scholarly journals Mechanical Fractionation Reveals Structural Requirements for Enteropathogenic Escherichia coli Tir Insertion into Host Membranes

2000 ◽  
Vol 68 (7) ◽  
pp. 4344-4348 ◽  
Author(s):  
Annick Gauthier ◽  
Myriam de Grado ◽  
B. Brett Finlay
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Hela Cells ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Host Cells ◽  
Host Cell Membrane ◽  
Bacterial Proteins ◽  
Host Cell Membranes ◽  
Triton X

ABSTRACT Enteropathogenic Escherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system. Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells. In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins. We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin.

scholarly journals The Hydrophilic Translocator for Vibrio parahaemolyticus, T3SS2, Is Also Translocated

2012 ◽  
Vol 80 (8) ◽  
pp. 2940-2947 ◽  
Author(s):  
Xiaohui Zhou ◽  
Jennifer M. Ritchie ◽  
Hirotaka Hiyoshi ◽  
Tetsuya Iida ◽  
Brigid M. Davis ◽  
...  
Keyword(s):  
Host Cell ◽  
Vibrio Parahaemolyticus ◽  
Diarrheal Disease ◽  
Host Cells ◽  
Cell Cytoplasm ◽  
Host Cell Membrane ◽  
Membrane Pore ◽  
Content Type ◽  
Bacterial Proteins ◽  
Host Cell Cytoplasm

ABSTRACTThe pathogenesis of the diarrheal disease caused byVibrio parahaemolyticus, a leading cause of seafood-associated enteritis worldwide, is dependent upon a type III secretion system, T3SS2. This apparatus enables the pathogen to inject bacterial proteins (effectors) into the cytosol of host cells and thereby modulate host processes. T3SS effector proteins transit into the host cell via a membrane pore (translocon) typically formed by 3 bacterial proteins. We have identified the third translocon protein for T3SS2: VopW, which was previously classified as an effector protein for a homologous T3SS inV. cholerae. VopW is a hydrophilic translocon protein; like other such proteins, it is not inserted into the host cell membrane but is required for insertion of the two hydrophobic translocators, VopB2 and VopD2, that constitute the membrane channel. VopW is not required for secretion of T3SS2 effectors into the bacterial culture medium; however, it is essential for transfer of these proteins into the host cell cytoplasm. Consequently, deletion ofvopWabrogates the virulence ofV. parahaemolyticusin several animal models of diarrheal disease. Unlike previously described hydrophilic translocators, VopW is itself translocated into the host cell cytoplasm, raising the possibility that it functions as both a translocator and an effector.

scholarly journals Induction of Epithelial Cell Death Including Apoptosis by Enteropathogenic Escherichia coli Expressing Bundle-Forming Pili

2001 ◽  
Vol 69 (12) ◽  
pp. 7356-7364 ◽  
Author(s):  
Maan Abul-Milh ◽  
Ying Wu ◽  
Bedy Lau ◽  
Clifford A. Lingwood ◽  
Debora Barnett Foster
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Host Cell ◽  
Morphological Changes ◽  
Laboratory Strain ◽  
Host Cells ◽  
Host Cell Membrane ◽  
Infantile Diarrhea ◽  
Enterocyte Effacement ◽  
Colonic Cells

ABSTRACT Infection with enteropathogenic Escherichia coli (EPEC) is a major cause of severe infantile diarrhea, particularly in parts of the developing world. The bundle-forming pilus (BFP) of EPEC is an established virulence factor encoded on the EPEC adherence factor plasmid (EAF) and has been implicated in both localized adherence to host cells and bacterial autoaggregation. We investigated the role of BFP in the ability of EPEC binding to kill host epithelial cells. BFP-expressing strains killed all three cell lines tested, comprising HEp-2 (laryngeal), HeLa (cervical), and Caco-2 (colonic) cells. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA, and morphological changes detected by electron microscopy indicated evidence of apoptosis. The extent of cell death was significantly greater for BFP-expressing strains, including E2348/69, a wild-type clinical isolate, as well as for a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP induced significantly less cell death, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, HB101, and HB101 complemented with the locus of enterocyte effacement pathogenicity island. These results indicate a direct correlation between BFP expression and induction of cell death, including apoptosis, an event which may involve the targeting of host cell membrane phosphatidylethanolamine.

scholarly journals Mycobacterium Survival Strategy Translated to Develop a Lipo-Peptide Based Fusion Inhibitor

2020 ◽  
Author(s):  
Avijit Sardar ◽  
Aritraa Lahiri ◽  
Amirul Islam Mallick ◽  
Pradip Kumar Tarafdar
Keyword(s):  
Membrane Fusion ◽  
Host Cell ◽  
Broad Spectrum ◽  
Antiviral Agent ◽  
Host Cells ◽  
Peptide Sequence ◽  
Fusion Inhibitor ◽  
Host Cell Membrane ◽  
Unique Structural Feature ◽  
Host Cell Membranes

