ,0303 health sciences ,Host cell cytosol ,biology ,Virulence ,Effector ,030302 biochemistry & molecular biology ,Enterobacteriaceae Infections ,Eukaryota ,Signaling cascades ,Animal Models ,Cell biology ,Edwardsiella ,Experimental Organism Systems ,Osteichthyes ,Vertebrates ,Cell lines ,Thioredoxin ,Biological cultures ,Research Article ,MAPK signaling cascades ,MAP Kinase Signaling System ,Virulence Factors ,QH301-705.5 ,Immunology ,Immunoblotting ,Molecular Probe Techniques ,MAP Kinase Kinase Kinase 5 ,Microbiology ,03 medical and health sciences ,Model Organisms ,Bacterial Proteins ,Virology ,Genetics ,Humans ,Animals ,HeLa cells ,Molecular Biology Techniques ,Molecular Biology ,030304 developmental biology ,Innate immune system ,Host Microbial Interactions ,Host Cells ,Organisms ,Biology and Life Sciences ,Proteins ,RC581-607 ,biology.organism_classification ,Cell cultures ,Immunity, Innate ,Research and analysis methods ,Fish ,Animal Studies ,Parasitology ,Immunologic diseases. Allergy ,Viral Transmission and Infection ,Developmental Biology - Abstract
It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection., Author summary Thioredoxin (Trx) is universally conserved thiol-oxidoreductase that regulates numerous cellular pathways under thiol-based redox control in both prokaryotic and eukaryotic organisms. Despite its central importance, the mechanism of bacterial Trx recognizes its target proteins in host cellular signaling remains unknown. Here, we uncover a bacterial thioredoxin-like protein that can be translocated into host cells and mimic the endogenous TRX1 to target ASK1-MAPK signaling, finally facilitating bacterial pathogenesis. This work expands our understanding of bacterial thioredoxins in manipulating host innate immunity.
- Published
- 2019
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