Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process.

PubMed

Forestier, Claire-Lise; Machu, Christophe; Loussert, Celine; Pescher, Pascale; Späth, Gerald F

2011-04-21

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry. Copyright © 2011 Elsevier Inc. All rights reserved.

Host cell processes that influence the intracellular survival of Legionella pneumophila.

PubMed

Shin, Sunny; Roy, Craig R

2008-06-01

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

PubMed

Hu, Zhilan; Guo, Donglin; Yip, Shirley S M; Zhan, Dejin; Misaghi, Shahram; Joly, John C; Snedecor, Bradley R; Shen, Amy Y

2013-01-01

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers.

Unique physiology of host-parasite interactions in microsporidia infections.

PubMed

Williams, Bryony A P

2009-11-01

Microsporidia are intracellular parasites of all major animal lineages and have a described diversity of over 1200 species and an actual diversity that is estimated to be much higher. They are important pathogens of mammals, and are now one of the most common infections among immunocompromised humans. Although related to fungi, microsporidia are atypical in genomic biology, cell structure and infection mechanism. Host cell infection involves the rapid expulsion of a polar tube from a dormant spore to pierce the host cell membrane and allow the direct transfer of the spore contents into the host cell cytoplasm. This intimate relationship between parasite and host is unique. It allows the microsporidia to be highly exploitative of the host cell environment and cause such diverse effects as the induction of hypertrophied cells to harbour prolific spore development, host sex ratio distortion and host cell organelle and microtubule reorganization. Genome sequencing has revealed that microsporidia have achieved this high level of parasite sophistication with radically reduced proteomes and with many typical eukaryotic pathways pared-down to what appear to be minimal functional units. These traits make microsporidia intriguing model systems for understanding the extremes of reductive parasite evolution and host cell manipulation.

Modulation of host cell function by Legionella pneumophila type IV effectors.

PubMed

Hubber, Andree; Roy, Craig R

2010-01-01

Macrophages and protozoa ingest bacteria by phagocytosis and destroy these microbes using a conserved pathway that mediates fusion of the phagosome with lysosomes. To survive within phagocytic host cells, bacterial pathogens have evolved a variety of strategies to avoid fusion with lysosomes. A virulence strategy used by the intracellular pathogen Legionella pneumophila is to manipulate host cellular processes using bacterial proteins that are delivered into the cytosolic compartment of the host cell by a specialized secretion system called Dot/Icm. The proteins delivered by the Dot/Icm system target host factors that play evolutionarily conserved roles in controlling membrane transport in eukaryotic cells, which enables L. pneumophila to create an endoplasmic reticulum-like vacuole that supports intracellular replication in both protozoan and mammalian host cells. This review focuses on intracellular trafficking of L. pneumophila and describes how bacterial proteins contribute to modulation of host processes required for survival within host cells.

Gene Expression Profiling of Monkeypox Virus-Infected Cells Reveals Novel Interfaces for Host-Virus Interactions

DTIC Science & Technology

2010-07-28

expression is plotted on Y -axis after normalization to mock-treated samples. Results plotted to compare calculated fold change in expression of each gene ...RESEARCH Open Access Gene expression profiling of monkeypox virus-infected cells reveals novel interfaces for host-virus interactions Abdulnaser...suppress antiviral cell defenses, exploit host cell machinery, and delay infection-induced cell death. However, a comprehensive study of all host genes

CD62L− memory T cells enhance T-cell regeneration after allogeneic stem cell transplantation by eliminating host resistance in mice

PubMed Central

Zhang, Jifeng; Barefoot, Brice E.; Mo, Wenjian; Deoliveira, Divino; Son, Jessica; Cui, Xiuyu; Ramsburg, Elizabeth

2012-01-01

A major challenge in allogeneic hematopoietic cell transplantation is how to transfer T-cell immunity without causing graft-versus-host disease (GVHD). Effector memory T cells (CD62L−) are a cell subset that can potentially address this challenge because they do not induce GVHD. Here, we investigated how CD62L− T cells contributed to phenotypic and functional T-cell reconstitution after transplantation. On transfer into allogeneic recipients, CD62L− T cells were activated and expressed multiple cytokines and cytotoxic molecules. CD62L− T cells were able to deplete host radioresistant T cells and facilitate hematopoietic engraftment, resulting in enhanced de novo T-cell regeneration. Enhanced functional immune reconstitution was demonstrated in CD62L− T-cell recipients using a tumor and an influenza virus challenge model. Even though CD62L− T cells are able to respond to alloantigens and deplete host radioresistant immune cells in GVHD recipients, alloreactive CD62L− T cells lost the reactivity over time and were eventually tolerant to alloantigens as a result of prolonged antigen exposure, suggesting a mechanism by which CD62L− T cells were able to eliminate host resistance without causing GVHD. These data further highlight the unique characteristics of CD62L− T cells and their potential applications in clinical hematopoietic cell transplantation. PMID:22596261

Trans-suppression of defense DEFB1 gene in intestinal epithelial cells following Cryptosporidium parvum infection is associated with host delivery of parasite Cdg7_FLc_1000 RNA.

PubMed

Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Dolata, Courtney E; Chen, Xian-Ming

2018-03-01

To counteract host immunity, Cryptosporidium parvum has evolved multiple strategies to suppress host antimicrobial defense. One such strategy is to reduce the production of the antimicrobial peptide beta-defensin 1 (DEFB1) by host epithelial cells but the underlying mechanisms remain unclear. Recent studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the expression profile of host beta-defensin genes in host cells following infection. We found that C. parvum infection caused a significant downregulation of the DEFB1 gene. Interestingly, downregulation of DEFB1 gene was associated with host delivery of Cdg7_FLc_1000 RNA transcript, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected host cells. Knockdown of Cdg7_FLc_1000 in host cells could attenuate the trans-suppression of host DEFB1 gene and decreased the parasite burden. Therefore, our data suggest that trans-suppression of DEFB1 gene in intestinal epithelial cells following C. parvum infection involves host delivery of parasite Cdg7_FLc_1000 RNA, a process that may be relevant to the epithelial defense evasion by C. parvum at the early stage of infection.

Regulation of the Host Antiviral State by Intercellular Communications

PubMed Central

Assil, Sonia; Webster, Brian; Dreux, Marlène

2015-01-01

Viruses usually induce a profound remodeling of host cells, including the usurpation of host machinery to support their replication and production of virions to invade new cells. Nonetheless, recognition of viruses by the host often triggers innate immune signaling, preventing viral spread and modulating the function of immune cells. It conventionally occurs through production of antiviral factors and cytokines by infected cells. Virtually all viruses have evolved mechanisms to blunt such responses. Importantly, it is becoming increasingly recognized that infected cells also transmit signals to regulate innate immunity in uninfected neighboring cells. These alternative pathways are notably mediated by vesicular secretion of various virus- and host-derived products (miRNAs, RNAs, and proteins) and non-infectious viral particles. In this review, we focus on these newly-described modes of cell-to-cell communications and their impact on neighboring cell functions. The reception of these signals can have anti- and pro-viral impacts, as well as more complex effects in the host such as oncogenesis and inflammation. Therefore, these “broadcasting” functions, which might be tuned by an arms race involving selective evolution driven by either the host or the virus, constitute novel and original regulations of viral infection, either highly localized or systemic. PMID:26295405

Synchronous induction of detachment and reattachment of symbiotic Chlorella spp. from the cell cortex of the host Paramecium bursaria.

PubMed

Kodama, Yuuki; Fujishima, Masahiro

2013-09-01

Paramecium bursaria harbor several hundred symbiotic Chlorella spp. Each alga is enclosed in a perialgal vacuole membrane, which can attach to the host cell cortex. How the perialgal vacuole attaches beneath the host cell cortex remains unknown. High-speed centrifugation (> 1000×g) for 1min induces rapid detachment of the algae from the host cell cortex and concentrates the algae to the posterior half of the host cell. Simultaneously, most of the host acidosomes and lysosomes accumulate in the anterior half of the host cell. Both the detached algae and the dislocated acidic vesicles recover their original positions by host cyclosis within 10min after centrifugation. These recoveries were inhibited if the host cytoplasmic streaming was arrested by nocodazole. Endosymbiotic algae during the early reinfection process also show the capability of desorption after centrifugation. These results demonstrate that adhesion of the perialgal vacuole beneath the host cell cortex is repeatedly inducible, and that host cytoplasmic streaming facilitates recovery of the algal attachment. This study is the first report to illuminate the mechanism of the induction to desorb for symbiotic algae and acidic vesicles, and will contribute to the understanding of the mechanism of algal and organelle arrangements in Paramecium. Copyright © 2013 Elsevier GmbH. All rights reserved.

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

PubMed

Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

2013-04-17

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

LAMP-2 absence interferes with plasma membrane repair and decreases T. cruzi host cell invasion.

PubMed

Couto, Natália Fernanda; Pedersane, Dina; Rezende, Luisa; Dias, Patrícia P; Corbani, Tayanne L; Bentini, Lívia C; Oliveira, Anny C S; Kelles, Ludmila F; Castro-Gomes, Thiago; Andrade, Luciana O

2017-06-01

Trypanosoma cruzi enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of T. cruzi into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2-/- cells, indicating that LAMP-2 is an important player during T. cruzi invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2-/- and LAMP1/2-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and T. cruzi ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in T. cruzi invasion.

LAMP-2 absence interferes with plasma membrane repair and decreases T. cruzi host cell invasion

PubMed Central

Rezende, Luisa; Bentini, Lívia C.; Oliveira, Anny C. S.

2017-01-01

Trypanosoma cruzi enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of T. cruzi into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2-/- cells, indicating that LAMP-2 is an important player during T. cruzi invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2-/- and LAMP1/2-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and T. cruzi ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in T. cruzi invasion. PMID:28586379

Host cell capable of producing enzymes useful for degradation of lignocellulosic material

DOE Office of Scientific and Technical Information (OSTI.GOV)

Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schoonneveld-Bergmans, Margot Elisabeth Francoise

The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes.

PubMed

Killackey, Samuel A; Sorbara, Matthew T; Girardin, Stephen E

2016-01-01

Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general.

Stem cell recruitment of newly formed host cells via a successful seduction? Filling the gap between neurogenic niche and injured brain site.

PubMed

Tajiri, Naoki; Kaneko, Yuji; Shinozuka, Kazutaka; Ishikawa, Hiroto; Yankee, Ernest; McGrogan, Michael; Case, Casey; Borlongan, Cesar V

2013-01-01

Here, we report that a unique mechanism of action exerted by stem cells in the repair of the traumatically injured brain involves their ability to harness a biobridge between neurogenic niche and injured brain site. This biobridge, visualized immunohistochemically and laser captured, corresponded to an area between the neurogenic subventricular zone and the injured cortex. That the biobridge expressed high levels of extracellular matrix metalloproteinases characterized initially by a stream of transplanted stem cells, but subsequently contained only few to non-detectable grafts and overgrown by newly formed host cells, implicates a novel property of stem cells. The transplanted stem cells manifest themselves as pathways for trafficking the migration of host neurogenic cells, but once this biobridge is formed between the neurogenic site and the injured brain site, the grafted cells disappear and relinquish their task to the host neurogenic cells. Our findings reveal that long-distance migration of host cells from the neurogenic niche to the injured brain site can be achieved through transplanted stem cells serving as biobridges for initiation of endogenous repair mechanisms. This is the first report of a stem cell-paved "biobridge". Indeed, to date the two major schools of discipline in stem cell repair mechanism primarily support the concept of "cell replacement" and bystander effects of "trophic factor secretion". The present novel observations of a stem cell seducing a host cell to engage in brain repair advances basic science concepts on stem cell biology and extracellular matrix, as well as provokes translational research on propagating this stem cell-paved biobridge beyond cell replacement and trophic factor secretion for the treatment of traumatic brain injury and other neurological disorders.

Interactions of Cryptococcus with Dendritic Cells

PubMed Central

Wozniak, Karen L.

2018-01-01

The fungal pathogens Cryptococcus neoformans and Cryptococcus gattii can cause life-threatening infections in immune compromised and immune competent hosts. These pathogens enter the host via inhalation, and respiratory tract innate immune cells such as dendritic cells (DCs) are one of the first host cells they encounter. The interactions between Cryptococcus and innate immune cells play a critical role in the progression of disease in the host. This review will focus specifically on the interactions between Cryptococcus and dendritic cells (DCs), including recognition/processing by DCs, effects of immune mediators on DC recruitment and activity, and the potential for DC vaccination against cryptococcosis. PMID:29543719

Interactions of Cryptococcus with Dendritic Cells.

PubMed

Wozniak, Karen L

2018-03-15

The fungal pathogens Cryptococcus neoformans and Cryptococcus gattii can cause life-threatening infections in immune compromised and immune competent hosts. These pathogens enter the host via inhalation, and respiratory tract innate immune cells such as dendritic cells (DCs) are one of the first host cells they encounter. The interactions between Cryptococcus and innate immune cells play a critical role in the progression of disease in the host. This review will focus specifically on the interactions between Cryptococcus and dendritic cells (DCs), including recognition/processing by DCs, effects of immune mediators on DC recruitment and activity, and the potential for DC vaccination against cryptococcosis.

A history of studies that examine the interactions of Toxoplasma with its host cell: Emphasis on in vitro models.

PubMed

Boyle, Jon P; Radke, Jay R

2009-07-01

This review is a historical look at work carried out over the past 50 years examining interactions of Toxoplasma with the host cell and attempts to focus on some of the seminal experiments in the field. This early work formed the foundation for more recent studies aimed at identifying the host and parasite factors mediating key Toxoplasma-host cell interactions. We focus especially on those studies that were performed in vitro and provide discussions of the following general areas: (i) establishment of the parasitophorous vacuole, (ii) the requirement of specific host cell molecules for parasite replication, (iii) the scenarios under which the host cell can resist parasite replication and/or persistence, (iv) host species-specific and host strain-specific responses to Toxoplasma infection, and (v) Toxoplasma-induced immune modulation.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.

PubMed

Wunsch, Christopher M; Lewis, Janina P

2015-12-17

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

PubMed Central

Wunsch, Christopher M.; Lewis, Janina P.

2015-01-01

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type. PMID:26709454

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis.

PubMed

Martín-Hernández, Raquel; Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E; Meana, Aránzazu; Boonham, Neil

2017-01-01

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host's cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite's survival within the cell.

Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes

PubMed Central

Killackey, Samuel A.; Sorbara, Matthew T.; Girardin, Stephen E.

2016-01-01

Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general. PMID:27066460

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

PubMed Central

Durrani, Zeeshan; Pillai, Sreerekha S.; Baird, Margaret; Shiels, Brian R.

2013-01-01

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner. PMID:23840536

Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

PubMed Central

Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

2015-01-01

Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

Cell wall composition profiling of parasitic giant dodder (Cuscuta reflexa) and its hosts: a priori differences and induced changes.

PubMed

Johnsen, Hanne R; Striberny, Bernd; Olsen, Stian; Vidal-Melgosa, Silvia; Fangel, Jonatan U; Willats, William G T; Rose, Jocelyn K C; Krause, Kirsten

2015-08-01

Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

The Influence of Programmed Cell Death in Myeloid Cells on Host Resilience to Infection with Legionella pneumophila or Streptococcus pyogenes

PubMed Central

Gamradt, Pia; Xu, Yun; Gratz, Nina; Duncan, Kellyanne; Kobzik, Lester; Högler, Sandra; Decker, Thomas

2016-01-01

Pathogen clearance and host resilience/tolerance to infection are both important factors in surviving an infection. Cells of the myeloid lineage play important roles in both of these processes. Neutrophils, monocytes, macrophages, and dendritic cells all have important roles in initiation of the immune response and clearance of bacterial pathogens. If these cells are not properly regulated they can result in excessive inflammation and immunopathology leading to decreased host resilience. Programmed cell death (PCD) is one possible mechanism that myeloid cells may use to prevent excessive inflammation. Myeloid cell subsets play roles in tissue repair, immune response resolution, and maintenance of homeostasis, so excessive PCD may also influence host resilience in this way. In addition, myeloid cell death is one mechanism used to control pathogen replication and dissemination. Many of these functions for PCD have been well defined in vitro, but the role in vivo is less well understood. We created a mouse that constitutively expresses the pro-survival B-cell lymphoma (bcl)-2 protein in myeloid cells (CD68(bcl2tg), thus decreasing PCD specifically in myeloid cells. Using this mouse model we explored the impact that decreased cell death of these cells has on infection with two different bacterial pathogens, Legionella pneumophila and Streptococcus pyogenes. Both of these pathogens target multiple cell death pathways in myeloid cells, and the expression of bcl2 resulted in decreased PCD after infection. We examined both pathogen clearance and host resilience and found that myeloid cell death was crucial for host resilience. Surprisingly, the decreased myeloid PCD had minimal impact on pathogen clearance. These data indicate that the most important role of PCD during infection with these bacteria is to minimize inflammation and increase host resilience, not to aid in the clearance or prevent the spread of the pathogen. PMID:27973535

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics.

PubMed

Cardoso, Rita; Soares, Helena; Hemphill, Andrew; Leitão, Alexandre

2016-07-01

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

PubMed Central

Woods, Kerry L.; Theiler, Romina; Mühlemann, Marcus; Segiser, Adrian; Huber, Sandra; Ansari, Hifzur R.; Pain, Arnab; Dobbelaere, Dirk A. E.

2013-01-01

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton. PMID:23675298

Host cell proteins in biotechnology-derived products: A risk assessment framework.

PubMed

de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

2015-11-01

To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

B Cell allogeneic responses after hematopoietic cell transplantation: is it time to address this issue?

PubMed

Perruche, Sylvain; Kleinclauss, François; Tiberghien, Pierre; Saas, Philippe

2005-02-15

To date, B cell responses have retained less attention than T, natural killer or dendritic cell responses in the alloreactive conflict after allogeneic hematopoietic cell transplantation (HCT). Here, we discuss recent clinical and experimental data supporting a role of allogeneic B cell responses in graft-host interactions after HCT. We report results in a murine model of reduced intensity conditioning transplantation (RICT) showing that host B cells can be involved in chronic graft-versus-host disease occurrence. We also describe the control of antidonor alloresponses by intravenous simultaneous infusion of apoptotic cells with allogeneic hematopoietic grafts.

Effects of long non-coding RNA HOST2 on cell migration and invasion by regulating MicroRNA let-7b in breast cancer.

PubMed

Lu, Peng-Wei; Li, Lin; Wang, Fang; Gu, Yuan-Ting

2018-06-01

The study intends to investigate the effects of long non-coding RNA HOST2 (lncRNA HOST2) on cell migration and invasion by regulating microRNA let-7b (let-7b) in breast cancer. Breast cancer and adjacent normal tissues were collected from 98 patients with breast cancer. Breast cancer MCF-7 cells were divided into the blank, negative control (NC), pcDNA3-Mock, siHOST2, let-7b inhibitor, pcDNA3-HOST2, let-7b mimic, pcDNA3-HOST2 + let-7b mimic, and siHOST2 + let-7b inhibitor groups. RT-qPCR was used to detect the mRNA expressions of HOST2, let-7b, and c-Myc. Western blotting was conducted to measure the c-Myc expression. Scratch test and Transwell assay were applied to detect the cell motility, migration, and invasion. Xenograft tumor in nude mice was performed to evaluate the effect of different transfection on the tumor growth. Compared with adjacent normal tissues, HOST2 expression was higher but let-7b expression lower in breast cancer tissues. HOST2 expression in breast cancer cells was remarkably increased compared with that in the normal breast epithelial MCF-10A cells. In MCF-7 cells, in comparison with the blank and NC groups, expressions of HOST2 and c-Myc were reduced, but let-7b expression was remarkably elevated in the siHOST2 and let-7b mimic groups; the let-7b inhibitor group exhibited higher expressions of HOST2 and c-Myc but lower let-7b expression. Overexpression of HOST2 could promote cell motility, migration and invasion, thus enhancing the growth of breast cancer tumor. By inhibiting HOST2, opposite trends were found. LncRNA HOST2 promotes cell migration and invasion by inhibiting let-7b in breast cancer patients. © 2017 Wiley Periodicals, Inc.

Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion

PubMed Central

Fernandes, Maria Cecilia; Cortez, Mauro; Flannery, Andrew R.; Tam, Christina; Mortara, Renato A.