The entry of enveloped viruses requires fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such virus-host cell membrane fusion may emerge as a broad-spectrum antiviral agent to neutralize the infection from an increasing diversity of harmful new viruses. Mycobacterium survives inside the phagosome of the host cells by inhibiting phagosome-lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40-repeat containing protein suggest that the tryptophan-aspartic acid (WD) sequence is placed at distorted β-meander motif (more exposed) whereas the WD resides in regular β-meander motif in other WD40 proteins. The unique structural feature of coronin 1 was explored to identify a simple lipo-peptide sequence (lipid-WD), which effectively inhibit the membrane fusion by increasing interfacial order and decreasing water penetration, surface potential. The effective fusion inhibitory role of mycobacterium inspired lipo-dipeptide was applied to combat type 1 influenza virus (H1N1) infection as a ‘broad spectrum’ antiviral agent.<br>

scholarly journals Mycobacterium Survival Strategy Translated to Develop a Lipo-Peptide Based Fusion Inhibitor

2020 ◽  
Author(s):  
Avijit Sardar ◽  
Aritraa Lahiri ◽  
Amirul Islam Mallick ◽  
Pradip Kumar Tarafdar
Keyword(s):  
Membrane Fusion ◽  
Host Cell ◽  
Broad Spectrum ◽  
Antiviral Agent ◽  
Host Cells ◽  
Peptide Sequence ◽  
Fusion Inhibitor ◽  
Host Cell Membrane ◽  
Unique Structural Feature ◽  
Host Cell Membranes

The entry of enveloped viruses requires fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such virus-host cell membrane fusion may emerge as a broad-spectrum antiviral agent to neutralize the infection from an increasing diversity of harmful new viruses. Mycobacterium survives inside the phagosome of the host cells by inhibiting phagosome-lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40-repeat containing protein suggest that the tryptophan-aspartic acid (WD) sequence is placed at distorted β-meander motif (more exposed) whereas the WD resides in regular β-meander motif in other WD40 proteins. The unique structural feature of coronin 1 was explored to identify a simple lipo-peptide sequence (lipid-WD), which effectively inhibit the membrane fusion by increasing interfacial order and decreasing water penetration, surface potential. The effective fusion inhibitory role of mycobacterium inspired lipo-dipeptide was applied to combat type 1 influenza virus (H1N1) infection as a ‘broad spectrum’ antiviral agent.<br>

scholarly journals Visualization and regulation of translocons inYersiniatype III protein secretion machines during host cell infection

2018 ◽  
Author(s):  
Theresa Nauth ◽  
Franziska Huschka ◽  
Michaela Schweizer ◽  
Jens B. Bosse ◽  
Andreas Diepold ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Host Cell ◽  
Target Cell ◽  
Hela Cells ◽  
Cell Membranes ◽  
Bacterial Pathogens ◽  
Stimulated Emission ◽  
Host Cells ◽  
Host Cell Membranes ◽  
Microscopy Techniques

AbstractType III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery - also named injectisome - assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their assembly have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualizeY. enterocoliticatranslocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the putatively operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology we provide novel information about the spatiotemporal organisation of T3SS translocons and their regulation by host cell factors.Author SummaryMany human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacteria’s infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogenYersinia enterocoliticaduring infection of human host cells. Thereby we could for the first time - with fluorescence microscopy - visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organisation and regulation of bacterial translocons and may thus open up new strategies to block the function of infectious bacteria.

scholarly journals Adhesion of Escherichia coli to HeLa Cells Mediated byTrypanosoma cruzi Surface Glycoprotein-Derived Peptides Inserted in the Outer Membrane Protein LamB

1999 ◽  
Vol 67 (9) ◽  
pp. 4908-4911 ◽  
Author(s):  
Cátia M. Pereira ◽  
Sílvio Favoreto ◽  
José Franco da Silveira ◽  
Nobuko Yoshida ◽  
Beatriz A. Castilho
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Cell Surface ◽  
Hela Cells ◽  
Outer Membrane Protein ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Parasite Invasion

ABSTRACT Peptides derived from the surface glycoprotein gp82 ofTrypanosoma cruzi, previously implicated in the parasite’s invasion of host cells, were expressed as fusions to the protein LamB of Escherichia coli in a region known to be exposed on the cell surface. Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells. Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by T. cruzi, supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

Interactions Between Salmonella Typhimurium, Enteropathogenic Escherichia Coli (EPEC), and Host Epithelial Cells

1995 ◽  
Vol 9 (1) ◽  
pp. 31-36 ◽  
Author(s):  
B.B. Finlay
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Salmonella Typhimurium ◽  
Host Cell ◽  
Inositol Phosphate ◽  
Enteric Pathogens ◽  
Host Protein ◽  
Host Cells ◽  
Enteropathogenic Escherichia Coli ◽  
Sequence Of Events

