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

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.

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

Infection and Immunity ◽

10.1128/iai.68.7.4344-4348.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 4344-4348 ◽

Cited By ~ 38

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.

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Two pathways for ATP release from host cells in enteropathogenicEscherichia coliinfection

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00137.2005 ◽

2005 ◽

Vol 289 (3) ◽

pp. G407-G417 ◽

Cited By ~ 19

Author(s):

John K. Crane ◽

Tonniele M. Naeher ◽

Shilpa S. Choudhari ◽

Elisa M. Giroux

Keyword(s):

Escherichia Coli ◽

Cell Death ◽

Host Cell ◽

Atp Release ◽

Host Cells ◽

Specific Cell ◽

Hypotonic Medium ◽

Enteropathogenic Escherichia Coli ◽

Extracellular Adenosine ◽

Transfected Cells

We previously reported that enteropathogenic Escherichia coli (EPEC) infection triggered a large release of ATP from the host cell that was correlated with and dependent on EPEC-induced killing of the host cell. We noted, however, that under some circumstances, EPEC-induced ATP release exceeded that which could be accounted for on the basis of host cell killing. For example, EPEC-induced ATP release was potentiated by noncytotoxic agents that elevate host cell cAMP, such as forskolin and cholera toxin, and by exposure to hypotonic medium. These findings and the performance of the EPEC espF mutant led us to hypothesize that the CFTR plays a role in EPEC-induced ATP release that is independent of cell death. We report the results of experiments using specific, cell-permeable CFTR activators and inhibitors, as well as transfection of the CFTR into non-CFTR-expressing cell lines, which incriminate the CFTR as a second pathway for ATP release from host cells. Increased ATP release via CFTR is not accompanied by an increase in EPEC adherence to transfected cells. The CFTR-dependent ATP release pathway becomes activated endogenously later in EPEC infection, and this activation is mediated, at least in part, by generation of extracellular adenosine from the breakdown of released ATP.

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The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.02876-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1139-1149 ◽

Cited By ~ 5

Author(s):

John Scott Wilbur ◽

Wyatt Byrd ◽

Shylaja Ramamurthy ◽

Hannah E. Ledvina ◽

Khaldoon Khirfan ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Death ◽

Epithelial Cell ◽

Host Cell ◽

Bacterial Colonization ◽

Clinical Signs ◽

Effector Proteins ◽

Host Cells ◽

Tunel Staining

Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenicespZdeletion strain (ΔespZ) and correspondingcis-complemented derivatives of rabbit enteropathogenicEscherichia coliand compared their abilities to regulate the T3SS and influence host cell survivalin vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports,espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZstrain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZstrain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZmutant fecal carriage progressively decreased on subsequent days. ΔespZmutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZstrain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.

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Shigella-induced necrosis and apoptosis of U937 cells and J774 macrophages

Microbiology ◽

10.1099/mic.0.26341-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2513-2527 ◽

Cited By ~ 34

Author(s):

Takashi Nonaka ◽

Taku Kuwabara ◽

Hitomi Mimuro ◽

Asaomi Kuwae ◽

Shinobu Imajoh-Ohmi

Keyword(s):

Cell Death ◽

Cell Membrane ◽

Host Cell ◽

Caspase 3 ◽

Cytochalasin D ◽

Host Cells ◽

U937 Cells ◽

Host Cell Membrane ◽

Caspase 1 ◽

Infected Cells

It is currently unclear whether Shigella kills its phagocytic host cells by apoptosis or necrosis. This study shows that rapid necrosis ensues in macrophage-like cell lines (U937 cells differentiated by all-trans-retinoic acid and J774 cells) infected with the Shigella flexneri strain YSH6000. The infected cells rapidly lose membrane integrity, a typical feature of necrosis, as indicated by the release of the cytoplasmic lactate dehydrogenase and the exposure of phosphatidylserine (PS) associated with the rapid uptake of propidium iodide (PI). The infected cells exhibit DNA fragmentation without nuclear condensation, and substantial involvement of either caspase-3/-7 or caspase-1 was not detected, which is also contrary to what is normally observed in apoptosis. Cytochalasin D potently inhibited Shigella-induced cell death, indicating that only internalized Shigella can cause necrosis. Osmoprotectants such as polyethylene glycols could suppress cell death, suggesting that insertion of a pore by Shigella into the host cell membrane induces the necrosis. The pore was estimated to be 2·87±0·4 nm in diameter. Shigella was also found to be able to induce apoptosis but only in one of the lines tested and under specific conditions, namely U937 cells differentiated with interferon-γ (U937IFN). Caspase-3/-7 but not caspase-1 activation was observed in these infected cells and the exposure of PS occurred without the uptake of PI. An avirulent Shigella strain, wild-type Shigella killed with gentamicin, and even Escherichia coli strain JM109, could also induce apoptosis in U937IFN cells, and cytochalasin D could not prevent apoptosis. It appears therefore that Shigella-induced apoptosis of U937IFN cells is unrelated to Shigella pathogenicity and does not require bacterial internalization. Thus, Shigella can induce rapid necrosis of macrophage-like cells in a virulence-related manner by forming pores in the host cell membrane while some cells can be killed through apoptosis in a virulence-independent fashion.