2011-01-01

Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca2+ transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear. We show that host cell entry by T. cruzi mimics a process of plasma membrane injury and repair that involves Ca2+-dependent exocytosis of lysosomes, delivery of acid sphingomyelinase (ASM) to the outer leaflet of the plasma membrane, and a rapid form of endocytosis that internalizes membrane lesions. Host cells incubated with T. cruzi trypomastigotes are transiently wounded, show increased levels of endocytosis, and become more susceptible to infection when injured with pore-forming toxins. Inhibition or depletion of lysosomal ASM, which blocks plasma membrane repair, markedly reduces the susceptibility of host cells to T. cruzi invasion. Notably, extracellular addition of sphingomyelinase stimulates host cell endocytosis, enhances T. cruzi invasion, and restores normal invasion levels in ASM-depleted cells. Ceramide, the product of sphingomyelin hydrolysis, is detected in newly formed parasitophorous vacuoles containing trypomastigotes but not in the few parasite-containing vacuoles formed in ASM-depleted cells. Thus, T. cruzi subverts the ASM-dependent ceramide-enriched endosomes that function in plasma membrane repair to infect host cells. PMID:21536739

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

PubMed

Esher, Shannon K; Ost, Kyla S; Kohlbrenner, Maria A; Pianalto, Kaila M; Telzrow, Calla L; Campuzano, Althea; Nichols, Connie B; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

2018-06-01

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

Effects of host cell sterol composition upon internalization of Yersinia pseudotuberculosis and clustered β1 integrin.

PubMed

Kim, JiHyun; Fukuto, Hana S; Brown, Deborah A; Bliska, James B; London, Erwin

2018-01-26

Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell β1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δ inv , Δ yadA , and Δ inv Δ yadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The Δ inv Δ yadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered β1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The Encapsidated Genome of Microplitis demolitor Bracovirus Integrates into the Host Pseudoplusia includens ▿ ‡

PubMed Central

Beck, Markus H.; Zhang, Shu; Bitra, Kavita; Burke, Gaelen R.; Strand, Michael R.

2011-01-01

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism. PMID:21880747

MHC-mismatched mixed chimerism restores peripheral tolerance of noncross-reactive autoreactive T cells in NOD mice

PubMed Central

Zhang, Mingfeng; Racine, Jeremy J.; Lin, Qing; Liu, Yuqing; Tang, Shanshan; Qin, Qi; Qi, Tong; Riggs, Arthur D.; Zeng, Defu

2018-01-01

Autoimmune type 1 diabetes (T1D) and other autoimmune diseases are associated with particular MHC haplotypes and expansion of autoreactive T cells. Induction of MHC-mismatched but not -matched mixed chimerism by hematopoietic cell transplantation effectively reverses autoimmunity in diabetic nonobese diabetic (NOD) mice, even those with established diabetes. As expected, MHC-mismatched mixed chimerism mediates deletion in the thymus of host-type autoreactive T cells that have T-cell receptor (TCR) recognizing (cross-reacting with) donor-type antigen presenting cells (APCs), which have come to reside in the thymus. However, how MHC-mismatched mixed chimerism tolerizes host autoreactive T cells that recognize only self-MHC–peptide complexes remains unknown. Here, using NOD.Rag1−/−.BDC2.5 or NOD.Rag1−/−.BDC12-4.1 mice that have only noncross-reactive transgenic autoreactive T cells, we show that induction of MHC-mismatched but not -matched mixed chimerism restores immune tolerance of peripheral noncross-reactive autoreactive T cells. MHC-mismatched mixed chimerism results in increased percentages of both donor- and host-type Foxp3+ Treg cells and up-regulated expression of programmed death-ligand 1 (PD-L1) by host-type plasmacytoid dendritic cells (pDCs). Furthermore, adoptive transfer experiments showed that engraftment of donor-type dendritic cells (DCs) and expansion of donor-type Treg cells are required for tolerizing the noncross-reactive autoreactive T cells in the periphery, which are in association with up-regulation of host-type DC expression of PD-L1 and increased percentage of host-type Treg cells. Thus, induction of MHC-mismatched mixed chimerism may establish a peripheral tolerogenic DC and Treg network that actively tolerizes autoreactive T cells, even those with no TCR recognition of the donor APCs. PMID:29463744

Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection.

PubMed

Yu, Qian; Xiong, Youhua; Gao, Hang; Liu, Jianliang; Chen, Zhiqiang; Wang, Qin; Wen, Dongling

2015-08-04

Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood. In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component. The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.

Changes in Reactivity In Vitro of CD4+CD25+ and CD4+CD25− T Cell Subsets in Transplant Tolerance

PubMed Central

Hall, Bruce M.; Robinson, Catherine M.; Plain, Karren M.; Verma, Nirupama D.; Tran, Giang T.; Nomura, Masaru; Carter, Nicole; Boyd, Rochelle; Hodgkinson, Suzanne J.

2017-01-01

Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4+CD25+ T cells, yet in many models, proliferation of CD4+ T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4+, CD4+CD25+, and CD4+CD25− T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG) cardiac allografts and from naïve DA rats were examined. Proliferation of CD4+ T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4+CD25− T cells was greater than naïve CD4+ T cells. In contrast, proliferation of CD4+CD25− T cells from tolerant hosts to specific-donor PVG was not greater than CD4+ T cells, whereas their response to Lewis and self-DA was greater than CD4+ T cells. Paradoxically, CD4+CD25+ T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4+CD25+ T cells proliferate to both PVG and Lewis but not to self-DA. CD4+CD25+ T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN)-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4+CD25+ T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4+CD25+ T cells that mediate transplant tolerance depend on IFN−γ or IL-5 from alloactivated Th1 and Th2 cells. PMID:28878770

Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis.

PubMed

Bevins, Charles L; Salzman, Nita H

2011-05-01

Building and maintaining a homeostatic relationship between a host and its colonizing microbiota entails ongoing complex interactions between the host and the microorganisms. The mucosal immune system, including epithelial cells, plays an essential part in negotiating this equilibrium. Paneth cells (specialized cells in the epithelium of the small intestine) are an important source of antimicrobial peptides in the intestine. These cells have become the focus of investigations that explore the mechanisms of host-microorganism homeostasis in the small intestine and its collapse in the processes of infection and chronic inflammation. In this Review, we provide an overview of the intestinal microbiota and describe the cell biology of Paneth cells, emphasizing the composition of their secretions and the roles of these cells in intestinal host defence and homeostasis. We also highlight the implications of Paneth cell dysfunction in susceptibility to chronic inflammatory bowel disease.

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

PubMed

Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Li, Yao; Pang, Jing; Dong, Stephanie; Strauss-Soukup, Juliane K; Chen, Xian-Ming

2018-05-01

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height, Cell-Substrate Interactions, and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

PubMed Central

Zlotkin-Rivkin, Efrat; Rund, David; Melamed-Book, Naomi; Zahavi, Eitan Erez; Perlson, Eran; Mercone, Silvana; Golosovsky, Michael; Davidov, Dan; Aroeti, Benjamin

2013-01-01

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity. PMID:24194932

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

PubMed Central

Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

2017-01-01

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

Graft-versus-host disease causes failure of donor hematopoiesis and lymphopoiesis in interferon-gamma receptor-deficient hosts.

PubMed

Delisle, Jean-Sébastien; Gaboury, Louis; Bélanger, Marie-Pier; Tassé, Eliane; Yagita, Hideo; Perreault, Claude

2008-09-01

The immunopathologic condition known as graft-versus-host disease (GVHD) results from a type I T-cell process. However, a prototypical type I cytokine, interferon-gamma (IFN-gamma), can protect against several manifestations of GVHD in recipients of major histocompatibility complex (MHC)-mismatched hematopoietic cells. We transplanted hematopoietic cells from C3H.SW donors in wild-type (wt) and IFN-gamma-receptor-deficient (IFN-gammaRKO) MHC-matched C57BL/6 recipients. In IFN-gammaRKO recipients, host cells were unresponsive to IFN-gamma, whereas wt donor cells were exposed to exceptionally high levels of IFN-gamma. From an IFN-gamma perspective, we could therefore evaluate the impact of a loss-of-function on host cells and gain-of-function on donor cells. We found that lack of IFN-gammaR prevented up-regulation of MHC proteins on host cells but did not mitigate damage to most target organs. Two salient phenotypes in IFN-gammaRKO recipients involved donor cells: lymphoid hypoplasia and hematopoietic failure. Lymphopenia was due to FasL-induced apoptosis and decreased cell proliferation. Bone marrow aplasia resulted from a decreased proliferation of hematopoietic stem/progenitor cells that was associated with down-regulation of 2 genes negatively regulated by IFN-gamma: Ccnd1 and Myc. We conclude that IFN-gamma produced by alloreactive T cells may entail a severe graft-versus-graft reaction and could be responsible for cytopenias that are frequently observed in subjects with GVHD.

Interactome of E. piscicida and grouper liver proteins reveals strategies of bacterial infection and host immune response.

PubMed

Li, Hui; Zhu, Qing-Feng; Peng, Xuan-Xian; Peng, Bo

2017-01-03

The occurrence of infectious diseases is related to heterogeneous protein interactions between a host and a microbe. Therefore, elucidating the host-pathogen interplay is essential. We previously revealed the protein interactome between Edwardsiella piscicida and fish gill cells, and the present study identified the protein interactome between E. piscicida and E. drummondhayi liver cells. E. drummondhayi liver cells and bacterial pull-down approaches were used to identify E. piscicida outer membrane proteins that bind to liver cells and fish liver cell proteins that interact with bacterial cells, respectively. Eight bacterial proteins and 11 fish proteins were characterized. Heterogeneous protein-protein interactions between these bacterial cells and fish liver cells were investigated through far-Western blotting and co-immunoprecipitation. A network was constructed based on 42 heterogeneous protein-protein interactions between seven bacterial proteins and 10 fish proteins. A comparison of the new interactome with the previously reported interactome showed that four bacterial proteins overlapped, whereas all of the identified fish proteins were new, suggesting a difference between bacterial tricks for evading host immunity and the host strategy for combating bacterial infection. Furthermore, these bacterial proteins were found to regulate the expression of host innate immune-related proteins. These findings indicate that the interactome contributes to bacterial infection and host immunity.

Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro

PubMed Central

Hegde, Shrilakshmi; Hegde, Shivanand Manjunath; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

2016-01-01

Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae’s induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection. PMID:27662492

Titan Cells Confer Protection from Phagocytosis in Cryptococcus neoformans Infections

PubMed Central

Okagaki, Laura H.

2012-01-01

The human fungal pathogen Cryptococcus neoformans produces an enlarged “titan” cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells. PMID:22544904

Titan cells confer protection from phagocytosis in Cryptococcus neoformans infections.

PubMed

Okagaki, Laura H; Nielsen, Kirsten

2012-06-01

The human fungal pathogen Cryptococcus neoformans produces an enlarged "titan" cell morphology when exposed to the host pulmonary environment. Titan cells exhibit traits that promote survival in the host. Previous studies showed that titan cells are not phagocytosed and that increased titan cell production in the lungs results in reduced phagocytosis of cryptococcal cells by host immune cells. Here, the effect of titan cell production on host-pathogen interactions during early stages of pulmonary cryptococcosis was explored. The relationship between titan cell production and phagocytosis was found to be nonlinear; moderate increases in titan cell production resulted in profound decreases in phagocytosis, with significant differences occurring within the first 24 h of the infection. Not only were titan cells themselves protected from phagocytosis, but titan cell formation also conferred protection from phagocytosis to normal-size cryptococcal cells. Large particles introduced into the lungs were not phagocytosed, suggesting the large size of titan cells protects against phagocytosis. The presence of large particles was unable to protect smaller particles from phagocytosis, revealing that titan cell size alone is not sufficient to provide the observed cross-protection of normal-size cryptococcal cells. These data suggest that titan cells play a critical role in establishment of the pulmonary infection by promoting the survival of the entire population of cryptococcal cells.

Bacterial effectors target the plant cell nucleus to subvert host transcription.

PubMed

Canonne, Joanne; Rivas, Susana

2012-02-01

In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells.

Evaluation of nanoparticles as endocytic tracers in cellular microbiology

NASA Astrophysics Data System (ADS)

Zhang, Yuying; Hensel, Michael

2013-09-01

The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology.The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr01550e

Protein kinase antagonists inhibit invasion of mammalian cells by Fonsecaea pedrosoi.

PubMed

Limongi, Cristiana L; De Souza, Wanderley; Rozental, Sonia

2003-03-01

The phosphorylation process is an important mechanism of cell signalling and regulation. It has been implicated recently in defence strategies against a variety of pathogens that alter host signalling pathways in order to facilitate their invasion and survival within host cells. In this study, the involvement of protein kinases (PKs) has been investigated in attachment and invasion by the pathogenic fungus Fonsecaea pedrosoi within epithelial cells and macrophages. The use of the PK inhibitors staurosporine, genistein and calphostin C prior to infection provided significant information about the role played by PKs in the F. pedrosoi-host cell interaction. All three PK inhibitors could reduce cell invasion by F. pedrosoi significantly. Pre-treatment of macrophages, epithelial cells or conidia with PK inhibitors decreased fungus invasion, and this effect could be overcome by okadaic acid, a phosphatase inhibitor. Immunofluorescence assays showed that tyrosine residues were phosphorylated in the first step of the interaction, while serine residues were phosphorylated in the subsequent step of entry of the parasite into the host cell. These results suggest that both host-cell and conidium PK activities are important in the interaction process, playing a significant role in cell invasion.

microRNA-Based Immunotherapy for Control of Early Stage Lung Cancer

DTIC Science & Technology

2016-09-01

in NSG hosts via subcutaneously injection. A549-Luc developed tumors successfully in NSG hosts and mice bearing A549-Luc tumors were the subject...activation of NK cells from whole blood. Next we evaluated NK cells and host interaction by transferring NK cells into A549-tumor bearing NSG host via...that did not receive NK cells. 4 At day 28, we harvested tumors, blood and tissues from tumor- bearing mice to analyze for NK presence in the

Accommodation of powdery mildew fungi in intact plant cells.

PubMed

Eichmann, Ruth; Hückelhoven, Ralph

2008-01-01

Parasitic powdery mildew fungi have to overcome basic resistance and manipulate host cells to establish a haustorium as a functional feeding organ in a host epidermal cell. Currently, it is of central interest how plant factors negatively regulate basal defense or whether they even support fungal development in compatible interactions. Additionally, creation of a metabolic sink in infected cells may involve host activity. Here, we review the current progress in understanding potential fungal targets for host reprogramming and nutrient acquisition.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise

The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells.

PubMed

Quentin, Michaëel; Abad, Pierre; Favery, Bruno

2013-01-01

Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells (FCs). Effectors synthesized in the esophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized FCs requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defense responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalized within FC nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesized roles in the unique feeding behavior of these pests.

Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

PubMed

Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

2014-03-01

Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression. © 2013 John Wiley & Sons Ltd.

Rapid Functional Decline of Activated and Memory Graft-vs-Host-Reactive T Cells Encountering Host Antigens in the Absence of Inflammation

PubMed Central

Li, Hao Wei; Andreola, Giovanna; Carlson, Alicia; Shao, Steven; Lin, Charles; Zhao, Guiling; Sykes, Megan

2015-01-01

Inflammation in the priming host environment has critical effects on the graft-vs-host (GVH) responses mediated by naïve donor T cells. However, it is unclear how a quiescent or inflammatory environment impacts the activity of GVH-reactive primed T and memory cells. We show here that GVH-reactive primed donor T cells generated in irradiated recipients had diminished ability compared to naïve T cells to increase donor chimerism when transferred to quiescent mixed allogeneic chimeras. GVH-reactive primed T cells showed marked loss of cytotoxic function and activation and delayed but not decreased proliferation or accumulation in lymphoid tissues when transferred to quiescent mixed chimeras compared to freshly irradiated secondary recipients. Primed CD4 and CD8 T cells provided mutual help to sustain these functions in both subsets. CD8 help for CD4 cells was largely IFN-γ-dependent. Toll-like receptor (TLR) stimulation following transfer of GVH-reactive primed T cells to mixed chimeras restored their cytotoxic effector function and permitted the generation of more effective T cell memory in association with reduced PD-1 expression on CD4 memory cells. Our data indicate that an inflammatory host environment is required for the maintenance of GVH-reactive primed T cell functions and the generation of memory T cells that can rapidly acquire effector functions. These findings have important implications for GVHD and T cell-mediated immunotherapies. PMID:26085679

Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement.

PubMed

Khang, Chang Hyun; Berruyer, Romain; Giraldo, Martha C; Kankanala, Prasanna; Park, Sook-Young; Czymmek, Kirk; Kang, Seogchan; Valent, Barbara

2010-04-01

Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.

The role of iron in cancer.

PubMed

Weinberg, E D

1996-02-01

Numerous laboratory and clinical investigations over the past few decades have observed that one of the dangers of iron is its ability to favour neoplastic cell growth. The metal is carcinogenic due to its catalytic effect on the formation of hydroxyl radicals, suppression of the activity of host defence cells and promotion of cancer cell multiplication. In both animals and humans, primary neoplasms develop at body sites of excessive iron deposits. The invaded host attempts to withhold iron from the cancer cells via sequestration of the metal in newly formed ferritin. The host also endeavours to withdraw the metal from cancer cells via macrophage synthesis of nitric oxide. Quantitative evaluation of body iron and of iron-withholding proteins has prognostic value in cancer patients. Procedures associated with lowering host iron intake and inducing host cell iron efflux can assist in prevention and management of neoplastic diseases. Pharmaceutical methods for depriving neoplastic cells of iron are being developed in experimental and clinical protocols.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication

PubMed Central

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K.; McCormick, Frank; Graeber, Thomas G.; Christofk, Heather R.

2014-01-01

SUMMARY Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. While recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. PMID:24703700

Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells

PubMed Central

Hadis, Mohammed; Alderwick, Luke

2017-01-01

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections. PMID:29186191

A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments.

PubMed

Munson-McGee, Jacob H; Peng, Shengyun; Dewerff, Samantha; Stepanauskas, Ramunas; Whitaker, Rachel J; Weitz, Joshua S; Young, Mark J

2018-06-01

The application of viral and cellular metagenomics to natural environments has expanded our understanding of the structure, functioning, and diversity of microbial and viral communities. The high diversity of many communities, e.g., soils, surface ocean waters, and animal-associated microbiomes, make it difficult to establish virus-host associations at the single cell (rather than population) level, assign cellular hosts, or determine the extent of viral host range from metagenomics studies alone. Here, we combine single-cell sequencing with environmental metagenomics to characterize the structure of virus-host associations in a Yellowstone National Park (YNP) hot spring microbial community. Leveraging the relatively low diversity of the YNP environment, we are able to overlay evidence at the single-cell level with contextualized viral and cellular community structure. Combining evidence from hexanucelotide analysis, single cell read mapping, network-based analytics, and CRISPR-based inference, we conservatively estimate that >60% of cells contain at least one virus type and a majority of these cells contain two or more virus types. Of the detected virus types, nearly 50% were found in more than 2 cellular clades, indicative of a broad host range. The new lens provided by the combination of metaviromics and single-cell genomics reveals a network of virus-host interactions in extreme environments, provides evidence that extensive virus-host associations are common, and further expands the unseen impact of viruses on cellular life.

At the Frontier; RXLR Effectors Crossing the Phytophthora-Host Interface.

PubMed

Bouwmeester, Klaas; Meijer, Harold J G; Govers, Francine

2011-01-01

Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens - like the potato late blight pathogen Phytophthora infestans - it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.

Th17 cell cytokine secretion profile in host defense and autoimmunity.

PubMed

Graeber, Kristen E; Olsen, Nancy J

2012-02-01

The goal of this review is to examine the effector functions of Th17 cells in host defense and autoimmunity. Published literature on Th17 cells was reviewed with a focus on the secreted products that mediate effector activities of these cells. Th17 cells secrete an array of cytokines that contribute to host defense and that bridge the innate and adaptive arms of the immune response. When this subset of T cells is dysregulated, autoimmune phenomena develop that contribute to the manifestations of many autoimmune diseases. Th17 cells are positioned at a crossroads between innate and adaptive immunity and provide mediators that are essential for host defense. Current interest in harnessing this system for treatment of autoimmune disease will be challenged by the need to avoid abrogating these many protective functions.