The interactions that occur between pathogenic micro-organisms and their host cells are complex and intimate. We have used two enteric pathogens, Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC), to examine the interactions that occur between these organisms and epithelial cells. Although these are enteric pathogens, the knowledge and techniques developed from these systems may be applied to the study of dental pathogens. Both S. typhimurium and EPEC disrupt epithelial monolayer integrity, although by different mechanisms. Both pathogens cause loss of microvilli and re-arrangement of the underlying host cytoskeleton. Despite these similarities, both organisms send different signals into the host cell. EPEC signal transduction involves generation of intracellular calcium and inositol phosphate fluxes, and activation of host tyrosine kinases that results in tyrosine phosphorylation of a 90-kDa host protein. Bacterial mutants have been identifed that are deficient in signaling to the host. We propose a sequence of events that occur when EPEC interacts with epithelial cells. Once inside a host cell, S. typhimurium remains within a vacuole. To define some of the parameters of the intracellular environment, we constructed genetic fusions of known genes with lacZ, and used these fusions as reporter probes of the intracellular vacuolar environment. We have also begun to examine the bacterial and host cell factors necessary for S. typhimurium to multiply within epithelial cells. We found that this organism triggers the formation of novel tubular lysosomes, and these structures are linked with intracellular replication.

Invasion of Toxoplasma gondii occurs by active penetration of the host cell

1995 ◽  
Vol 108 (6) ◽  
pp. 2457-2464 ◽  
Author(s):  
J.H. Morisaki ◽  
J.E. Heuser ◽  
L.D. Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Tyrosine Phosphorylation ◽  
Host Cells ◽  
The Novel ◽  
Host Proteins ◽  
Host Cell Membrane ◽  
Membrane Ruffling ◽  
Obligate Intracellular ◽  
Obligate Intracellular Parasite

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of vertebrate cells including macrophages. We have used a combination of video microscopy and fluorescence localization to examine the entry of Toxoplasma into macrophages and nonphagocytic host cells. Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. Invasion occurred rapidly and within 25–40 seconds the parasite penetrated into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting phagosome that formed over a period of 2–4 minutes. Phagocytosis involved both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins. In some cases, parasites that were first internalized by phagocytosis, were able to escape from the phagosome by a process analogous to invasion. These studies reveal that active penetration of the host cell by Toxoplasma is fundamentally different from phagocytosis or induced endocytic uptake. The novel ability to penetrate the host cell likely contributes to the capability of Toxoplasma-containing vacuoles to avoid endocytic processing.

scholarly journals Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs.

1996 ◽  
Vol 40 (11) ◽  
pp. 2455-2458 ◽  
Author(s):  
J Nakajima-Shimada ◽  
Y Hirota ◽  
T Aoki
Keyword(s):  
Trypanosoma Cruzi ◽  
Growth Inhibition ◽  
Host Cell ◽  
Culture System ◽  
Mammalian Cells ◽  
Screening Method ◽  
Host Cells ◽  
Mammalian Host ◽  
Dependent Manner ◽  
Pyrimidine Analogs

Trypanosoma cruzi, the causative agent of Chagas' disease, exhibits two different developmental stages in mammals, the amastigote, an intracellular form that proliferates in the cytoplasm of host cells, and the trypomastigote, an extracellular form that circulates in the bloodstream. We have already established an in vitro culture system using mammalian host cells (HeLa) infected with T. cruzi in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of anti-T. cruzi agents that would ideally prove to be effective against trypanosomes with no toxicity to the host cell. Of the purine analogs tested, allopurinol markedly inhibited the growth of amastigotes in a dose-dependent manner, with no lethal effect on trypomastigotes. 3'-Deoxyinosine and 3'-deoxyadenosine also suppressed T. cruzi growth inside the host cell, with the concentrations causing 50% growth inhibition being 10 and 5 microM, respectively, in contrast to a concentration causing 50% growth inhibition of 3 microM for allopurinol. Among the pyrimidine analogs examined, 3'-azido-3'-deoxythymidine (zidovudine) significantly reduced the growth of the parasite at concentrations as low as 1 microM. The anti-human immunodeficiency virus agents 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine caused a decrease in amastigote growth, while 2',3'-dideoxycytidine and 2',3'-dideoxyuridine had no inhibitory effect. When Swiss 3T3 fibroblasts were used as host cells, allopurinol, 3'-deoxyinosine, 3'-deoxyadenosine, and 3'-azid-3'-deoxythymidine also markedly inhibited T. cruzi proliferation. These results indicate that our culture system is useful as a primary screening method for candidate compounds against T. cruzi on the basis of two criteria, namely, intracellular replication by the parasite and host-cell infection rate.

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