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Pathogen induced subversion of NAD+ metabolism mediating host cell death: a target for development of chemotherapeutics

Cell Death Discovery ◽

10.1038/s41420-020-00366-z ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Ayushi Chaurasiya ◽

Swati Garg ◽

Ashish Khanna ◽

Chintam Narayana ◽

Ved Prakash Dwivedi ◽

...

Keyword(s):

Cell Death ◽

Host Cell ◽

Nicotinamide Adenine Dinucleotide ◽

Malaria Parasite ◽

Host Cells ◽

Nicotinamide Adenine ◽

Targeted Therapeutics ◽

Nad Metabolism ◽

Host Cell Death ◽

Nanomolar Range

AbstractHijacking of host metabolic status by a pathogen for its regulated dissemination from the host is prerequisite for the propagation of infection. M. tuberculosis secretes an NAD+-glycohydrolase, TNT, to induce host necroptosis by hydrolyzing Nicotinamide adenine dinucleotide (NAD+). Herein, we expressed TNT in macrophages and erythrocytes; the host cells for M. tuberculosis and the malaria parasite respectively, and found that it reduced the NAD+ levels and thereby induced necroptosis and eryptosis resulting in premature dissemination of pathogen. Targeting TNT in M. tuberculosis or induced eryptosis in malaria parasite interferes with pathogen dissemination and reduction in the propagation of infection. Building upon our discovery that inhibition of pathogen-mediated host NAD+ modulation is a way forward for regulation of infection, we synthesized and screened some novel compounds that showed inhibition of NAD+-glycohydrolase activity and pathogen infection in the nanomolar range. Overall this study highlights the fundamental importance of pathogen-mediated modulation of host NAD+ homeostasis for its infection propagation and novel inhibitors as leads for host-targeted therapeutics.

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

Advances in Dental Research ◽

10.1177/08959374950090010601 ◽

1995 ◽

Vol 9 (1) ◽

pp. 31-36 ◽

Cited By ~ 5

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.

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

Journal of Cell Science ◽

10.1242/jcs.108.6.2457 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2457-2464 ◽

Cited By ~ 4

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.

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Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death

mBio ◽

10.1128/mbio.00711-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Eun Jung Thak ◽

Su-Bin Lee ◽

Shengjie Xu-Vanpala ◽

Dong-Jik Lee ◽

Seung-Yeon Chung ◽

...

Keyword(s):

Cell Death ◽

Fungal Infection ◽

Host Cell ◽

Host Cells ◽

Glycan Structure ◽

Macrophage Cell ◽

Content Type ◽

Host Cell Death ◽

Intact Core

ABSTRACT Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformans. IMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans. Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

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Characterization of Stages of Hepatozoon americanum and of Parasitized Canine Host Cells

Veterinary Pathology ◽

10.1354/vp.42-6-788 ◽

2005 ◽

Vol 42 (6) ◽

pp. 788-796 ◽

Cited By ~ 20

Author(s):

C. A. Cummings ◽

R. J. Panciera ◽

K. M. Kocan ◽

J. S. Mathew ◽

S. A. Ewing

Keyword(s):

Cell Membrane ◽

Host Cell ◽

Striated Muscle ◽

Parasitophorous Vacuole ◽

Protozoan Parasite ◽

Host Cells ◽

Blood Leukocytes ◽

Host Cell Membrane ◽

Asexual Multiplication ◽

Hepatozoon Americanum

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in “onion skin” cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a “tail” in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.

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Sequence, expression and localization of calmodulin-domain protein kinases inEimeria tenellaandEimeria maxima

Parasitology ◽

10.1017/s0031182000081506 ◽

1996 ◽

Vol 113 (5) ◽

pp. 439-448 ◽

Cited By ~ 21

Author(s):

P. P. J. Dunn ◽

J. M. Bumstead ◽

F. M. Tomley

Keyword(s):

Host Cell ◽

Protein Kinases ◽

Catalytic Domain ◽

Sequence Data ◽

Host Cells ◽

Cdna Clones ◽

Host Cell Membrane ◽

Amino Terminal ◽

Domain Protein

SUMMARYWe have isolated and sequenced cDNA clones fromEimeria tenellaandEimeria maximawhich encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of theE. tenellacalmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells inin vitroculture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells byE. tenella.

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