In vivo Host Environment Alters Pseudomonas aeruginosa Susceptibility to Aminoglycoside Antibiotics

PubMed Central

Pan, Xiaolei; Dong, Yuanyuan; Fan, Zheng; Liu, Chang; Xia, Bin; Shi, Jing; Bai, Fang; Jin, Yongxin; Cheng, Zhihui; Jin, Shouguang; Wu, Weihui

2017-01-01

During host infection, Pseudomonas aeruginosa coordinately regulates the expression of numerous genes to adapt to the host environment while counteracting host clearance mechanisms. As infected patients take antibiotics, the invading bacteria encounter antibiotics in the host milieu. P. aeruginosa is highly resistant to antibiotics due to multiple chromosomally encoded resistant determinants. And numerous in vitro studies have demonstrated the regulatory mechanisms of antibiotic resistance related genes in response to antibiotics. However, it is not well-known how host environment affects bacterial response to antibiotics. In this study, we found that P. aeruginosa cells directly isolated from mice lungs displayed higher susceptibility to tobramycin than in vitro cultured bacteria. In vitro experiments demonstrated that incubation with A549 and differentiated HL60 (dHL60) cells sensitized P. aeruginosa to tobramycin. Further studies revealed that reactive oxygen species produced by the host cells contributed to the increased bacterial susceptibility. At the same concentration of tobramycin, presence of A549 and dHL60 cells resulted in higher expression of heat shock proteins, which are known inducible by tobramycin. Further analyses revealed decreased membrane potential upon incubation with the host cells and modification of lipopolysaccharide, which contributed to the increased susceptibility to tobramycin. Therefore, our results demonstrate that contact with host cells increased bacterial susceptibility to tobramycin. PMID:28352614

How pathogens use linear motifs to perturb host cell networks.

PubMed

Via, Allegra; Uyar, Bora; Brun, Christine; Zanzoni, Andreas

2015-01-01

Molecular mimicry is one of the powerful stratagems that pathogens employ to colonise their hosts and take advantage of host cell functions to guarantee their replication and dissemination. In particular, several viruses have evolved the ability to interact with host cell components through protein short linear motifs (SLiMs) that mimic host SLiMs, thus facilitating their internalisation and the manipulation of a wide range of cellular networks. Here we present convincing evidence from the literature that motif mimicry also represents an effective, widespread hijacking strategy in prokaryotic and eukaryotic parasites. Further insights into host motif mimicry would be of great help in the elucidation of the molecular mechanisms behind host cell invasion and the development of anti-infective therapeutic strategies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Leishmania Hijacks Myeloid Cells for Immune Escape

PubMed Central

Martínez-López, María; Soto, Manuel; Iborra, Salvador; Sancho, David

2018-01-01

Protozoan parasites of the Leishmania genus are the causative agents of leishmaniasis, a group of neglected tropical diseases whose clinical manifestations vary depending on the infectious Leishmania species but also on host factors. Recognition of the parasite by host myeloid immune cells is a key to trigger an effective Leishmania-specific immunity. However, the parasite is able to persist in host myeloid cells by evading, delaying and manipulating host immunity in order to escape host resistance and ensure its transmission. Neutrophils are first in infiltrating infection sites and could act either favoring or protecting against infection, depending on factors such as the genetic background of the host or the parasite species. Macrophages are the main host cells where the parasites grow and divide. However, macrophages are also the main effector population involved in parasite clearance. Parasite elimination by macrophages requires the priming and development of an effector Th1 adaptive immunity driven by specific subtypes of dendritic cells. Herein, we will provide a comprehensive outline of how myeloid cells regulate innate and adaptive immunity against Leishmania, and the mechanisms used by the parasites to promote their evasion and sabotage. Understanding the interactions between Leishmania and the host myeloid cells may lead to the development of new therapeutic approaches and improved vaccination to leishmaniases, an important worldwide health problem in which current therapeutic or preventive approaches are limited. PMID:29867798

Counting Legionella cells within single amoeba host cells

EPA Science Inventory

Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

Butanol tolerance in microorganisms

DOEpatents

Bramucci, Michael G.; Nagarajan, Vasantha

2016-03-01

Provided herein are recombinant yeast host cells and methods for their use for production of fermentation products from a pyruvate utilizing pathway. Yeast host cells provided herein comprise reduced pyruvate decarboxylase activity and modified adenylate cyclase activity. In embodiments, yeast host cells provided herein comprise resistance to butanol and increased biomass production.

Methods for production of proteins in host cells

DOEpatents

Donnelly, Mark; Joachimiak, Andrzej

2004-01-13

The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

DOEpatents

Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL

2008-02-05

The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

PubMed Central

Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

2015-01-01

Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

The Use of High Pressure Freezing and Freeze Substitution to Study Host-Pathogen Interactions in Fungal Diseases of Plants

NASA Astrophysics Data System (ADS)

Mims, C. W.; Celio, Gail J.; Richardson, Elizabeth A.

2003-12-01

This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host pathogen interactions in fungal diseases of plants. The specific host pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.

Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor precursors

PubMed Central

Pearson, R. A.; Gonzalez-Cordero, A.; West, E. L.; Ribeiro, J. R.; Aghaizu, N.; Goh, D.; Sampson, R. D.; Georgiadis, A.; Waldron, P. V.; Duran, Y.; Naeem, A.; Kloc, M.; Cristante, E.; Kruczek, K.; Warre-Cornish, K.; Sowden, J. C.; Smith, A. J.; Ali, R. R.

2016-01-01

Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor–host nuclear or cell–cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders. PMID:27701378

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

PubMed

Asmat, Tauseef M; Tenenbaum, Tobias; Jonsson, Ann-Beth; Schwerk, Christian; Schroten, Horst

2014-01-01

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Comparative Transcriptomic Analysis of Race 1 and Race 4 of Fusarium oxysporum f. sp. cubense Induced with Different Carbon Sources

PubMed Central

Qin, Shiwen; Ji, Chunyan; Li, Yunfeng; Wang, Zhenzhong

2017-01-01

The fungal pathogen Fusarium oxysporum f. sp. cubense causes Fusarium wilt, one of the most destructive diseases in banana and plantain cultivars. Pathogenic race 1 attacks the “Gros Michel” banana cultivar, and race 4 is pathogenic to the Cavendish banana cultivar and those cultivars that are susceptible to Foc1. To understand the divergence in gene expression modules between the two races during degradation of the host cell wall, we performed RNA sequencing to compare the genome-wide transcriptional profiles of the two races grown in media containing banana cell wall, pectin, or glucose as the sole carbon source. Overall, the gene expression profiles of Foc1 and Foc4 in response to host cell wall or pectin appeared remarkably different. When grown with host cell wall, a much larger number of genes showed altered levels of expression in Foc4 in comparison with Foc1, including genes encoding carbohydrate-active enzymes (CAZymes) and other virulence-related genes. Additionally, the levels of gene expression were higher in Foc4 than in Foc1 when grown with host cell wall or pectin. Furthermore, a great majority of genes were differentially expressed in a variety-specific manner when induced by host cell wall or pectin. More specific CAZymes and other pathogenesis-related genes were expressed in Foc4 than in Foc1 when grown with host cell wall. The first transcriptome profiles obtained for Foc during degradation of the host cell wall may provide new insights into the mechanism of banana cell wall polysaccharide decomposition and the genetic basis of Foc host specificity. PMID:28468818

Comparative Transcriptomic Analysis of Race 1 and Race 4 of Fusarium oxysporum f. sp. cubense Induced with Different Carbon Sources.

PubMed

Qin, Shiwen; Ji, Chunyan; Li, Yunfeng; Wang, Zhenzhong

2017-07-05

The fungal pathogen Fusarium oxysporum f. sp. cubense causes Fusarium wilt, one of the most destructive diseases in banana and plantain cultivars. Pathogenic race 1 attacks the "Gros Michel" banana cultivar, and race 4 is pathogenic to the Cavendish banana cultivar and those cultivars that are susceptible to Foc1. To understand the divergence in gene expression modules between the two races during degradation of the host cell wall, we performed RNA sequencing to compare the genome-wide transcriptional profiles of the two races grown in media containing banana cell wall, pectin, or glucose as the sole carbon source. Overall, the gene expression profiles of Foc1 and Foc4 in response to host cell wall or pectin appeared remarkably different. When grown with host cell wall, a much larger number of genes showed altered levels of expression in Foc4 in comparison with Foc1, including genes encoding carbohydrate-active enzymes (CAZymes) and other virulence-related genes. Additionally, the levels of gene expression were higher in Foc4 than in Foc1 when grown with host cell wall or pectin. Furthermore, a great majority of genes were differentially expressed in a variety-specific manner when induced by host cell wall or pectin. More specific CAZymes and other pathogenesis-related genes were expressed in Foc4 than in Foc1 when grown with host cell wall. The first transcriptome profiles obtained for Foc during degradation of the host cell wall may provide new insights into the mechanism of banana cell wall polysaccharide decomposition and the genetic basis of Foc host specificity. Copyright © 2017 Qin et al.

Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

PubMed

Vester, Diana; Rapp, Erdmann; Gade, Dörte; Genzel, Yvonne; Reichl, Udo

2009-06-01

Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

A Viral Pilot for HCMV Navigation?

PubMed

Adler, Barbara

2015-07-15

gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host.

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

PubMed Central

Kuçi, Zyrafete; Bönig, Halvard; Kreyenberg, Hermann; Bunos, Milica; Jauch, Anna; Janssen, Johannes W.G.; Škifić, Marijana; Michel, Kristina; Eising, Ben; Lucchini, Giovanna; Bakhtiar, Shahrzad; Greil, Johann; Lang, Peter; Basu, Oliver; von Luettichau, Irene; Schulz, Ansgar; Sykora, Karl-Walter; Jarisch, Andrea; Soerensen, Jan; Salzmann-Manrique, Emilia; Seifried, Erhard; Klingebiel, Thomas; Bader, Peter; Kuçi, Selim

2016-01-01

To circumvent donor-to-donor heterogeneity which may lead to inconsistent results after treatment of acute graft-versus-host disease with mesenchymal stromal cells generated from single donors we developed a novel approach by generating these cells from pooled bone marrow mononuclear cells of 8 healthy “3rd-party” donors. Generated cells were frozen in 209 vials and designated as mesenchymal stromal cell bank. These vials served as a source for generation of clinical grade mesenchymal stromal cell end-products, which exhibited typical mesenchymal stromal cell phenotype, trilineage differentiation potential and at later passages expressed replicative senescence-related markers (p21 and p16). Genetic analysis demonstrated their genomic stability (normal karyotype and a diploid pattern). Importantly, clinical end-products exerted a significantly higher allosuppressive potential than the mean allosuppressive potential of mesenchymal stromal cells generated from the same donors individually. Administration of 81 mesenchymal stromal cell end-products to 26 patients with severe steroid-resistant acute graft-versus-host disease in 7 stem cell transplant centers who were refractory to many lines of treatment, induced a 77% overall response at the primary end point (day 28). Remarkably, although the cohort of patients was highly challenging (96% grade III/IV and only 4% grade II graft-versus-host disease), after treatment with mesenchymal stromal cell end-products the overall survival rate at two years follow up was 71±11% for the entire patient cohort, compared to 51.4±9.0% in graft-versus-host disease clinical studies, in which mesenchymal stromal cells were derived from single donors. Mesenchymal stromal cell end-products may, therefore, provide a novel therapeutic tool for the effective treatment of severe acute graft-versus-host disease. PMID:27175026

Influence of Stress and Antibiotic Resistance on Cell-Length Distribution in Mycobacterium tuberculosis Clinical Isolates

PubMed Central

Vijay, Srinivasan; Vinh, Dao N.; Hai, Hoang T.; Ha, Vu T. N.; Dung, Vu T. M.; Dinh, Tran D.; Nhung, Hoang N.; Tram, Trinh T. B.; Aldridge, Bree B.; Hanh, Nguyen T.; Thu, Do D. A.; Phu, Nguyen H.; Thwaites, Guy E.; Thuong, Nguyen T. T.

2017-01-01

Mycobacterial cellular variations in growth and division increase heterogeneity in cell length, possibly contributing to cell-to-cell variation in host and antibiotic stress tolerance. This may be one of the factors influencing Mycobacterium tuberculosis persistence to antibiotics. Tuberculosis (TB) is a major public health problem in developing countries, antibiotic persistence, and emergence of antibiotic resistance further complicates this problem. We wanted to investigate the factors influencing cell-length distribution in clinical M. tuberculosis strains. In parallel we examined M. tuberculosis cell-length distribution in a large set of clinical strains (n = 158) from ex vivo sputum samples, in vitro macrophage models, and in vitro cultures. Our aim was to understand the influence of clinically relevant factors such as host stresses, M. tuberculosis lineages, antibiotic resistance, antibiotic concentrations, and disease severity on the cell size distribution in clinical M. tuberculosis strains. Increased cell size and cell-to-cell variation in cell length were associated with bacteria in sputum and infected macrophages rather than liquid culture. Multidrug-resistant (MDR) strains displayed increased cell length heterogeneity compared to sensitive strains in infected macrophages and also during growth under rifampicin (RIF) treatment. Importantly, increased cell length was also associated with pulmonary TB disease severity. Supporting these findings, individual host stresses, such as oxidative stress and iron deficiency, increased cell-length heterogeneity of M. tuberculosis strains. In addition we also observed synergism between host stress and RIF treatment in increasing cell length in MDR-TB strains. This study has identified some clinical factors contributing to cell-length heterogeneity in clinical M. tuberculosis strains. The role of these cellular adaptations to host and antibiotic tolerance needs further investigation. PMID:29209302

Tumor cells versus host immune cells: whose PD-L1 contributes to PD-1/PD-L1 blockade mediated cancer immunotherapy?

PubMed

Tang, Fei; Zheng, Pan

2018-01-01

Antibody blockade of the PD-1/PD-L1 pathway has elicited durable antitumor responses in the therapy of a broad spectrum of cancers. PD-L1 is constitutively expressed in certain tumors and host immune cells, and its expression can be induced or maintained by many factors. The expression of PD-L1 on tumor tissues has been reported to be positively correlated with the efficacy of anti-PD-1/PD-L1 therapy in patients. However, multiple clinical trials indicate that patients with PD-L1-negative tumors also respond to this blockade therapy, which suggests the potential contribution of PD-L1 from host immune cells. Recently, six articles independently evaluated and verified the contributions of PD-L1 from tumor versus non-tumor cells in various mouse tumor models. These studies confirmed that PD-L1 on either tumor cells or host immune cells contributes to tumor escape, and the relative contributions of PD-L1 on these cells seem to be context-dependent. While both tumor- and host-derived PD-L1 can play critical roles in immune suppression, differences in tumor immunogenicity appear to underlie their relative importance. Notably, these reports highlight the essential roles of PD-L1 from host myeloid cells in negatively regulating T cell activation and limiting T cell trafficking. Therefore, comprehensive evaluating the global PD-L1 expression, rather than monitoring PD-L1 expression on tumor cells alone, should be a more accurate way for predicting responses in PD-1/PD-L1 blockade therapy in cancer patients.

Central importance of immunoglobulin A in host defense against Giardia spp.

PubMed

Langford, T Dianne; Housley, Michael P; Boes, Marianne; Chen, Jianzhu; Kagnoff, Martin F; Gillin, Frances D; Eckmann, Lars

2002-01-01

The protozoan pathogen Giardia is an important cause of parasitic diarrheal disease worldwide. It colonizes the lumen of the small intestine, suggesting that effective host defenses must act luminally. Immunoglobulin A (IgA) antibodies are presumed to be important for controlling Giardia infection, but direct evidence for this function is lacking. B-cell-independent effector mechanisms also exist and may be equally important for antigiardial host defense. To determine the importance of the immunoglobulin isotypes that are transported into the intestinal lumen, IgA and IgM, for antigiardial host defense, we infected gene-targeted mice lacking IgA-expressing B-cells, IgM-secreting B-cells, or all B-cells as controls with Giardia muris or Giardia lamblia GS/M-83-H7. We found that IgA-deficient mice could not eradicate either G. muris or G. lamblia infection, demonstrating that IgA is required for their clearance. Furthermore, although neither B-cell-deficient nor IgA-deficient mice could clear G. muris infections, IgA-deficient mice controlled infection significantly better than B-cell-deficient mice, suggesting the existence of B-cell-dependent but IgA-independent antigiardial defenses. In contrast, mice deficient for secreted IgM antibodies cleared G. muris infection normally, indicating that they have no unique functions in antigiardial host defense. These data, together with the finding that B-cell-deficient mice have some, albeit limited, residual capacity to control G. muris infection, show that IgA-dependent host defenses are central for eradicating Giardia spp. Moreover, B-cell-dependent but IgA-independent and B-cell-independent antigiardial host defenses exist but are less important for controlling infection.

Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface.

PubMed

Tan, Shumin; Noto, Jennifer M; Romero-Gallo, Judith; Peek, Richard M; Amieva, Manuel R

2011-05-01

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche.

Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

PubMed Central

Tan, Shumin; Noto, Jennifer M.; Romero-Gallo, Judith; Peek, Richard M.; Amieva, Manuel R.

2011-01-01

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche. PMID:21589900

Chlamydia Infection Across Host Species Boundaries Promotes Distinct Sets of Transcribed Anti-Apoptotic Factors

PubMed Central

Messinger, Joshua E.; Nelton, Emmalin; Feeney, Colleen; Gondek, David C.

2015-01-01

Chlamydiae, obligate intracellular bacteria, cause significant human and veterinary associated diseases. Having emerged an estimated 700-million years ago, these bacteria have twice adapted to humans as a host species, causing sexually transmitted infection (C. trachomatis) and respiratory associated disease (C. pneumoniae). The principle mechanism of host cell defense against these intracellular bacteria is the induction of cell death via apoptosis. However, in the “arms race” of co-evolution, Chlamydiae have developed mechanisms to promote cell viability and inhibit cell death. Herein we examine the impact of Chlamydiae infection across multiple host species on transcription of anti-apoptotic genes. We found mostly distinct patterns of gene expression (Mcl1 and cIAPs) elicited by each pathogen-host pair indicating Chlamydiae infection across host species boundaries does not induce a universally shared host response. Understanding species specific host-pathogen interactions is paramount to deciphering how potential pathogens become emerging diseases. PMID:26779446

Influenza A virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein Clusterin

PubMed Central

Tripathi, S; Batra, J; Cao, W; Sharma, K; Patel, J R; Ranjan, P; Kumar, A; Katz, J M; Cox, N J; Lal, R B; Sambhara, S; Lal, S K

2013-01-01

Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process. PMID:23538443

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus

PubMed Central

Duperthuy, Marylise; Schmitt, Paulina; Garzón, Edwin; Caro, Audrey; Rosa, Rafael D.; Le Roux, Frédérique; Lautrédou-Audouy, Nicole; Got, Patrice; Romestand, Bernard; de Lorgeril, Julien; Kieffer-Jaquinod, Sylvie; Bachère, Evelyne; Destoumieux-Garzón, Delphine

2011-01-01

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg-EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD. PMID:21282662

Host cells and methods for production of isobutanol

DOEpatents

Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

2017-10-17

Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

Host cells and methods for production of isobutanol

DOEpatents

Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

2016-08-23

Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.

PubMed Central

Moulder, J W; Levy, N J; Zeichner, S L; Lee, C K

1981-01-01

Almost all the cells in populations of mouse fibroblasts (L cells) persistently infected with the 6BC strain of Chlamydia psittaci were immune to superinfection with high multiplicities of C. psittaci, whether or not the L cells contained visible chlamydial inclusions. As ascertained by experiments with 14C-labeled C. psittaci, immunity to superinfection resulted from the failure of added chlamydiae to attach to persistently infected host cells. However, when exogenous C. psittaci was introduced into persistently infected L cells by centrifuging the inoculum onto host cell monolayers or by pretreating the monolayers with diethylaminoethyl-dextran, these chlamydiae produced expected numbers of infectious progeny. Persistently infected L cells were associated in an unknown way with a C. psittaci population that entered the host cells only with the aid of centrifugation or pretreatment with diethylaminoethyl-dextran. Inclusion-free, persistently infected L cells appeared to present at least two separate hindrances to chlamydial activity: blockage of the attachment of exogenous elementary bodies to persistently infected host cells and prevention of the initiation of chlamydial multiplication by means of a normal developmental cycle in the absence of added C. psittaci. Images PMID:7298188

Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media

PubMed Central

Stone, Melani C.; Borman, Jon; Ferreira, Gisela

2017-01-01

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process‐related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ‐650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141–149, 2018 PMID:28884511

The Frustrated Host Response to Legionella pneumophila Is Bypassed by MyD88-Dependent Translation of Pro-inflammatory Cytokines

PubMed Central

Asrat, Seblewongel; Dugan, Aisling S.; Isberg, Ralph R.

2014-01-01

Many pathogens, particularly those that require their host for survival, have devised mechanisms to subvert the host immune response in order to survive and replicate intracellularly. Legionella pneumophila, the causative agent of Legionnaires' disease, promotes intracellular growth by translocating proteins into its host cytosol through its type IV protein secretion machinery. At least 5 of the bacterial translocated effectors interfere with the function of host cell elongation factors, blocking translation and causing the induction of a unique host cell transcriptional profile. In addition, L. pneumophila also interferes with translation initiation, by preventing cap-dependent translation in host cells. We demonstrate here that protein translation inhibition by L. pneumophila leads to a frustrated host MAP kinase response, where genes involved in the pathway are transcribed but fail to be translated due to the bacterium-induced protein synthesis inhibition. Surprisingly, few pro-inflammatory cytokines, such as IL-1α and IL-1β, bypass this inhibition and get synthesized in the presence of Legionella effectors. We show that the selective synthesis of these genes requires MyD88 signaling and takes place in both infected cells that harbor bacteria and neighboring bystander cells. Our findings offer a perspective of how host cells are able to cope with pathogen-encoded activities that disrupt normal cellular process and initiate a successful inflammatory response. PMID:25058342

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

PubMed

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

2014-04-01

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

Foxp3+ regulatory T cells, immune stimulation and host defence against infection

PubMed Central

Rowe, Jared H; Ertelt, James M; Way, Sing Sing

2012-01-01

The immune system is intricately regulated allowing potent effectors to expand and become rapidly mobilized after infection, while simultaneously silencing potentially detrimental responses that averts immune-mediated damage to host tissues. This relies in large part on the delicate interplay between immune suppressive regulatory CD4+ T (Treg) cells and immune effectors that without active suppression by Treg cells cause systemic and organ-specific autoimmunity. Although these beneficial roles have been classically described as counterbalanced by impaired host defence against infection, newfound protective roles for Treg cells against specific viral pathogens (e.g. herpes simplex virus 2, lymphocytic choriomeningitis virus, West Nile virus) have been uncovered using transgenic mice that allow in vivo Treg-cell ablation based on Foxp3 expression. In turn, Foxp3+ Treg cells also provide protection against some parasitic (Plasmodium sp., Toxoplasma gondii) and fungal (Candida albicans) pathogens. By contrast, for bacterial and mycobacterial infections (e.g. Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis), experimental manipulation of Foxp3+ cells continues to indicate detrimental roles for Treg cells in host defence. This variance is probably related to functional plasticity in Treg cell suppression that shifts discordantly following infection with different types of pathogens. Furthermore, the efficiency whereby Treg cells silence immune activation coupled with the plasticity in Foxp3+ cell activity suggest that overriding Treg-mediated suppression represents a prerequisite ‘signal zero’ that together with other stimulation signals [T-cell receptor (signal 1), co-stimulation (signal 2), inflammatory cytokines (signal 3)] are essential for T-cell activation in vivo. Herein, the importance of Foxp3+ Treg cells in host defence against infection, and the significance of infection-induced shifts in Treg-cell suppression are summarized. PMID:22211994

Susceptibility to Plasmodium liver stage infection is altered by hepatocyte polyploidy.

PubMed

Austin, Laura S; Kaushansky, Alexis; Kappe, Stefan H I

2014-05-01

Plasmodium parasites infect hepatocytes of their mammalian hosts and undergo obligate liver stage development. The specific host cell attributes that are important for liver infection remain largely unknown. Several host signalling pathways are perturbed in infected hepatocytes, some of which are important in the generation of hepatocyte polyploidy. To test the functional consequence of polyploidy on liver infection, we infected hepatocytes with the rodent malaria parasite Plasmodium yoelii both in vitro and in vivo and examined the ploidy of infected and uninfected hepatocytes by flow cytometry. In both hepatoma cell lines and in the mouse liver, the fraction of polyploid cells was higher in the infected cell population than in the uninfected cell population. When the data were reanalysed by comparing the extent of Plasmodium infection within each ploidy subset, we found that infection rates were elevated in more highly polyploid cells and lower in diploid cells. Furthermore, we found that the parasite's preference for host cells with high ploidy is conserved among rodent malaria species and the human malaria parasite Plasmodium falciparum. This parasite preference for host cells of high ploidy cannot be explained by differences in hepatocyte size or DNA replication. We conclude that Plasmodium preferentially infects and develops in polyploid hepatocytes. © 2014 John Wiley & Sons Ltd.

Color-Coded Imaging of Breast Cancer Metastatic Niche Formation in Nude Mice.

PubMed

Suetsugu, Atsushi; Momiyama, Masashi; Hiroshima, Yukihiko; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2015-12-01

We report here a color-coded imaging model in which metastatic niches in the lung and liver of breast cancer can be identified. The transgenic green fluorescent protein (GFP)-expressing nude mouse was used as the host. The GFP nude mouse expresses GFP in all organs. However, GFP expression is dim in the liver parenchymal cells. Mouse mammary tumor cells (MMT 060562) (MMT), expressing red fluorescent protein (RFP), were injected in the tail vein of GFP nude mice to produce experimental lung metastasis and in the spleen of GFP nude mice to establish a liver metastasis model. Niche formation in the lung and liver metastasis was observed using very high resolution imaging systems. In the lung, GFP host-mouse cells accumulated around as few as a single MMT-RFP cell. In addition, GFP host cells were observed to form circle-shaped niches in the lung even without RFP cancer cells, which was possibly a niche in which future metastasis could be formed. In the liver, as with the lung, GFP host cells could form circle-shaped niches. Liver and lung metastases were removed surgically and cultured in vitro. MMT-RFP cells and GFP host cells resembling cancer-associated fibroblasts (CAFs) were observed interacting, suggesting that CAFs could serve as a metastatic niche. © 2015 Wiley Periodicals, Inc.

Susceptibility to Plasmodium liver stage infection is altered by hepatocyte polyploidy

PubMed Central

Austin, Laura S.; Kaushansky, Alexis; Kappe, Stefan H.I.

2014-01-01

Summary Plasmodium parasites infect hepatocytes of their mammalian hosts and within undergo obligate liver stage development. The specific host cell attributes that are important for liver infection remain largely unknown. Several host signaling pathways are perturbed in infected hepatocytes, some of which are important in the generation of hepatocyte polyploidy. To test the functional consequence of polyploidy in liver infection, we infected hepatocytes with the rodent malaria parasite Plasmodium yoelii both in vitro and in vivo and examined the ploidy of infected and uninfected hepatocytes by flow cytometry. In both hepatoma cell lines and in the mouse liver, the fraction of polyploid cells was higher in the infected cell population than in the uninfected cell population. When the data were reanalyzed by comparing the extent of Plasmodium infection within each ploidy subset, we found that infection rates were elevated in more highly polyploid cells and lower in diploid cells. Furthermore, we found that the parasite’s preference for host cells with high ploidy is conserved among rodent malaria species and the human malaria parasite Plasmodium falciparum. This parasite preference for host cells of high ploidy cannot be explained by differences in hepatocyte size or DNA replication. We conclude that Plasmodium preferentially infects and develops in polyploid hepatocytes. PMID:24612025

Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes, and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease.

PubMed

Kawasaki, Yasufumi; Sato, Kazuya; Hayakawa, Hiroko; Takayama, Norihito; Nakano, Hirofumi; Ito, Ryoji; Mashima, Kiyomi; Oh, Iekuni; Minakata, Daisuke; Yamasaki, Ryoko; Morita, Kaoru; Ashizawa, Masahiro; Yamamoto, Chihiro; Hatano, Kaoru; Fujiwara, Shin-Ichiro; Ohmine, Ken; Muroi, Kazuo; Kanda, Yoshinobu

2018-04-17

Xenogeneic graft-versus-host disease (GVHD) models in highly immunodeficient mice are currently being used worldwide to investigate human immune responses against foreign antigens in vivo. However, the individual roles of CD4 + and CD8 + T cells, and donor/host hematopoietic and nonhematopoietic antigen-presenting cells (APCs) in the induction and development of GVHD have not been fully investigated. In the present study, we comprehensively investigated the immune responses of human T cells and the antigen presentation capacity of donor/host hematopoietic and nonhematopoietic APCs in xenogeneic GVHD models using nonobese diabetic/Shi-scid-IL2rg null mice. CD4 + T cells and, to a lesser extent, CD8 + T cells individually mediated potentially lethal GVHD. In addition to inflammatory cytokine production, CD4 + T cells also supported the activation and proliferation of CD8 + T cells. Using bone marrow chimeras, we demonstrated that host hematopoietic, but not nonhematopoietic, APCs play a critical role in the development of CD4 + T cell-mediated GVHD. During early GVHD, we detected 2 distinct populations in memory CD4 + T cells. One population was highly activated and proliferated in major histocompatibility complex antigen (MHC) +/+ mice but not in MHC -/- mice, indicating alloreactive T cells. The other population showed a less activated and slowly proliferative status regardless of host MHC expression, and was associated with higher susceptibility to apoptosis, indicating nonalloreactive T cells in homeostasis-driven proliferation. These observations are clinically relevant to donor T cell response after allogeneic hematopoietic stem cell transplantation. Our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD. Copyright © 2018. Published by Elsevier Inc.

The effect of enterohemorrhagic E. coli infection on the cell mechanics of host cells.

PubMed

Chen, Yin-Quan; Su, Pin-Tzu; Chen, Yu-Hsuan; Wei, Ming-Tzo; Huang, Chien-Hsiu; Osterday, Kathryn; del Álamo, Juan C; Syu, Wan-Jr; Chiou, Arthur

2014-01-01

Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity.

Cell wall glycoproteins at interaction sites between parasitic giant dodder (Cuscuta reflexa) and its host Pelargonium zonale

PubMed Central

Striberny, Bernd; Krause, Kirsten

2015-01-01

The process of host plant penetration by parasitic dodder (genus Cuscuta) is accompanied by molecular and structural changes at the host/parasite interface. Recently, changes in pectin methyl esterification levels in the host cell walls abutting parasitic cells in established infection sites were reported. In addition to that, we show here that the composition of cell wall glycoproteins in Cuscuta-infected Pelargonium zonale undergoes substantial changes. While several arabinogalactan protein epitopes exhibit decreased abundances in the vicinity of the Cuscuta reflexa haustorium, extensins tend to increase in the infected areas. PMID:26367804

Cell wall glycoproteins at interaction sites between parasitic giant dodder (Cuscuta reflexa) and its host Pelargonium zonale.

PubMed

Striberny, Bernd; Krause, Kirsten

2015-01-01

The process of host plant penetration by parasitic dodder (genus Cuscuta) is accompanied by molecular and structural changes at the host/parasite interface. Recently, changes in pectin methyl esterification levels in the host cell walls abutting parasitic cells in established infection sites were reported. In addition to that, we show here that the composition of cell wall glycoproteins in Cuscuta-infected Pelargonium zonale undergoes substantial changes. While several arabinogalactan protein epitopes exhibit decreased abundances in the vicinity of the Cuscuta reflexa haustorium, extensins tend to increase in the infected areas.

Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin.

PubMed

Bastounis, Effie E; Yeh, Yi-Ting; Theriot, Julie A

2018-05-02

Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been previously studied, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens was hitherto unexplored. To address this open question, we manufactured hydrogels of varying physiologically-relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm), and found that adhesion of Lm onto host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.

Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

PubMed Central

Romano, Julia D.; de Beaumont, Catherine; Carrasco, Jose A.; Ehrenman, Karen; Bavoil, Patrik M.

2013-01-01

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients. PMID:23243063

Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

PubMed

Romano, Julia D; de Beaumont, Catherine; Carrasco, Jose A; Ehrenman, Karen; Bavoil, Patrik M; Coppens, Isabelle

2013-02-01

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections

PubMed Central

El-Aouar Filho, Rachid A.; Nicolas, Aurélie; De Paula Castro, Thiago L.; Deplanche, Martine; De Carvalho Azevedo, Vasco A.; Goossens, Pierre L.; Taieb, Frédéric; Lina, Gerard; Le Loir, Yves; Berkova, Nadia

2017-01-01

Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host. PMID:28589102

Glycosylation-dependent galectin-receptor interactions promote Chlamydia trachomatis infection.

PubMed

Lujan, Agustin L; Croci, Diego O; Gambarte Tudela, Julián A; Losinno, Antonella D; Cagnoni, Alejandro J; Mariño, Karina V; Damiani, María T; Rabinovich, Gabriel A

2018-06-11

Chlamydia trachomatis ( Ct ) constitutes the most prevalent sexually transmitted bacterium worldwide. Chlamydial infections can lead to severe clinical sequelae including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. As an obligate intracellular pathogen, Ct has evolved multiple strategies to promote adhesion and invasion of host cells, including those involving both bacterial and host glycans. Here, we show that galectin-1 (Gal1), an endogenous lectin widely expressed in female and male genital tracts, promotes Ct infection. Through glycosylation-dependent mechanisms involving recognition of bacterial glycoproteins and N -glycosylated host cell receptors, Gal1 enhanced Ct attachment to cervical epithelial cells. Exposure to Gal1, mainly in its dimeric form, facilitated bacterial entry and increased the number of infected cells by favoring Ct - Ct and Ct -host cell interactions. These effects were substantiated in vivo in mice lacking Gal1 or complex β1-6-branched N -glycans. Thus, disrupting Gal1- N -glycan interactions may limit the severity of chlamydial infection by inhibiting bacterial invasion of host cells.

Intravenous apoptotic spleen cell infusion induces a TGF-beta-dependent regulatory T-cell expansion

PubMed Central

Kleinclauss, François M.; Perruche, Sylvain; Masson, Emeline; De Carvalho Bittencourt, Marcelo; Biichle, Sabeha; Remy-Martin, Jean-Paul; Ferrand, Christophe; Martin, Mael; Bittard, Hugues; Chalopin, Jean-Marc; Seilles, Estelle; Tiberghien, Pierre; Saas, Philippe

2006-01-01

Apoptotic leukocytes are endowed with immunomodulatory properties that can be used to enhance hematopoietic engraftment and prevent graft-versus-host disease. This apoptotic cell-induced tolerogenic effect is mediated by host macrophages and not recipient dendritic cells or donor phagocytes present in the bone marrow graft as evidenced by selective cell depletion and trafficking experiments. Furthermore, apoptotic cell infusion is associated with TGF-β-dependent donor CD4+CD25+ T cell expansion. Such cells have a regulatory phenotype (CD62Lhigh and intracellular CTLA-4+), express high levels of Foxp3 mRNA and exert ex vivo suppressive activity through a cell-to-cell contact mechanism. In vivo CD25 depletion after apoptotic cell infusion prevents the apoptotic spleen cell-induced beneficial effects on engraftment and graft-versus-host disease occurrence. This highlights the role of regulatory T cells in the tolerogenic effect of apoptotic spleen cell infusion. This novel association between apoptosis and regulatory T cell expansion may also contribute to preventing deleterious auto-immune responses during normal turnover. PMID:15962005

Graft-versus-host disease is independent of innate signaling pathways triggered by pathogens in host hematopoietic cells.

PubMed

Li, Hongmei; Matte-Martone, Catherine; Tan, Hung Sheng; Venkatesan, Srividhya; McNiff, Jennifer; Demetris, Anthony J; Jain, Dhanpat; Lakkis, Fadi; Rothstein, David; Shlomchik, Warren D

2011-01-01

Graft-versus-host disease (GVHD) is initiated by APCs that prime alloreactive donor T cells. In antipathogen responses, Ag-bearing APCs receive signals through pattern-recognition receptors, including TLRs, which induce the expression of costimulatory molecules and production of inflammatory cytokines, which in turn mold the adaptive T cell response. However, in allogeneic hematopoietic stem cell transplantation (alloSCT), there is no specific pathogen, alloantigen is ubiquitous, and signals that induce APC maturation are undefined. To investigate APC activation in GVHD, we used recipient mice with hematopoietic cells genetically deficient in pathways critical for APC maturation in models in which host APCs are absolutely required. Strikingly, CD8-mediated and CD4-mediated GVHD were similar whether host APCs were wild-type or deficient in MyD88, TRIF, or MyD88 and TRIF, which excludes essential roles for TLRs and IL-1β, the key product of inflammasome activation. Th1 differentiation was if anything augmented when APCs were MyD88/TRIF(-/-), and T cell production of IFN-γ did not require host IL-12. GVHD was also intact when APCs lacked the type I IFNR, which amplifies APC activation pathways that induce type I IFNs. Thus in GVHD, alloreactive T cells can be activated when pathways critical for antipathogen T cell responses are impaired.

A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell

PubMed Central

Herbert, Kristina M.; Nag, Anita

2016-01-01

Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage. PMID:27271653

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

Yersinia virulence factors - a sophisticated arsenal for combating host defences

PubMed Central

Atkinson, Steve; Williams, Paul

2016-01-01

The human pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica cause enterocolitis, while Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six ‘effector’ proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen. PMID:27347390

Genetic reprogramming of host cells by bacterial pathogens.

PubMed

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach also...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide, inflammatory

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

both pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...from live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach...Bioorthogonal click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide

Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line.

PubMed

Rasmussen, B; Davis, R; Thomas, J; Reddy, P

1998-11-01

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.

Paucity of CD4+CCR5+ T cells is a typical feature of natural SIV hosts

PubMed Central

Pandrea, Ivona; Apetrei, Cristian; Gordon, Shari; Barbercheck, Joseph; Dufour, Jason; Bohm, Rudolf; Sumpter, Beth; Roques, Pierre; Marx, Preston A.; Hirsch, Vanessa M.; Kaur, Amitinder; Lackner, Andrew A.; Veazey, Ronald S.; Silvestri, Guido

2007-01-01

In contrast to lentiviral infections of humans and macaques, simian immunodeficiency virus (SIV) infection of natural hosts is nonpathogenic despite high levels of viral replication. However, the mechanisms underlying this absence of disease are unknown. Here we report that natural hosts for SIV infection express remarkably low levels of CCR5 on CD4+ T cells isolated from blood, lymph nodes, and mucosal tissues. Given that this immunologic feature is found in 5 different species of natural SIV hosts (sooty mangabeys, African green monkeys, mandrills, sun-tailed monkeys, and chimpanzees) but is absent in 5 nonnatural/recent hosts (humans, rhesus, pigtail, cynomolgus macaques, and baboons), it may represent a key feature of the coevolution between the virus and its natural hosts that led to a nonpathogenic infection. Beneficial effects of low CCR5 expression on CD4+ T cells may include the reduction of target cells for viral replication and a decreased homing of activated CD4+ T cells to inflamed tissue. PMID:17003371

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

PubMed Central

Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

2017-01-01

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

PubMed Central

Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

2016-01-01

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

A Viral Pilot for HCMV Navigation?

PubMed Central

Adler, Barbara

2015-01-01

gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host. PMID:26184287

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle

PubMed Central

Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A.; Moore, Christina A.; Vella, Stephen A.; Hortua Triana, Miryam A.; Liu, Jing; Garcia, Celia R. S.; Pace, Douglas A.; Moreno, Silvia N. J.

2015-01-01

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca2+ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca2+ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca2+ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca2+ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca2+ influx. This is the first study showing, in real time, Ca2+ signals preceding egress and their direct link with motility, an essential virulence trait. PMID:26374900

Graft-versus-host disease

MedlinePlus

GVHD; Bone marrow transplant - graft-versus-host disease; Stem cell transplant - graft-versus-host disease; Allogeneic transplant - ... GVHD may occur after a bone marrow, or stem cell, transplant in which someone receives bone marrow ...

Host-microbiota interactions: Epigenomic regulation

PubMed Central

Woo, Vivienne; Alenghat, Theresa

2016-01-01

The coevolution of mammalian hosts and their commensal microbiota has led to the development of complex symbiotic relationships between resident microbes and mammalian cells. Epigenomic modifications enable host cells to alter gene expression without modifying the genetic code, and therefore represent potent mechanisms by which mammalian cells can transcriptionally respond, transiently or stably, to environmental cues. Advances in genome-wide approaches are accelerating our appreciation of microbial influences on host physiology, and increasing evidence highlights that epigenomics represent a level of regulation by which the host integrates and responds to microbial signals. In particular, bacterial-derived short chain fatty acids have emerged as one clear link between how the microbiota intersects with host epigenomic pathways. Here we review recent findings describing crosstalk between the microbiota and epigenomic pathways in multiple mammalian cell populations. Further, we discuss interesting links that suggest that the scope of our understanding of epigenomic regulation in the host-microbiota relationship is still in its infancy. PMID:28103497

Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15.

PubMed

Liu, Qing; Gao, Wen-Wei; Elsheikha, Hany M; He, Jun-Jun; Li, Fa-Cai; Yang, Wen-Bin; Zhu, Xing-Quan

2018-06-19

Growth and replication of the protozoan parasite Toxoplasma gondii within host cell entail the production of several effector proteins, which the parasite exploits for counteracting the host's immune response. Despite considerable research to define the host signaling pathways manipulated by T. gondii and their effectors, there has been limited progress into understanding how individual members of the dense granule proteins (GRAs) modulate gene expression within host cells. The aim of this study was to evaluate whether T. gondii GRA15 protein plays any role in regulating host gene expression. Baby hamster kidney cells (BHK-21) were transfected with plasmids encoding GRA15 genes of either type I GT1 strain (GRA15 I ) or type II PRU strain (GRA15 II ). Gene expression patterns of transfected and nontransfected BHK-21 cells were investigated using RNA-sequencing analysis. GRA15 I and GRA15 II induced both known and novel transcriptional changes in the transfected BHK-21 cells compared with nontransfected cells. Pathway analysis revealed that GRA15 II was mainly involved in the regulation of tumor necrosis factor (TNF), NF-κB, HTLV-I infection, and NOD-like receptor signaling pathways. GRA15 I preferentially influenced the synthesis of unsaturated fatty acids in host cells. Our findings support the hypothesis that certain functions of GRA15 protein are strain dependent and that GRA15 modulates the expression of signaling pathways and genes with important roles in T. gondii pathophysiology. A greater understanding of host signaling pathways influenced by T. gondii effectors would allow the development of more efficient anti-T. gondii therapeutic schemes, capitalizing on disrupting parasite virulence factors to advance the treatment of toxoplasmosis.

Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

PubMed Central

Shiokawa, Mai; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Okamoto, Toru; Watanabe, Noriyuki; Wakita, Takaji

2014-01-01

ABSTRACT Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV. IMPORTANCE Previous studies have shown that liver-specific host factors are required for efficient replication of HCV RNA and formation of infectious particles. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified novel permissive cell lines for complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV life cycle, as well as the development of therapeutic agents for chronic hepatitis C. PMID:24599999

A Reinterpretation of Cell Transplantation: GFP Transfer From Donor to Host Photoreceptors.

PubMed

Ortin-Martinez, Arturo; Tsai, En Leh Samuel; Nickerson, Philip E; Bergeret, Miriam; Lu, Yao; Smiley, Sheila; Comanita, Lacrimioara; Wallace, Valerie A

2017-04-01

The utilization of fluorescent reporter transgenes to discriminate donor versus host cells has been a mainstay of photoreceptor transplantation research, the assumption being that the presence of reporter+ cells in outer nuclear layer (ONL) of transplant recipients represents the integration of donor photoreceptors. We previously reported that GFP + cells in the ONL of cone-GFP transplanted retinas exhibited rod-like characteristics, raising the possibility that GFP signal in recipient tissue may not be a consequence of donor cell integration. To investigate the basis for this mismatch, we performed a series of transplantations using multiple transgenic donor and recipient models, and assessed cell identity using nuclear architecture, immunocytochemistry, and DNA prelabeling. Our results indicate that GFP + cells in the ONL fail to exhibit hallmark elements of donor cells, including nuclear hetero/euchromatin architecture. Furthermore, GFP signal does not appear to be a consequence of classic donor/host cell fusion or transfating post-transplant, but is most likely due to material exchange between donor and host photoreceptors. This transfer can be mediated by rods and cones, is bidirectional between donor and host cells, requires viable photoreceptors, occurs preferentially at sites of outer limiting membrane disruption and can be detected in second-order retinal neurons and Müller glia. Collectively, these data warrant re-evaluation of the use of lineage tracing fluorescent reporters in transplantation studies involving the retina and other CNS tissues. Furthermore, the reinterpretation of previous functional rescue data, based on material exchange, rather than cell integration, may offer a novel approach to vision rescue. Stem Cells 2017;35:932-939. © 2016 AlphaMed Press.

Herpes B Virus, Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

PubMed Central

Vasireddi, Mugdha

2012-01-01

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8+ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions. PMID:22973043

Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells

PubMed Central

Garner, Omai B.; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C.; Park, Arnold; Bowden, Thomas A.; Freiberg, Alexander N.

2014-01-01

ABSTRACT Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. IMPORTANCE Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. PMID:25505064

The Impact of Prophage on the Equilibria and Stability of Phage and Host

NASA Astrophysics Data System (ADS)

Yu, Pei; Nadeem, Alina; Wahl, Lindi M.

2017-06-01

In this paper, we present a bacteriophage model that includes prophage, that is, phage genomes that are incorporated into the host cell genome. The general model is described by an 18-dimensional system of ordinary differential equations. This study focuses on asymptotic behaviour of the model, and thus the system is reduced to a simple six-dimensional model, involving uninfected host cells, infected host cells and phage. We use dynamical system theory to explore the dynamic behaviour of the model, studying in particular the impact of prophage on the equilibria and stability of phage and host. We employ bifurcation and stability theory, centre manifold and normal form theory to show that the system has multiple equilibrium solutions which undergo a series of bifurcations, finally leading to oscillating motions. Numerical simulations are presented to illustrate and confirm the analytical predictions. The results of this study indicate that in some parameter regimes, the host cell population may drive the phage to extinction through diversification, that is, if multiple types of host emerge; this prediction holds even if the phage population is likewise diverse. This parameter regime is restricted, however, if infecting phage are able to recombine with prophage sequences in the host cell genome.

Legionella phospholipases implicated in virulence.

PubMed

Kuhle, Katja; Flieger, Antje

2013-01-01

Phospholipases are diverse enzymes produced in eukaryotic hosts and their bacterial pathogens. Several pathogen phospholipases have been identified as major virulence factors acting mainly in two different modes: on the one hand, they have the capability to destroy host membranes and on the other hand they are able to manipulate host signaling pathways. Reaction products of bacterial phospholipases may act as secondary messengers within the host and therefore influence inflammatory cascades and cellular processes, such as proliferation, migration, cytoskeletal changes as well as membrane traffic. The lung pathogen and intracellularly replicating bacterium Legionella pneumophila expresses a variety of phospholipases potentially involved in disease-promoting processes. So far, genes encoding 15 phospholipases A, three phospholipases C, and one phospholipase D have been identified. These cell-associated or secreted phospholipases may contribute to intracellular establishment, to egress of the pathogen from the host cell, and to the observed lung pathology. Due to the importance of phospholipase activities for host cell processes, it is conceivable that the pathogen enzymes may mimic or substitute host cell phospholipases to drive processes for the pathogen's benefit. The following chapter summarizes the current knowledge on the L. pneumophila phospholipases, especially their substrate specificity, localization, mode of secretion, and impact on host cells.

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

PubMed Central

2012-01-01

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection. PMID:22857383

A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

PubMed Central

Sateriale, Adam; Huston, Christopher D.

2011-01-01

The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model. PMID:21331284

HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

Cancer.gov

When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with

Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

1985-11-01

The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitismmore » of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.« less

Origin of Langerhans cells in normal skin and chronic GVHD after hematopoietic stem-cell transplantation.

PubMed

Andani, Rafiq; Robertson, Ivan; Macdonald, Kelli P A; Durrant, Simon; Hill, Geoffrey R; Khosrotehrani, Kiarash

2014-01-01

Chronic graft-versus-host disease (cGVHD) is a common complication following allogeneic stem-cell transplantation (SCT). Past studies have implicated the persistence of host antigen-presenting cells (APCs) in GVHD. Our objective was to determine the frequency of host Langerhans cells (LCs) in normal skin post-SCT and ask if their persistence could predict cGVHD. Biopsies of normal skin from 124 sex-mismatched T-cell-replete allogenic SCT recipients were taken 100 days post-transplant. Patients with acute GVHD and those with <9 months of follow-up were excluded and prospective follow-up information was collected from remaining 22 patients. CD1a staining and X and Y chromosome in-situ hybridization were performed to label LCs and to identify their host or donor origin. At 3 months, 59 ± 5% of LCs were host derived. The density of LCs and the proportion of host-derived LCs were similar between patients that did or did not develop cGVHD. Most LCs in the skin remained of host origin 3 months after SCT regardless of cGVHD status. This finding is in line with the redundant role of LCs in acute GVHD initiation uncovered in recent experimental models. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pathobiology of Pneumocystis pneumonia: life cycle, cell wall and cell signal transduction.

PubMed

Skalski, Joseph H; Kottom, Theodore J; Limper, Andrew H

2015-09-01

Pneumocystis is a genus of ascomycetous fungi that are highly morbid pathogens in immunosuppressed humans and other mammals. Pneumocystis cannot easily be propagated in culture, which has greatly hindered understanding of its pathobiology. The Pneumocystis life cycle is intimately associated with its mammalian host lung environment, and life cycle progression is dependent on complex interactions with host alveolar epithelial cells and the extracellular matrix. The Pneumocystis cell wall is a varied and dynamic structure containing a dominant major surface glycoprotein, β-glucans and chitins that are important for evasion of host defenses and stimulation of the host immune system. Understanding of Pneumocystis cell signaling pathways is incomplete, but much has been deduced by comparison of the Pneumocystis genome with homologous genes and proteins in related fungi. In this mini-review, the pathobiology of Pneumocystis is reviewed, with particular focus on the life cycle, cell wall components and cell signal transduction. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

PubMed

Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

2010-07-23

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues. Copyright 2010 Elsevier Inc. All rights reserved.

Utilization of human DC-SIGN and L-SIGN for entry and infection of host cells by the New World arenavirus, Junín virus

PubMed Central

Belouzard, Sandrine; Cordo, Sandra M.; Candurra, Nélida A.; Whittaker, Gary R.

2014-01-01

The target cell tropism of enveloped viruses is regulated by interactions between viral proteins and cellular receptors determining susceptibility at a host cell, tissue or species level. However, a number of additional cell-surface moieties can also bind viral envelope glycoproteins and could act as capture receptors, serving as attachment factors to concentrate virus particles on the cell surface, or to disseminate the virus infection to target organs or susceptible cells within the host. Here, we used Junín virus (JUNV) or JUNV glycoprotein complex (GPC)-pseudotyped particles to study their ability to be internalized by the human C-type lectins hDC- or hL-SIGN. Our results provide evidence that hDC- and hL-SIGN can mediate the entry of Junín virus into cells, and may play an important role in virus infection and dissemination in the host. PMID:24183720

Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

PubMed

Tissot, Tazzio; Arnal, Audrey; Jacqueline, Camille; Poulin, Robert; Lefèvre, Thierry; Mery, Frédéric; Renaud, François; Roche, Benjamin; Massol, François; Salzet, Michel; Ewald, Paul; Tasiemski, Aurélie; Ujvari, Beata; Thomas, Frédéric

2016-03-01

Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research. © 2016 WILEY Periodicals, Inc.

Reconstituted normal human breast in nude mice: effect of host pregnancy environment and human chorionic gonadotropin on proliferation.

PubMed

Popnikolov, N; Yang, J; Liu, A; Guzman, R; Nandi, S

2001-03-01

The proliferation of normal human breast epithelial cells in women is highest during the first trimester of pregnancy. In an attempt to analyze this hormonal environment in a model system, the effect of host mouse pregnancy and the administration of human chorionic gonadotropin (hCG) were assessed in normal human breast epithelial cells transplanted into athymic nude mice. Human breast epithelial cells, dissociated from reduction mammoplasty specimens and embedded inside the extracellular matrices comprised of collagen gel and Matrigel, were transplanted into nude mice. Proliferation was measured in vivo by BrdU labeling followed by immunostaining of sections from recovered gels in response to an altered hormonal environment of the host animal. The host animal was mated to undergo pregnancy and the complex hormonal environment of the host animal pregnancy stimulated growth of transplanted human cells. This effect increased with progression of pregnancy and reached the maximum during late pregnancy prior to parturition. In order to determine whether additional stimulation could be achieved, the transplanted human cells were exposed to a second cycle of host mouse pregnancy by immediately mating the animal after parturition. This additional exposure of host mouse pregnancy did not result in further increase of proliferation. The effect of hCG administration on transplanted human cells was also tested, since hCG level is highest during the first trimester of human pregnancy and coincides with the maximal breast cell proliferation. Administration of hCG alone stimulated proliferation of human cells in a dose-dependent manner, and could further enhance stimulation achieved with estrogen. The host mouse mammary gland also responded to hCG treatment resulting in increased branching and lobulo-alveolar development. However, the hCG effect on both human and mouse cells was dependent on intact ovary since the stimulation did not occur in ovariectomized animals. Although hCG receptor transcripts were detected in human breast epithelial cells, raising the possibility of a direct mitogenic action, the hCG effect observed in this study may have been mediated via the ovary by increased secretion of ovarian steroids. In summary, using our in vivo nude mice system, the proliferation of normal human breast epithelial cells could be stimulated by host mouse pregnancy and by administration of hCG.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

PubMed

Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

2017-07-01

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence.

PubMed

Perruche, Sylvain; Marandin, Aliette; Kleinclauss, François; Angonin, Régis; Fresnay, Stéphanie; Baron, Marie Hélène; Tiberghien, Pierre; Saas, Philippe

2006-02-27

Use of a reduced-intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a coexistence of both host and donor hematopoietic cells may influence posttransplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced-intensity conditioning transplantation (RICT), autoantibody production, and chronic GVHD (cGVHD)-related pathology. Chimerism state, circulating anticardiolipin, and antidouble stranded DNA autoantibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. We observed a novel association between mixed chimerism state, high levels of pathogenic IgG autoantibodies, and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of autoantibodies. Recipient B cell persistence may contribute to the frequency and/or severity of cGVHD after RICT.

The Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion

PubMed Central

Jackson, Allison J.; Whitelaw, Jamie A.; Pall, Gurman; Black, Jennifer Ann; Ferguson, David J. P.; Tardieux, Isabelle; Mogilner, Alex; Meissner, Markus

2014-01-01

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasite's own actomyosin system, and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility. PMID:24632839

Simultaneous Evaluation of Life Cycle Dynamics between a Host Paramecium and the Endosymbionts of Paramecium bursaria Using Capillary Flow Cytometry.

PubMed

Takahashi, Toshiyuki

2016-08-17

Endosymbioses are driving forces underlying cell evolution. The endosymbiosis exhibited by Paramecium bursaria is an excellent model with which to study symbiosis. A single-cell microscopic analysis of P. bursaria reveals that endosymbiont numbers double when the host is in the division phase. Consequently, endosymbionts must arrange their cell cycle schedule if the culture-condition-dependent change delays the generation time of P. bursaria. However, it remains poorly understood whether endosymbionts keep pace with the culture-condition-dependent behaviors of P. bursaria, or not. Using microscopy and flow cytometry, this study investigated the life cycle behaviors occurring between endosymbionts and the host. To establish a connection between the host cell cycle and endosymbionts comprehensively, multivariate analysis was applied. The multivariate analysis revealed important information related to regulation between the host and endosymbionts. Results show that dividing endosymbionts underwent transition smoothly from the division phase to interphase, when the host was in the logarithmic phase. In contrast, endosymbiont division stagnated when the host was in the stationary phase. This paper explains that endosymbionts fine-tune their cell cycle pace with their host and that a synchronous life cycle between the endosymbionts and the host is guaranteed in the symbiosis of P. bursaria.

Simultaneous Evaluation of Life Cycle Dynamics between a Host Paramecium and the Endosymbionts of Paramecium bursaria Using Capillary Flow Cytometry

PubMed Central

Takahashi, Toshiyuki

2016-01-01

Endosymbioses are driving forces underlying cell evolution. The endosymbiosis exhibited by Paramecium bursaria is an excellent model with which to study symbiosis. A single-cell microscopic analysis of P. bursaria reveals that endosymbiont numbers double when the host is in the division phase. Consequently, endosymbionts must arrange their cell cycle schedule if the culture-condition-dependent change delays the generation time of P. bursaria. However, it remains poorly understood whether endosymbionts keep pace with the culture-condition-dependent behaviors of P. bursaria, or not. Using microscopy and flow cytometry, this study investigated the life cycle behaviors occurring between endosymbionts and the host. To establish a connection between the host cell cycle and endosymbionts comprehensively, multivariate analysis was applied. The multivariate analysis revealed important information related to regulation between the host and endosymbionts. Results show that dividing endosymbionts underwent transition smoothly from the division phase to interphase, when the host was in the logarithmic phase. In contrast, endosymbiont division stagnated when the host was in the stationary phase. This paper explains that endosymbionts fine-tune their cell cycle pace with their host and that a synchronous life cycle between the endosymbionts and the host is guaranteed in the symbiosis of P. bursaria. PMID:27531180

Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

PubMed

Licona-Limón, Paula; Henao-Mejia, Jorge; Temann, Angela U; Gagliani, Nicola; Licona-Limón, Ileana; Ishigame, Harumichi; Hao, Liming; Herbert, De'broski R; Flavell, Richard A

2013-10-17

Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection. Copyright © 2013 Elsevier Inc. All rights reserved.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

PubMed

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis -specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

PubMed Central

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria. PMID:28638803

Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

PubMed Central

Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

2016-01-01

Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

PubMed

Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

2016-04-01

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

PubMed Central

Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric

2017-01-01

ABSTRACT   Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. PMID:28119472

Transcriptional profiling of Epstein–Barr virus (EBV) genes and host cellular genes in nasal NK/T-cell lymphoma and chronic active EBV infection

PubMed Central

Zhang, Y; Ohyashiki, J H; Takaku, T; Shimizu, N; Ohyashiki, K

2006-01-01

Nasal NK/T-cell lymphoma is an aggressive subtype of non-Hodgkin lymphoma (NHL) that is closely associated with Epstein–Barr virus (EBV). The clonal expansion of EBV-infected NK or T cells is also seen in patients with chronic active EBV (CAEBV) infection, suggesting that two diseases might share a partially similar mechanism by which EBV affects host cellular gene expression. To understand the pathogenesis of EBV-associated NK/T-cell lymphoproliferative disorders (LPD) and design new therapies, we employed a novel EBV DNA microarray to compare patterns of EBV expression in six cell lines established from EBV-associated NK/T-cell LPD. We found that expression of BZLF1, which encodes the immediate-early gene product Zta, was expressed in SNK/T cells and the expression levels were preferentially high in cell lines from CAEBV infection. We also analyzsd the gene expression patterns of host cellular genes using a human oligonucleotide DNA microarray. We identified a subset of pathogenically and clinically relevant host cellular genes, including TNFRSF10D, CDK2, HSPCA, IL12A as a common molecular biological properties of EBV-associated NK/T-cell LPD and a subset of genes, such as PDCD4 as a putative contributor for disease progression. This study describes a novel approach from the aspects of viral and host gene expression, which could identify novel therapeutic targets in EBV-associated NK/T-cell LPD. PMID:16449999

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope*

PubMed Central

Kim, Jiyeun Kate; Son, Dae Woo; Kim, Chan-Hee; Cho, Jae Hyun; Marchetti, Roberta; Silipo, Alba; Sturiale, Luisa; Park, Ha Young; Huh, Ye Rang; Nakayama, Hiroshi; Fukatsu, Takema; Molinaro, Antonio; Lee, Bok Luel

2015-01-01

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis. PMID:26116716

SIV Coreceptor Specificity in Natural and Non-Natural Host Infection: Implications for Cell Targeting and Differential Outcomes from Infection.

PubMed

Wetzel, Katherine S; Elliott, Sarah T C; Collman, Ronald G

2018-01-01

Pathogenic HIV-1 infection of humans and SIVmac infection of macaques are the result of zoonotic transfer of primate immunodeficiency viruses from their natural hosts into non-natural host species. Natural host infections do not result in pathogenesis despite high levels of virus replication, and evidence suggests that differences in anatomical location and specific subsets of CD4+ T cells infected may underlie distinct outcomes from infection. The coreceptor CCR5 has long been considered the sole pathway for SIV entry and the key determinant of CD4+ cell targeting, but it has also been known that natural hosts express exceedingly low levels of CCR5 despite maintaining high levels of virus replication. This review details emerging data indicating that in multiple natural host species, CCR5 is dispensable for SIV infection ex vivo and/or in vivo and, contrary to the established dogma, alternative coreceptors, particularly CXCR6, play a central role in infection and cell targeting. Infections of non-natural hosts, however, are characterized by CCR5-exclusive entry. These findings suggest that alternative coreceptor-mediated cell targeting in natural hosts, combined with low CCR5 expression, may direct the virus to distinct populations of cells that are dispensable for immune homeostasis, particularly extralymphoid and more differentiated CD4+ T cells. In contrast, CCR5-mediated entry in non-natural hosts results in targeting of CD4+ T cells that are located in lymphoid tissues, critical for immune homeostasis, or necessary for gut barrier integrity. Thus, fundamental differences in viral entry coreceptor use may be central determinants of infection outcome. These findings redefine the normal SIV/host relationship in natural host species, shed new light on key features linked to zoonotic immunodeficiency virus transfer, and highlight important questions regarding how and why this coreceptor bottleneck occurs and the coevolutionary equilibrium is lost following cross-species transfer that results in AIDS. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Manipulation of intestinal epithelial cell function by the cell contact-dependent type III secretion systems of Vibrio parahaemolyticus

PubMed Central

O'Boyle, Nicky; Boyd, Aoife

2013-01-01

Vibrio parahaemolyticus elicits gastroenteritis by deploying Type III Secretion Systems (TTSS) to deliver effector proteins into epithelial cells of the human intestinal tract. The bacteria must adhere to the human cells to allow colonization and operation of the TTSS translocation apparatus bridging the bacterium and the host cell. This article first reviews recent advances in identifying the molecules responsible for intercellular adherence. V. parahaemolyticus possesses two TTSS, each of which delivers an exclusive set of effectors and mediates unique effects on the host cell. TTSS effectors primarily target and alter the activation status of host cell signaling proteins, thereby bringing about changes in the regulation of cellular behavior. TTSS1 is responsible for the cytotoxicity of V. parahaemolyticus, while TTSS2 is necessary for the enterotoxicity of the pathogen. Recent publications have elucidated the function of several TTSS effectors and their importance in the virulence of the bacterium. This review will explore the ability of the TTSS to manipulate activities of human intestinal cells and how this modification of cell function favors bacterial colonization and persistence of V. parahaemolyticus in the host. PMID:24455490

Significant differences in genotoxicity induced by retrovirus integration in human T cells and induced pluripotent stem cells.

PubMed

Zheng, Weiyan; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

2013-04-25

Retrovirus is frequently used in the genetic modification of mammalian cells and the establishment of induced pluripotent stem cells (iPSCs) via cell reprogramming. Vector-induced genotoxicity could induce profound effect on the physiology and function of these stem cells and their differentiated progeny. We analyzed retrovirus-induced genotoxicity in somatic cell Jurkat and two iPSC lines. In Jurkat cells, retrovirus frequently activated host gene expression and gene activation was not dependent on the distance between the integration site and the transcription start site of the host gene. In contrast, retrovirus frequently down-regulated host gene expression in iPSCs, possibly due to the action of chromatin silencing that spreads from the provirus to the nearby host gene promoter. Our data raises the issue that some of the phenotypic variability observed among iPSC clones derived from the same parental cell line may be caused by retrovirus-induced gene expression changes rather than by the reprogramming process itself. It also underscores the importance of characterizing retrovirus integration and carrying out risk assessment of iPSCs before they can be applied in basic research and clinics. Copyright © 2013 Elsevier B.V. All rights reserved.

A Color-coded Imageable Syngeneic Mouse Model of Stromal-cell Recruitment by Metastatic Lymphoma.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Shibata, Yuhei; Nakamura, Nobuhiko; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2015-09-01

A syngeneic color-coded imageable lymphoma model has been developed to visualize recruitment of host stromal cells by malignant lymphoma during metastasis. The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were initially established. EL4-RFP cells were subsequently injected into the tail vein of C57/BL6-GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the upper mediastinum and in the liver 28 days after cell injection. Large EL4-RFP liver metastases in C57/BL6-GFP mice contained GFP-expressing stromal cells derived from the host. In addition, EL4-RFP lymphoma metastasis was formed in peri-gastric lymph nodes, which were also enriched in host GFP-expressing cells. Furthermore, EL4-RFP lymphoma cells were also observed in the peripheral blood and bone marrow of C57/BL6-GFP transgenic mice, where they were associated with GFP-expressing host cells. Lymph node, liver and bone marrow metastases were found approximately 4 weeks after transplantation and all RFP-expressing metastases were highly enriched in GFP-expressing host stromal cells. This model of malignant lymphoma can be used to study early tumor development, metastasis, and the role of the stroma, as well as for discovery and evaluation of novel therapeutics for this treatment-resistant disease. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Notable Aspects of Glycan-Protein Interactions

PubMed Central

Cohen, Miriam

2015-01-01

This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

Optimizing eukaryotic cell hosts for protein production through systems biotechnology and genome-scale modeling.

PubMed

Gutierrez, Jahir M; Lewis, Nathan E

2015-07-01

Eukaryotic cell lines, including Chinese hamster ovary cells, yeast, and insect cells, are invaluable hosts for the production of many recombinant proteins. With the advent of genomic resources, one can now leverage genome-scale computational modeling of cellular pathways to rationally engineer eukaryotic host cells. Genome-scale models of metabolism include all known biochemical reactions occurring in a specific cell. By describing these mathematically and using tools such as flux balance analysis, the models can simulate cell physiology and provide targets for cell engineering that could lead to enhanced cell viability, titer, and productivity. Here we review examples in which metabolic models in eukaryotic cell cultures have been used to rationally select targets for genetic modification, improve cellular metabolic capabilities, design media supplementation, and interpret high-throughput omics data. As more comprehensive models of metabolism and other cellular processes are developed for eukaryotic cell culture, these will enable further exciting developments in cell line engineering, thus accelerating recombinant protein production and biotechnology in the years to come. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

PubMed Central

Mostowy, Serge; Shenoy, Avinash R.

2016-01-01

Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. PMID:26292640

Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.

PubMed Central

Su, H; Watkins, N G; Zhang, Y X; Caldwell, H D

1990-01-01

The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions. Images PMID:2318528

Staphylococcus aureus-Induced G2/M Phase Transition Delay in Host Epithelial Cells Increases Bacterial Infective Efficiency

PubMed Central

Almeida, Sintia; Legembre, Patrick; Edmond, Valérie; Azevedo, Vasco; Miyoshi, Anderson; Even, Sergine; Taieb, Frédéric; Arlot-Bonnemains, Yannick; Le Loir, Yves; Berkova, Nadia

2013-01-01

Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration. PMID:23717407

In Vitro Alterations Do Not Reflect a Requirement for Host Cell Cycle Progression during Plasmodium Liver Stage Infection

PubMed Central

Hanson, Kirsten K.; March, Sandra; Ng, Shengyong; Bhatia, Sangeeta N.

2014-01-01

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection. PMID:25416236

Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures

DOEpatents

Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

2014-09-30

A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures

DOEpatents

Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

2016-04-19

A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

Structure and biophysics of type III secretion in bacteria.

PubMed

Chatterjee, Srirupa; Chaudhury, Sukanya; McShan, Andrew C; Kaur, Kawaljit; De Guzman, Roberto N

2013-04-16

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

The parasitophorous vacuole of Encephalitozoon cuniculi: biogenesis and characteristics of the host cell-pathogen interface.

PubMed

Bohne, Wolfgang; Böttcher, Karin; Gross, Uwe

2011-06-01

Microsporidia are obligate intracellular fungal pathogens of increasing importance in immunocompromised patients. They have developed a unique invasion mechanism, which is based on the explosive discharge of a hollow tubulus, the so-called polar tube. The infectious sporoplasm is subsequently extruded through this flexible tube and injected into the host cell. The model microsporidium Encephalitozoon cuniculi is a paradigm of a fungus with an extreme host cell dependency. This human pathogen possesses one of the smallest eukaryotic genomes (<3MB) identified so far and has reduced its own biosynthetic pathways to a minimum, thus depending on an efficient supply of metabolites from the host cell. E. cuniculi spends its entire intracellular life cycle inside a parasitophorous vacuole (PV), which is formed during invasion. We have provided here an overview of the biogenesis and characteristics of this important host cell-pathogen interface and suggest in this context a modified model for E. cuniculi invasion. According to the model, the host cell plasma membrane is not pierced by the polar tube, but is pushed at the contact site into the cell interior by the mechanical force of the expelled polar tube. This results in a channel-like invagination of the plasma membrane, from which finally the parasitophorous vacuole is pinched-off. Copyright © 2011 Elsevier GmbH. All rights reserved.

Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K–powered ER/actin network

PubMed Central

Yang, Qinghua; Li, Xiaoyang; Tu, Haitao; Pan, Shen Q.

2017-01-01

Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium-delivered factors are trafficked through the cytoplasm. In this study, we monitored the movement of Agrobacterium-delivered VirE2 inside plant cells by using a split-GFP approach in real time. Agrobacterium-delivered VirE2 trafficked via the endoplasmic reticulum (ER) and F-actin network inside plant cells. During this process, VirE2 was aggregated as filamentous structures and was present on the cytosolic side of the ER. VirE2 movement was powered by myosin XI-K. Thus, exogenously produced and delivered VirE2 protein can use the endogenous host ER/actin network for movement inside host cells. The A. tumefaciens pathogen hijacks the conserved host infrastructure for virulence trafficking. Well-conserved infrastructure may be useful for Agrobacterium to target a wide range of recipient cells and achieve a high efficiency of transformation. PMID:28242680

Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome.

PubMed

van der Meer-Janssen, Ynske P M; van Galen, Josse; Batenburg, Joseph J; Helms, J Bernd

2010-01-01

Lipids were long believed to have a structural role in biomembranes and a role in energy storage utilizing cellular lipid droplets and plasma lipoproteins. Research over the last decades has identified an additional role of lipids in cellular signaling, membrane microdomain organization and dynamics, and membrane trafficking. These properties make lipids an attractive target for pathogens to modulate host cell processes in order to allow their survival and replication. In this review we will summarize the often ingenious strategies of pathogens to modify the lipid homeostasis of host cells, allowing them to divert cellular processes. To this end pathogens take full advantage of the complexity of the lipidome. The examples are categorized in generalized and emerging principles describing the involvement of lipids in host-pathogen interactions. Several pathogens are described that simultaneously induce multiple changes in the host cell signaling and trafficking mechanisms. Elucidation of these pathogen-induced changes may have important implications for drug development. The emergence of high-throughput lipidomic techniques will allow the description of changes of the host cell lipidome at the level of individual molecular lipid species and the identification of lipid biomarkers.

Responses triggered in chloroplast of Chlorella variabilis NC64A by long-term association with Paramecium bursaria.

PubMed

Minaeva, Ekaterina; Ermilova, Elena

2017-07-01

The unicellular green alga Chlorella variabilis NC64A is an endosymbiont of the ciliate Paramecium bursaria. The host's control, including the transfer of biochemical substrates from P. bursaria to C. variabilis, is involved in symbiotic relationships. C. variabilis NC64A that had been re-infected to P. bursaria for more than 1 year and isolated from the host showed higher chlorophyll levels compared to those in free-living cells. Unlike the host, the expression of C. variabilis NC64A heat shock 70 kDa protein was independent of establishment of endosymbiosis. In symbiotic cells, the levels of PII signal transduction protein (CvPII) that coordinate the central C/N anabolic metabolism were slightly higher than those in free-living cells. Furthermore, the environmental cues (light and host food bacteria availability) affected the abundance of CvPII, suggesting that synthesis of the protein was influenced by the host. Moreover, arginine concentrations in the symbiotic algae of P. bursaria were also controlled by the host's nutritional conditions. Together, our results imply that signal substrates and/or products of metabolism in host cells might act as messengers mediating the regulation of key events in symbiont cells.

Infection by Toxoplasma gondii Specifically Induces Host c-Myc and the Genes This Pivotal Transcription Factor Regulates

PubMed Central

Franco, Magdalena; Shastri, Anjali J.

2014-01-01

Toxoplasma gondii infection has previously been described to cause dramatic changes in the host transcriptome by manipulating key regulators, including STATs, NF-κB, and microRNAs. Here, we report that Toxoplasma tachyzoites also mediate rapid and sustained induction of another pivotal regulator of host cell transcription, c-Myc. This induction is seen in cells infected with all three canonical types of Toxoplasma but not the closely related apicomplexan parasite Neospora caninum. Coinfection of cells with both Toxoplasma and Neospora still results in an increase in the level of host c-Myc, showing that c-Myc is actively upregulated by Toxoplasma infection (rather than repressed by Neospora). We further demonstrate that this upregulation may be mediated through c-Jun N-terminal protein kinase (JNK) and is unlikely to be a nonspecific host response, as heat-killed Toxoplasma parasites do not induce this increase and neither do nonviable parasites inside the host cell. Finally, we show that the induced c-Myc is active and that transcripts dependent on its function are upregulated, as predicted. Hence, c-Myc represents an additional way in which Toxoplasma tachyzoites have evolved to specifically alter host cell functions during intracellular growth. PMID:24532536

p53 Is a Host Cell Regulator during Herpes Simplex Encephalitis.

PubMed

Maruzuru, Yuhei; Koyanagi, Naoto; Takemura, Naoki; Uematsu, Satoshi; Matsubara, Daisuke; Suzuki, Yutaka; Arii, Jun; Kato, Akihisa; Kawaguchi, Yasushi

2016-08-01

p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knockout reduced viral replication in the brains of mice and led to significantly reduced rates of mortality due to herpes simplex encephalitis. These results suggest that p53 is an important host cell regulator of HSV-1 replication and pathogenesis in the central nervous system (CNS). HSV-1 causes sporadic cases of encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism, which may suggest novel clinical treatment options for herpes simplex encephalitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Bistable Expression of CsgD in Salmonella enterica Serovar Typhimurium Connects Virulence to Persistence

PubMed Central

MacKenzie, Keith D.; Wang, Yejun; Shivak, Dylan J.; Wong, Cynthia S.; Hoffman, Leia J. L.; Lam, Shirley; Kröger, Carsten; Cameron, Andrew D. S.; Townsend, Hugh G. G.; Köster, Wolfgang

2015-01-01

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment. PMID:25824832

How Stem Cells Speak with Host Immune Cells in Inflammatory Brain Diseases

PubMed Central

Pluchino, Stefano; Cossetti, Chiara

2014-01-01

Advances in stem cell biology have raised great expectations that diseases and injuries of the central nervous system (CNS) may be ameliorated by the development of non-hematopoietic stem cell medicines. Yet, the application of adult stem cells as CNS therapeutics is challenging and the interpretation of some of the outcomes ambiguous. In fact, the initial idea that stem cell transplants work only via structural cell replacement has been challenged by the observation of consistent cellular signaling between the graft and the host. Cellular signaling is the foundation of coordinated actions and flexible responses, and arises via networks of exchanging and interacting molecules that transmit patterns of information between cells. Sustained stem cell graft-to-host communication leads to remarkable trophic effects on endogenous brain cells and beneficial modulatory actions on innate and adaptive immune responses in vivo, ultimately promoting the healing of the injured CNS. Among a number of adult stem cell types, mesenchymal stem cells (MSCs) and neural stem/precursor cells (NPCs) are being extensively investigated for their ability to signal to the immune system upon transplantation in experimental CNS diseases. Here, we focus on the main cellular signaling pathways that grafted MSCs and NPCs use to establish a therapeutically relevant cross talk with host immune cells, while examining the role of inflammation in regulating some of the bidirectionality of these communications. We propose that the identification of the players involved in stem cell signaling might contribute to the development of innovative, high clinical impact therapeutics for inflammatory CNS diseases. PMID:23633288

Modeling conduction in host-graft interactions between stem cell grafts and cardiomyocytes.

PubMed

Chen, Michael Q; Yu, Jin; Whittington, R Hollis; Wu, Joseph C; Kovacs, Gregory T A; Giovangrandi, Laurent

2009-01-01

Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Kupffer cell complement receptor clearance function and host defense.

PubMed

Loegering, D J

1986-01-01

Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.

Live-cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae.

PubMed

Mochizuki, Susumu; Minami, Eiichi; Nishizawa, Yoko

2015-12-01

The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

A touch of sleep: biophysical model of contact-mediated dormancy of archaea by viruses.

PubMed

Gulbudak, Hayriye; Weitz, Joshua S

2016-09-28

The canonical view of the interactions between viruses and their microbial hosts presumes that changes in host and virus fate requires the initiation of infection of a host by a virus. Infection may lead to the death of the host cell and release of viruses, to the elimination of the viral genome through cellular defence mechanisms or the integration of the viral genome with the host as a chromosomal or extrachromosomal element. Here, we revisit this canonical view, inspired by recent experimental findings in which the majority of target host cells can be induced into a dormant state when exposed to either active or deactivated viruses, even when viruses are present at low relative titre. We propose that both the qualitative phenomena and the quantitative timescales of dormancy induction are consistent with the hypothesis that cellular physiology can be altered by contact on the surface of host cells rather than strictly by infection In order to test this hypothesis, we develop and study a biophysical model of contact-mediated dynamics involving virus particles and target cells. We show how virus particles can catalyse cellular transformations among many cells, even if they ultimately infect only one (or none). We also find that population-scale dormancy is robust to variation in the representation of model dynamics, including cell growth, death and recovery. © 2016 The Author(s).

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells.

PubMed

Garner, Omai B; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C; Park, Arnold; Bowden, Thomas A; Freiberg, Alexander N; Lee, Benhur; Baum, Linda G

2015-03-01

Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Salmonella modulation of host cell gene expression promotes its intracellular growth.

PubMed

Hannemann, Sebastian; Gao, Beile; Galán, Jorge E

2013-01-01

Salmonella Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS), which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that Salmonella Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by Salmonella T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the Salmonella-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for Salmonella pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

PubMed

Naor, Adit; Panas, Michael W; Marino, Nicole; Coffey, Michael J; Tonkin, Christopher J; Boothroyd, John C

2018-04-03

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins. Copyright © 2018 Naor et al.

Glycosides derived from Hemidesmus indicus R. Br. root inhibit adherence of Salmonella typhimurium to host cells: receptor mimicry.

PubMed

Das, Sarita; Devaraj, S Niranjali

2006-09-01

For centuries, indigenous plants have been used against enteritis but their molecular targets and mode of action remain obscure. The present study was carried out to elucidate the protective and therapeutic role, if any, of glycosides from Hemidesmus indicus against S. typhimurium-induced pathogenesis. Studies were carried out in a human intestinal cell line (Int 407) and a murine macrophage cell line (P388D1) in order to evaluate its potency in local as well as systemic infections. The inhibitory role of the glycosides present in Hemidesmus indicus root extract (GHI) were tested by pre-coating the cells (both Int 407 and P388D1) with GHI prior to infection, and by neutralizing the wild-type bacteria with GHI before cell infection. In both cases, GHI protected the host cells from the cytotoxic effects of the wild S. typhimurium. This suggests that the biologically significant sugars (hexose, hexosamine, fucose and sialic acid etc) present in GHI might be mimicking host cell receptor saccharides and thereby blocking the bacterial ligands from binding to the host cells. Int 407 cells infected with wild-type bacteria had a diffused adherence pattern after 4 h incubation, but this typical character was not observed in cells infected with GHI-treated bacteria and the cells were normal in appearance at 4 h. After 18 h cells infected with wild-type bacteria were hypertrophoid with a disintegrated membrane and wrapped in a bacterial coat, whereas cells infected with treated bacteria had comparatively less morphological changes and few defective shrunken rods adhered locally. This suggests that the glycosides can change the adherence pattern of S. typhimurium from diffused to local. Treated bacteria had less adherence and invasion capability in Int 407 as well as P388D1 cells. The results show the decreased ability of adherence of GHI-treated S. typhimurium was due to a loss of surface hydrophobicity. A nonspecific binding between S. typhimurium and the glycosides was confirmed using ELISA. In summary, the glycosides of H. indicus root inhibited S. typhimurium induced pathogenesis nonspecifically, by reducing bacterial surface hydrophobicity and perhaps also by mimicking host cell receptors, thereby blocking its attachment to host cell and further pathological effects. Copyright (c) 2006 John Wiley & Sons, Ltd.

Toxoplasma gondii Infection Is Associated with Mitochondrial Dysfunction in-Vitro

PubMed Central

Syn, Genevieve; Anderson, Denise; Blackwell, Jenefer M.; Jamieson, Sarra E.

2017-01-01

Upon invasion of host cells, the ubiquitous pathogen Toxoplasma gondii manipulates several host processes, including re-organization of host organelles, to create a replicative niche. Host mitochondrial association to T. gondii parasitophorous vacuoles is rapid and has roles in modulating host immune responses. Here gene expression profiling of T. gondii infected cells reveals enrichment of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial dysfunction 6 h post-infection. We identified 11 hub genes (HIF-1α, CASP8, FN1, POU5F1, CD44, ISG15, HNRNPA1, MDM2, RPL35, VHL, and NUPR1) and 10 predicted upstream regulators, including 4 endogenous regulators RICTOR, KDM5A, RB1, and D-glucose. We characterized a number of mitochondrial parameters in T. gondii infected human foreskin fibroblast cells over a 36 h time-course. In addition to the usual rapid recruitment and apparent enlargement of mitochondria around the parasitophorous vacuole we observed fragmented host mitochondria in infected cells, not linked to cellular apoptosis, from 24 h post-infection. An increase in mitochondrial superoxide levels in T. gondii infected cells was observed that required active parasite invasion and peaked at 30 h post-infection. Measurement of OXPHOS proteins showed decreased expression of Complex IV in infected cells at 24 h post-infection, followed by decreased expression of Complexes I and II at 36 h post-infection. No change occurred in Complex V. No difference in host mitochondrial membrane potential between infected and mock-infected cells was observed at any time. Our results show perturbation of host mitochondrial function following T. gondii infection that likely impacts on pathogenesis of disease. PMID:29312892

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

PubMed

Fineran, Paul; Lloyd-Evans, Emyr; Lack, Nathan A; Platt, Nick; Davis, Lianne C; Morgan, Anthony J; Höglinger, Doris; Tatituri, Raju Venkata V; Clark, Simon; Williams, Ian M; Tynan, Patricia; Al Eisa, Nada; Nazarova, Evgeniya; Williams, Ann; Galione, Antony; Ory, Daniel S; Besra, Gurdyal S; Russell, David G; Brenner, Michael B; Sim, Edith; Platt, Frances M

2016-11-18

Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis , achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

PubMed Central

Fineran, Paul; Lloyd-Evans, Emyr; Lack, Nathan A.; Platt, Nick; Davis, Lianne C.; Morgan, Anthony J.; Höglinger, Doris; Tatituri, Raju Venkata V.; Clark, Simon; Williams, Ian M.; Tynan, Patricia; Al Eisa, Nada; Nazarova, Evgeniya; Williams, Ann; Galione, Antony; Ory, Daniel S.; Besra, Gurdyal S.; Russell, David G.; Brenner, Michael B.; Sim, Edith; Platt, Frances M.

2017-01-01

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC.  Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed.  Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells.  Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies. PMID:28008422

Gut Immune Maturation Depends on Colonization with a Host-Specific Microbiota

PubMed Central

Chung, Hachung; Pamp, Sünje J.; Hill, Jonathan A.; Surana, Neeraj K.; Edelman, Sanna M.; Troy, Erin B.; Reading, Nicola C.; Villablanca, Eduardo J.; Wang, Sen; Mora, Jorge R.; Umesaki, Yoshinori; Mathis, Diane; Benoist, Christophe; Relman, David A.; Kasper, Dennis L.

2012-01-01

SUMMARY Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4+ and CD8+ T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression–all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system. PMID:22726443

Reduced-intensity conditioning for the treatment of malignant and life-threatening non-malignant disorders.

PubMed

Slavin, Shimon; Aker, Mehmet; Shapira, Michael Y; Resnick, Igor; Bitan, Menachem; Or, Reuven

2003-01-01

Allogeneic bone marrow or blood stem cell transplantation (BMT) represents an important therapeutic tool for the treatment of an otherwise incurable broad spectrum of malignant and non-malignant diseases. Until recently, BMT was used primarily to replace a malignant, genetically abnormal or deficient immunohematopoietic compartment and therefore, highly toxic myeloablative regimens were considered mandatory for more effective eradication of all undesirable host-derived hematopoietic cells, including stem cells and their progeny. Our preclinical and ongoing clinical studies indicated that much more effective eradication of host immunohematopoietic system cells can be mediated by donor lymphocytes in the process of adoptive allogeneic cell therapy following BMT. Thus, eradication of all malignant cells, especially in patients with CML and, to a lesser extent, in patients with other hematologic malignancies can be accomplished despite complete resistance of puch tumor cells to maximally tolerated doses of chemoradiotherapy. Our cumulative experience suggested that graft-versus-malignancy effects might be used as a tool for eradication of otherwise resistant tumor cells of host origin. We speculated that the therapeutic benefit of BMT may be improved by using safer conditioning for engraftment of donor stem cells induce host-versus-graft unresponsiveness to enable engraftment of donor lymphocytes for subsequent induction of graft-versus-malignancy effects, or even graft-versus-autoimmunity and graft-versus-genetically abnormal cells. In other words, focusing on more selective and smarter rather than stronger modalities. Effective BMT procedures may be accomplished without lethal conditioning of the host, using a new, well-tolerated and user-friendly non-myeloablative regimen, thus eliminating or minimizing immediate and late procedure-related toxicity and mortality. It appears that initial induction of graft tolerance, mediated by engraftment of donor stem cells, leads to durable engraftment of immunocompetent donor lymphocytes, which may be necessary for induction of effective biologic warfare against host-type immunohematopoietic cells. Consequently, stem-cell therapy following induction of transplantation tolerance by selective elimination of alloreactive donor lymphocytes may represent the treatment of choice for a wide range of otherwise incurable diseases, including cancer (hematologic malignancies and certain metastatic solid tumors), genetic disorders (hemoglobinopathies and enzyme deficiency disorders), diseases caused by self-reactive lymphocytes (autoimmune diseases such as multiple sclerosis, rheumatoid arthritis) to mention just a few. Using reduced intensity conditioning, non-myeloablative stem cell transplantation (NST) can be accomplished with no major procedure-related toxicity or mortality. Thus, NST offers the feasibility of safe stem cell transplantation and cell-mediated procedures for a large and constantly growing spectrum of clinical indications for all patients in need without lower or upper age limit. Future strategies currently under investigation include developing new approaches for control of alloreactivity of host-versus-graft and graft-versus host reactivity reactions and developing better approaches for maximizing the capacity of donor lymphocytes to eliminate cancer cells more selectively, while avoiding or minimizing GVHD for safer and more effective treatment of patients in need of BMT.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, Graham, E-mail: gsimmons@bloodsystems.or; Bertram, Stephanie; Glowacka, Ilona

Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a proteasemore » essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.« less

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

PubMed

Tyler, Brett M; Kale, Shiv D; Wang, Qunqing; Tao, Kai; Clark, Helen R; Drews, Kelly; Antignani, Vincenzo; Rumore, Amanda; Hayes, Tristan; Plett, Jonathan M; Fudal, Isabelle; Gu, Biao; Chen, Qinghe; Affeldt, Katharyn J; Berthier, Erwin; Fischer, Gregory J; Dou, Daolong; Shan, Weixing; Keller, Nancy P; Martin, Francis; Rouxel, Thierry; Lawrence, Christopher B

2013-06-01

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction.

PubMed

Nievas, Yesica R; Coceres, Veronica M; Midlej, Victor; de Souza, Wanderley; Benchimol, Marlene; Pereira-Neves, Antonio; Vashisht, Ajay A; Wohlschlegel, James A; Johnson, Patricia J; de Miguel, Natalia

2018-06-01

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.

Host parasite communications-Messages from helminths for the immune system: Parasite communication and cell-cell interactions.

PubMed

Coakley, Gillian; Buck, Amy H; Maizels, Rick M

2016-07-01

Helminths are metazoan organisms many of which have evolved parasitic life styles dependent on sophisticated manipulation of the host environment. Most notably, they down-regulate host immune responses to ensure their own survival, by exporting a range of immuno-modulatory mediators that interact with host cells and tissues. While a number of secreted immunoregulatory parasite proteins have been defined, new work also points to the release of extracellular vesicles, or exosomes, that interact with and manipulate host gene expression. These recent results are discussed in the overall context of how helminths communicate effectively with the host organism. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Transfer and detection of freshly isolated or cultured chicken (Gallus gallus) and exotic species' embryonic gonadal germ stem cells in host embryos.

PubMed

Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

2014-06-01

The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ovo sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds.

Comparative venom toxicity between Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) toward the hemocytes of their natural hosts, non-target insects and cultured insect cells.

PubMed

Zhang, Zhong; Ye, Gong-Yin; Cai, Jun; Hu, Cui

2005-09-01

Crude venoms from two parasitoid species, Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) were assayed for biological activities toward hemocytes from two species of their natural hosts and eight species of their non-natural hosts as well as two lines of cultured Lepidoptera cells, respectively. By inhibiting the spreading and viability of insect hemocytes, the venom from P. puparum displayed significantly higher activities toward plasmatocytes and granular cells from both larvae and pupae of two natural hosts, Pieris rapae and Papilio xuthus, and lower activity toward those from Spodoptera litura, Musca domestica and Sarcophaga peregrina. However, no effect was found towards any type of hemocytes from other five insects tested, namely, Ectropis oblique, Galleria mellonella, Sesamia inferens, Bombyx mori and Parnara guttata. In contrast, the venom from N. vitripennis showed a narrower range of targeted insects. It appeared to have highly adverse effects on the spreading and viability of plasmatocytes and granular cells only from the natural hosts, M. domestica and S. peregrina, little toxicity to cells from P. rapae and P. xuthus, and no effect on any of the other insects tested. Pteromalus puparum venom also apparently presented a high ability to block the spreading of Tn-5B1-4 cells derived from Trichoplusia ni, and high cytotoxicity to the cells and Ha cells derived from Helicoverpa armigera. Nasonia vitripennis venom, however, only had a marked lethal effect to Ha cells. In addition, the possibility that the host range of a defined parasitoid could be assessed using our method of treating hemocytes from candidate insects with venom in vitro, and the potential of our venoms tested in the development of bio-insecticides, insect-resistant transgenic plants, are discussed.

Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles

PubMed Central

Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E.

2015-01-01

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol. PMID:25822753

How do viruses control mitochondria-mediated apoptosis?

PubMed

Neumann, Simon; El Maadidi, Souhayla; Faletti, Laura; Haun, Florian; Labib, Shirin; Schejtman, Andrea; Maurer, Ulrich; Borner, Christoph

2015-11-02

There is no doubt that viruses require cells to successfully reproduce and effectively infect the next host. The question is what is the fate of the infected cells? All eukaryotic cells can "sense" viral infections and exhibit defence strategies to oppose viral replication and spread. This often leads to the elimination of the infected cells by programmed cell death or apoptosis. This "sacrifice" of infected cells represents the most primordial response of multicellular organisms to viruses. Subverting host cell apoptosis, at least for some time, is therefore a crucial strategy of viruses to ensure their replication, the production of essential viral proteins, virus assembly and the spreading to new hosts. For that reason many viruses harbor apoptosis inhibitory genes, which once inside infected cells are expressed to circumvent apoptosis induction during the virus reproduction phase. On the other hand, viruses can take advantage of stimulating apoptosis to (i) facilitate shedding and hence dissemination, (ii) to prevent infected cells from presenting viral antigens to the immune system or (iii) to kill non-infected bystander and immune cells which would limit viral propagation. Hence the decision whether an infected host cell undergoes apoptosis or not depends on virus type and pathogenicity, its capacity to oppose antiviral responses of the infected cells and/or to evade any attack from immune cells. Viral genomes have therefore been adapted throughout evolution to satisfy the need of a particular virus to induce or inhibit apoptosis during its life cycle. Here we review the different strategies used by viruses to interfere with the two major apoptosis as well as with the innate immune signaling pathways in mammalian cells. We will focus on the intrinsic mitochondrial pathway and discuss new ideas about how particular viruses could activately engage mitochondria to induce apoptosis of their host. Copyright © 2015 Elsevier B.V. All rights reserved.

Multifaceted Activity of Listeriolysin O, the Cholesterol-Dependent Cytolysin of Listeria monocytogenes

PubMed Central

2014-01-01

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by numerous Gram-positive bacterial pathogens. These toxins are released in the extracellular environment as water-soluble monomers or dimers that bind to cholesterol-rich membranes and assemble into large pore complexes. Depending upon their concentration, the nature of the host cell and membrane (cytoplasmic or intracellular) they target, the CDCs can elicit many different cellular responses. Among the CDCs, listeriolysin O (LLO), which is a major virulence factor of the facultative intracellular pathogen Listeria monocytogenes, is involved in several stages of the intracellular lifecycle of the bacterium and displays unique characteristics. It has long been known that following L. monocytogenes internalization into host cells, LLO disrupts the internalization vacuole, enabling the bacterium to replicate into the host cell cytosol. LLO is then used by cytosolic bacteria to spread from cell to cell, avoiding bacterial exposure to the extracellular environment. Although LLO is continuously produced during the intracellular lifecycle of L. monocytogenes, several processes limit its toxicity to ensure the survival of infected cells. It was previously thought that LLO activity was limited to mediating vacuolar escape during bacterial entry and cell to cell spreading. This concept has been challenged by compelling evidence suggesting that LLO secreted by extracellular L. monocytogenes perforates the host cell plasma membrane, triggering important host cell responses. This chapter provides an overview of the well-established intracellular activity of LLO and the multiple roles attributed to LLO secreted by extracellular L. monocytogenes. PMID:24798012

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence

PubMed Central

Perruche, Sylvain; Marandin, Aliette; Kleinclauss, François M.; Angonin, Régis; Fresnay, Stéphanie; Baron, Marie Hélène; Tiberghien, Pierre; Saas, Philippe

2006-01-01

Background Use of a reduced intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a co-existence of both host and donor hematopoietic cells may influence post-transplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced intensity conditioning transplantation (RICT), auto-antibody production and chronic GVHD (cGVHD)-related pathology. Methods Chimerism state, circulating anti-cardiolipin and anti-double stranded DNA auto-antibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. Results We observed a novel association between mixed chimerism state, high levels of pathogenic IgG auto-Abs and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of auto-Abs. Conclusions Recipient B cell persistence may therefore contribute to the frequency and/or severity of cGVHD after RICT. PMID:16495806

Reduction of fatal graft-versus-host disease by /sup 3/H--thymidine suicide of donor cells cultured with host cells. [Mice, gamma radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheever, M.A.; Einstein, A.B. Jr.; Kempf, R.A.

The effect of the tritiated thymidine (/sup 3/H-TdR) suicide technique on the ability of donor cells to induce fatal graft-versus-host disease (GVHD) was studied. C57BL/6 (H-2/sup b/) spleen cells were stimulated in vitro with irradiated BALB/c (H-2/sup d/) Moloney lymphoma cells in mixed culture and /sup 3/H-TdR of high-specific activity added to eliminate proliferating cells. The ability of such cells to induce fatal GVHD was assayed by injecting them i.v. into adult BALB/c mice immunosuppressed with cyclophosphamide (180 mg/kg). These cells induced fatal GVHD in fewer mice (52 percent) than did C57BL/6 cells cultured with BALB/c lymphoma cells but withoutmore » /sup 3/H-TdR (87 percent) and C57BL/6 cells cultured with irradiated C57BL/6 cells with (95 percent) or without /sup 3/H-TdR (86 percent). Thus, the /sup 3/H-TdR suicide technique greatly diminished the ability of cells to induce lethal GVHD.« less

Stromal cells in breast cancer as a potential therapeutic target

PubMed Central

Dykes, Samantha S.; Hughes, Veronica S.; Wiggins, Jennifer M.; Fasanya, Henrietta O.; Tanaka, Mai; Siemann, Dietmar

2018-01-01

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Cell Surface Changes Associated with Cellular Immune Reactions in Drosophila

NASA Astrophysics Data System (ADS)

Nappi, Anthony J.; Silvers, Michael

1984-09-01

In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts.

The Unique Molecular and Cellular Microenvironment of Ovarian Cancer

PubMed Central

Worzfeld, Thomas; Pogge von Strandmann, Elke; Huber, Magdalena; Adhikary, Till; Wagner, Uwe; Reinartz, Silke; Müller, Rolf

2017-01-01

The reciprocal interplay of cancer cells and host cells is an indispensable prerequisite for tumor growth and progression. Cells of both the innate and adaptive immune system, in particular tumor-associated macrophages (TAMs) and T cells, as well as cancer-associated fibroblasts enter into a malicious liaison with tumor cells to create a tumor-promoting and immunosuppressive tumor microenvironment (TME). Ovarian cancer, the most lethal of all gynecological malignancies, is characterized by a unique TME that enables specific and efficient metastatic routes, impairs immune surveillance, and mediates therapy resistance. A characteristic feature of the ovarian cancer TME is the role of resident host cells, in particular activated mesothelial cells, which line the peritoneal cavity in huge numbers, as well as adipocytes of the omentum, the preferred site of metastatic lesions. Another crucial factor is the peritoneal fluid, which enables the transcoelomic spread of tumor cells to other pelvic and peritoneal organs, and occurs at more advanced stages as a malignancy-associated effusion. This ascites is rich in tumor-promoting soluble factors, extracellular vesicles and detached cancer cells as well as large numbers of T cells, TAMs, and other host cells, which cooperate with resident host cells to support tumor progression and immune evasion. In this review, we summarize and discuss our current knowledge of the cellular and molecular interactions that govern this interplay with a focus on signaling networks formed by cytokines, lipids, and extracellular vesicles; the pathophysiologial roles of TAMs and T cells; the mechanism of transcoelomic metastasis; and the cell type selective processing of signals from the TME. PMID:28275576

Relationships between host and symbiont cell cycles in sea anemones and their symbiotic dinoflagellates.

PubMed

Dimond, James L; Pineda, Rea R; Ramos-Ascherl, Zullaylee; Bingham, Brian L

2013-10-01

The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbiotic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastrodermal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across species and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

PubMed Central

Szaszák, Márta; Steven, Philipp; Shima, Kensuke; Orzekowsky-Schröder, Regina; Hüttmann, Gereon; König, Inke R.; Solbach, Werner; Rupp, Jan

2011-01-01

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity. PMID:21779161

Potential role of chitinases and chitin-binding proteins in host-microbial interactions during the development of intestinal inflammation

PubMed Central

Tran, Hoa T.; Barnich, Nicolas; Mizoguchi, Emiko

2011-01-01

Summary The small and large intestines contain an abundance of luminal antigens derived from food products and enteric microorganisms. The function of intestinal epithelial cells is tightly regulated by several factors produced by enteric bacteria and the epithelial cells themselves. Epithelial cells actively participate in regulating the homeostasis of intestine, and failure of this function leads to abnormal and host-microbial interactions resulting in the development of intestinal inflammation. Major determinants of host susceptibility against luminal commensal bacteria include genes regulating mucosal immune responses, intestinal barrier function and microbial defense. Of note, it has been postulated that commensal bacterial adhesion and invasion on/into host cells may be strongly involved in the pathogenesis of inflammatory bowel disease (IBD). During the intestinal inflammation, the composition of the commensal flora is altered, with increased population of aggressive and detrimental bacteria and decreased populations of protective bacteria. In fact, some pathogenic bacteria, including Adherent Invasive Escherichia coli, Listeria monocytogenes and Vibrio cholerae are likely to initiate their adhesion to the host cells by expressing accessory molecules such as chitinases and/or chitin-binding proteins on themselves. In addition, several inducible molecules (e.g., chitinase 3-like-1, CEACAM6) are also induced on the host cells (e.g. epithelial cells, lamina proprial macrophages) under inflammatory conditions, and are actively participated in the host-microbial interactions. In this review, we will summarize and discuss the potential roles of these important molecules during the development of acute and chronic inflammatory conditions. PMID:21938682

Arabinogalactan Proteins Accumulate in the Cell Walls of Searching Hyphae of the Stem Parasitic Plants, Cuscuta campestris and Cuscuta japonica.

PubMed

Hozumi, Akitaka; Bera, Subhankar; Fujiwara, Daiki; Obayashi, Takeshi; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Aoki, Koh

2017-11-01

Stem parasitic plants (Cuscuta spp.) develop a specialized organ called a haustorium to penetrate their hosts' stem tissues. To reach the vascular tissues of the host plant, the haustorium needs to overcome the physical barrier of the cell wall, and the parasite-host interaction via the cell wall is a critical process. However, the cell wall components responsible for the establishment of parasitic connections have not yet been identified. In this study, we investigated the spatial distribution patterns of cell wall components at a parasitic interface using parasite-host complexes of Cuscuta campestris-Arabidopsis thaliana and Cuscuta japonica-Glycine max. We focused on arabinogalactan proteins (AGPs), because AGPs accumulate in the cell walls of searching hyphae of both C. campestris and C. japonica. We found more AGPs in elongated haustoria than in pre haustoria, indicating that AGP accumulation is developmentally regulated. Using in situ hybridization, we identified five genes in C. campestris that encode hyphal-expressed AGPs that belong to the fasciclin-like AGP (FLA) family, which were named CcFLA genes. Three of the five CcFLA genes were expressed in the holdfast, which develops on the Cuscuta stem epidermis at the attachment site for the host's stem epidermis. Our results suggest that AGPs are involved in hyphal elongation and adhesion to host cells, and in the adhesion between the epidermal tissues of Cuscuta and its host. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Enhanced renewal of regulatory T cells in relation to CD4(+) conventional T lymphocytes in the peripheral compartment.

PubMed

Nogueira, Jeane de Souza; Canto, Fábio Barrozo do; Nunes, Caroline Fraga Cabral Gomes; Vianna, Pedro Henrique Oliveira; Paiva, Luciana de Souza; Nóbrega, Alberto; Bellio, Maria; Fucs, Rita

2016-02-01

CD4(+) Foxp3(+) regulatory T (Treg) cells are necessary for the maintenance of self-tolerance and T-cell homeostasis. This population is kept at stable frequencies in secondary lymphoid organs for the majority of the lifetime, despite permanent thymic emigration or in the face of thymic involution. Continuous competition is expected to occur between recently thymus-emigrated and resident Treg cells (either natural or post-thymically induced). In the present work, we analysed the renewal dynamics of Treg cells compared with CD4(+) Foxp3- conventional T cells (Tconv), using protocols of single or successive T-cell transfers into syngeneic euthymic or lymphopenic (nu/nu or RAG2(-/-)) mice, respectively. Our results show a higher turnover for Treg cells in the peripheral compartment, compared with Tconv cells, when B cell-sufficient euthymic or nude hosts are studied. This increased renewal within the Treg pool, shown by the greater replacement of resident Treg cells by donor counterparts, correlates with augmented rates of proliferation and is not modified following temporary environmental perturbations induced by inflammatory state or microbiota alterations. Notably, the preferential substitution of Treg lymphocytes was not observed in RAG2(-/-) hosts. We showed that limited B-cell replenishment in the RAG2(-/-) hosts decisively contributed to the altered peripheral T-cell homeostasis. Accordingly, weekly transfers of B cells to RAG2(-/-) hosts rescued the preferential substitution of Treg lymphocytes. Our study discloses a new aspect of T-cell homeostasis that depends on the presence of B lymphocytes to regulate the relative incorporation of recently arrived Treg and Tconv cells in the peripheral compartment. © 2015 John Wiley & Sons Ltd.

Pathogenic mechanisms of intracellular bacteria.

PubMed

Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

2017-06-01

We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Structural basis for Epstein–Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sathiyamoorthy, Karthik; Hu, Yao Xiong; Möhl, Britta S.

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein–Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, in this paper we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator ofmore » EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. Finally, these observations clarify key determinants of EBV host cell tropism.« less

Structural basis for Epstein–Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins

DOE PAGES

Sathiyamoorthy, Karthik; Hu, Yao Xiong; Möhl, Britta S.; ...

2016-12-08

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein–Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, in this paper we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator ofmore » EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. Finally, these observations clarify key determinants of EBV host cell tropism.« less

Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

NASA Astrophysics Data System (ADS)

Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

2005-04-01

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

Symbiotic Origin of Aging.

PubMed

Greenberg, Edward F; Vatolin, Sergei

2018-06-01

Normally aging cells are characterized by an unbalanced mitochondrial dynamic skewed toward punctate mitochondria. Genetic and pharmacological manipulation of mitochondrial fission/fusion cycles can contribute to both accelerated and decelerated cellular or organismal aging. In this work, we connect these experimental data with the symbiotic theory of mitochondrial origin to generate new insight into the evolutionary origin of aging. Mitochondria originated from autotrophic α-proteobacteria during an ancient endosymbiotic event early in eukaryote evolution. To expand beyond individual host cells, dividing α-proteobacteria initiated host cell lysis; apoptosis is a product of this original symbiont cell lytic exit program. Over the course of evolution, the host eukaryotic cell attenuated the harmful effect of symbiotic proto-mitochondria, and modern mitochondria are now functionally interdependent with eukaryotic cells; they retain their own circular genomes and independent replication timing. In nondividing differentiated or multipotent eukaryotic cells, intracellular mitochondria undergo repeated fission/fusion cycles, favoring fission as organisms age. The discordance between cellular quiescence and mitochondrial proliferation generates intracellular stress, eventually leading to a gradual decline in host cell performance and age-related pathology. Hence, aging evolved from a conflict between maintenance of a quiescent, nonproliferative state and the evolutionarily conserved propagation program driving the life cycle of former symbiotic organisms: mitochondria.

Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

PubMed Central

Yu, Song-Hee; Jang, Yu-Jin; Lee, Eun-Shil; Hwang, Dong-Youn; Jeon, Chang-Jin

2010-01-01

Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell. PMID:21245978

Identification of the cell binding domain in Nipah virus G glycoprotein using a phage display system.

PubMed

Lam, Chui-Wan; AbuBakar, Sazaly; Chang, Li-Yen

2017-05-01

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with unusual broad host tropism and is designated as a Category C pathogen by the U.S. National Institute of Allergy and Infectious Diseases. NiV infection is initiated after binding of the viral G glycoprotein to the host cell receptor. The aim of this study was to map the NiV G glycoprotein cell binding domain using a phage display system. The NiV G extracellular domain was truncated and displayed as attachment proteins on M13 phage g3p minor coat protein. The binding efficiency of recombinant phages displaying different regions of NiV G to mammalian cells was evaluated. Results showed that regions of NiV G consisting of amino acids 396-602 (recombinant phage G4) and 498-602 (recombinant phage G5) demonstrated the highest binding to both Vero (5.5×10 3 cfu/ml and 5.6×10 3 cfu/ml) and THP-1 cells (3.5×10 3 cfu/ml and 2.9×10 3 cfu/ml). However, the binding of both of these recombinant phages to THP-1 cells was significantly lower than to Vero cells, and this could be due to the lack of primary host cell receptor expression on THP-1 cells. Furthermore, the binding between these two recombinant phages was competitive suggesting that there was a common host cell attachment site. This study employed an approach that is suitable for use in a biosafety level 2 containment laboratory without the need to use live virus to show that NiV G amino acids 498-602 play an important role for attachment to host cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

PubMed

Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

2016-08-05

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Single-cell transcriptional dynamics of flavivirus infection

PubMed Central

Bekerman, Elena

2018-01-01

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level. PMID:29451494

Probiotics production and alternative encapsulation methodologies to improve their viabilities under adverse environmental conditions.

PubMed

Coghetto, Chaline Caren; Brinques, Graziela Brusch; Ayub, Marco Antônio Záchia

2016-12-01

Probiotic products are dietary supplements containing live microorganisms producing beneficial health effects on the host by improving intestinal balance and nutrient absorption. Among probiotic microorganisms, those classified as lactic acid bacteria are of major importance to the food and feed industries. Probiotic cells can be produced using alternative carbon and nitrogen sources, such as agroindustrial residues, at the same time contributing to reduce process costs. On the other hand, the survival of probiotic cells in formulated food products, as well as in the host gut, is an essential nutritional aspect concerning health benefits. Therefore, several cell microencapsulation techniques have been investigated as a way to improve cell viability and survival under adverse environmental conditions, such as the gastrointestinal milieu of hosts. In this review, different aspects of probiotic cells and technologies of their related products are discussed, including formulation of culture media, and aspects of cell microencapsulation techniques required to improve their survival in the host.

A statistical approach to determining criticality of residual host cell DNA.

PubMed

Yang, Harry; Wei, Ziping; Schenerman, Mark

2015-01-01

We propose a method for determining the criticality of residual host cell DNA, which is characterized through two attributes, namely the size and amount of residual DNA in biopharmaceutical product. By applying a mechanistic modeling approach to the problem, we establish the linkage between residual DNA and product safety measured in terms of immunogenicity, oncogenicity, and infectivity. Such a link makes it possible to establish acceptable ranges of residual DNA size and amount. Application of the method is illustrated through two real-life examples related to a vaccine manufactured in Madin Darby Canine Kidney cell line and a monoclonal antibody using Chinese hamster ovary (CHO) cell line as host cells.

Challenges and future perspectives of T cell immunotherapy in cancer

PubMed Central

de Aquino, Maria Teresa P; Malhotra, Anshu; Mishra, Manoj K; Shanker, Anil

2015-01-01

Since the formulation of the tumour immunosurveillance theory, considerable focus has been on enhancing the effectiveness of host antitumour immunity, particularly with respect to T cells. A cancer evades or alters the host immune response by various ways to ensure its development and survival. These include modifications of the immune cell metabolism and T cell signaling. An inhibitory cytokine milieu in the tumour microenvironment also leads to immune suppression and tumour progression within a host. This review traces the development in the field and attempts to summarize the hurdles that the approach of adoptive T cell immunotherapy against cancer faces, and discusses the conditions that must be improved to allow effective eradication of cancer. PMID:26096822

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

PubMed

Todeschi, Maria R; El Backly, Rania M; Varghese, Oommen P; Hilborn, Jöns; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-07-01

This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

Alemtuzumab and Glucocorticoids in Treating Newly Diagnosed Acute Graft-Versus-Host Disease in Patients Who Have Undergone a Donor Stem Cell Transplant

ClinicalTrials.gov

2010-05-12

Breast Cancer; Chronic Myeloproliferative Disorders; Gestational Trophoblastic Tumor; Graft Versus Host Disease; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Neuroblastoma; Ovarian Cancer; Testicular Germ Cell Tumor

High Dose Cyclophosphamide, Tacrolimus, and Mycophenolate Mofetil in Preventing Graft Versus Host Disease in Patients With Hematological Malignancies Undergoing Myeloablative or Reduced Intensity Donor Stem Cell Transplant

ClinicalTrials.gov

2018-01-24

Acute Leukemia; Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Diffuse Large B-Cell Lymphoma; Follicular Lymphoma; Graft Versus Host Disease; Hodgkin Lymphoma; Mantle Cell Lymphoma; Marginal Zone Lymphoma; Myelodysplastic Syndrome; Myeloproliferative Neoplasm; Recurrent Acute Myeloid Leukemia With Myelodysplasia-Related Changes; Recurrent Plasma Cell Myeloma; Refractory Plasma Cell Myeloma; Secondary Myelodysplastic Syndrome

Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

PubMed Central

Saxena, Kapil; Blutt, Sarah E.; Ettayebi, Khalil; Zeng, Xi-Lei; Broughman, James R.; Crawford, Sue E.; Karandikar, Umesh C.; Sastri, Narayan P.; Conner, Margaret E.; Opekun, Antone R.; Graham, David Y.; Qureshi, Waqar; Sherman, Vadim; Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga; Zachos, Nicholas C.; Donowitz, Mark

2015-01-01

ABSTRACT Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures. PMID:26446608

Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.

PubMed

Nyholm, S V; McFall-Ngai, M J

1998-10-01

The symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri has a pronounced diel rhythm, one component of which is the venting of the contents of the light organ into the surrounding seawater each day at dawn. In this study, we explored the use of this behavior to sample the microenvironment of the light-organ crypts. Intact crypt contents, which emerge from the lateral pores of the organ as a thick paste-like exudate, were collected from anesthetized host animals that had been exposed to a light cue. Microscopy revealed that the expelled material is composed of a conspicuous population of host cells in association with the bacterial symbionts, all of which are embedded in a dense acellular matrix that strongly resembles the bacteria-based biofilms described in other systems. Assays of the viability of expelled crypt cells revealed no dead bacterial symbionts and a mixture of live and dead host cells. Analyses of the ultrastructure, biochemistry, and phagocytic activity of a subset of the host cell population suggested that some of these cells are macrophage-like molluscan hemocytes.

HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

Cancer.gov

When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

Cancer.gov

Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures.

PubMed

Forsberg, David; Thonabulsombat, Charoensri; Jäderstad, Johan; Jäderstad, Linda Maria; Olivius, Petri; Herlenius, Eric

2017-08-14

Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neural network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell-cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment.

PubMed

Guzman-Rojas, Liliana; Rangel, Roberto; Salameh, Ahmad; Edwards, Julianna K; Dondossola, Eleonora; Kim, Yun-Gon; Saghatelian, Alan; Giordano, Ricardo J; Kolonin, Mikhail G; Staquicini, Fernanda I; Koivunen, Erkki; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2012-01-31

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Parvoviral nuclear import: bypassing the host nuclear-transport machinery.

PubMed

Cohen, Sarah; Behzad, Ali R; Carroll, Jeffrey B; Panté, Nelly

2006-11-01

The parvovirus Minute virus of mice (MVM) is a small DNA virus that replicates in the nucleus of its host cells. However, very little is known about the mechanisms underlying parvovirus' nuclear import. Recently, it was found that microinjection of MVM into the cytoplasm of Xenopus oocytes causes damage to the nuclear envelope (NE), suggesting that the nuclear-import mechanism of MVM involves disruption of the NE and import through the resulting breaks. Here, fluorescence microscopy and electron microscopy were used to examine the effect of MVM on host-cell nuclear structure during infection of mouse fibroblast cells. It was found that MVM caused dramatic changes in nuclear shape and morphology, alterations of nuclear lamin immunostaining and breaks in the NE of infected cells. Thus, it seems that the unusual nuclear-import mechanism observed in Xenopus oocytes is in fact used by MVM during infection of host cells.

Henneguya laterocapsulata Landsberg, 1987 (Myxosporea, Myxozoa) in cultured hybrid African catfish: Ultrastructure of the parasite-host interface.

PubMed

Obiekezie, A; Schmahl, G

1993-02-19

The ultrastructure of the host-parasite interface was studied in Henneguya laterocapsulata, parasitizing the skin of hybrid catfishes (Clarias gariepinus × Heterobranchus bidorsalis) in Nigeria. The plasmodia were located between malpighian cells, which are the main elements of the multilayered fish epidermis, and were bordered by a single cell membrane. The desmosomal junctions between the malpighian cells were forced apart by finger-like protrusions of the Plasmodium. These plasmodial protrusions finally ran into the host cell without disrupting of the host cell membrane and formed network-like extensions. At the margin of the plasmodium an extensive vacuolization occurred, leading to a wavy surface. Infections with H. laterocapsulata may be an adverse factor in the large-scale production of hybrid catfish fingerlings used for aquaculture in Africa. Copyright © 1993 Gustav Fischer Verlag · Stuttgart · Jena · New York. Published by Elsevier GmbH.. All rights reserved.

Candida albicans triggers interleukin-6 and interleukin-8 responses by oral fibroblasts in vitro.

PubMed

Dongari-Bagtzoglou, A; Wen, K; Lamster, I B

1999-12-01

Oral candidiasis is the most frequent opportunistic infection associated with an immunocompromised host. Production of proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, by host cells in response to Candida albicans can be expected to have a major impact in the activation of immune effector cells against the invading microorganism. Using a human cell--C. albicans coculture model system, we determined that this microorganism can trigger secretion of these potent chemoattractant and proinflammatory cytokines by oral mucosal fibroblasts. This response varied depending on the infecting strain and required fungal viability, germination of yeast into hyphae and mannose-mediated direct contact between the host cell and Candida. The secretion of proinflammatory cytokines by oral mucosal fibroblasts in response to C. albicans suggests that these cells have the potential to enhance the host defense against this organism in vivo. This may have important implications in controlling fungal overgrowth in the oral cavity.

Host cells and methods for producing isoprenyl alkanoates

DOEpatents

Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

2015-12-01

The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

Intracellular staphylococcus aureus: Live-in and let die

PubMed Central

Fraunholz, Martin; Sinha, Bhanu

2012-01-01

Staphylococcus aureus uses a plethora of virulence factors to accommodate a diversity of niches in its human host. Aside from the classical manifestations of S. aureus-induced diseases, the pathogen also invades and survives within mammalian host cells.The survival strategies of the pathogen are as diverse as strains or host cell types used. S. aureus is able to replicate in the phagosome or freely in the cytoplasm of its host cells. It escapes the phagosome of professional and non-professional phagocytes, subverts autophagy, induces cell death mechanisms such as apoptosis and pyronecrosis, and even can induce anti-apoptotic programs in phagocytes. The focus of this review is to present a guide to recent research outlining the variety of intracellular fates of S. aureus. PMID:22919634

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

PubMed

Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

2016-06-01

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

PubMed Central

Rac, Jürgen; Haas, Florian; Schumacher, Andrina; Middeldorp, Jaap M.; Delecluse, Henri-Jacques; Speck, Roberto F.

2015-01-01

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva. PMID:25856387

Potential Sabotage of Host Cell Physiology by Apicomplexan Parasites for Their Survival Benefits

PubMed Central

Chakraborty, Shalini; Roy, Sonti; Mistry, Hiral Uday; Murthy, Shweta; George, Neena; Bhandari, Vasundhra; Sharma, Paresh

2017-01-01

Plasmodium, Toxoplasma, Cryptosporidium, Babesia, and Theileria are the major apicomplexan parasites affecting humans or animals worldwide. These pathogens represent an excellent example of host manipulators who can overturn host signaling pathways for their survival. They infect different types of host cells and take charge of the host machinery to gain nutrients and prevent itself from host attack. The mechanisms by which these pathogens modulate the host signaling pathways are well studied for Plasmodium, Toxoplasma, Cryptosporidium, and Theileria, except for limited studies on Babesia. Theileria is a unique pathogen taking into account the way it modulates host cell transformation, resulting in its clonal expansion. These parasites majorly modulate similar host signaling pathways, however, the disease outcome and effect is different among them. In this review, we discuss the approaches of these apicomplexan to manipulate the host–parasite clearance pathways during infection, invasion, survival, and egress. PMID:29081773

Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling.

PubMed

Dong, Jun; Dai, Xing-liang; Lu, Zhao-hui; Fei, Xi-feng; Chen, Hua; Zhang, Quan-bin; Zhao, Yao-dong; Wang, Zhi-min; Wang, Ai-dong; Lan, Qing; Huang, Qiang

2012-12-01

The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells. However, these tumor cells are hard to be visualized directly in histopathological preparations, or in experimental glioma models. Therefore, we developed an experimental human dual-color in vivo glioma model, which made tracking solitary invasive glioma cells possible, for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells. This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling. Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein (EGFP) were crossed with male Balb/c nude mice. Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive. Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein (RFP) gene, and a rat C6 glioma cell line was stained directly with CM-DiI, to establish three glioma cell lines emitting red fluorescence (SU3-RFP, U87-RFP, and C6-CM-DiI). Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice. Tumor-bearing mice were sacrificed when their clinical symptoms appeared, and the whole brain was harvested and snap frozen for further analysis. Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells. Almost all the essential tissues of the established EGFP athymic Balb/c nude mice, except hair and erythrocytes, fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm, approximately 50% of the offsprings were nu/nu EGFP+. SU3-RFP, U87-RFP, and C6-CM-DiI almost 100% expressed red fluorescence under the fluorescence microscope. Under fluorescence microscopic view, RFP+ cells were observed growing wherever they arrived at, locating in the brain parenchyma, ventricles, and para-vascular region. The interactions between the transplanted tumor cells and host adjacent cells could be classified into three types: (1) interweaving; (2) mergence; and (3) fusion. Interweaving was observed in the early stage of tumor remodeling, in which both transplantable tumor cells and host cells were observed scattered in the tumor invading and spreading area without organic connections. Mergence was defined as mutual interactions between tumor cells and host stroma during tumorigenesis. Direct cell fusion between transplantable tumor cells and host cells could be observed occasionally. This study showed that self-established EGFP athymic nude mice offered the possibility of visualizing tumorigenesis of human xenograft tumor, and the dual-color xenograft glioma model was of considerable utility in studying the process of tumor remodeling. Based on this platform, mutual interactions between glioma cells and host tissues could be observed directly to further elucidate the development of tumor microenvironment.

Abundance and Relative Distribution of Frankia Host Infection Groups Under Actinorhizal Alnus glutinosa and Non-actinorhizal Betula nigra Trees.

PubMed

Samant, Suvidha; Huo, Tian; Dawson, Jeffrey O; Hahn, Dittmar

2016-02-01

Quantitative polymerase chain reaction (qPCR) was used to assess the abundance and relative distribution of host infection groups of the root-nodule forming, nitrogen-fixing actinomycete Frankia in four soils with similar physicochemical characteristics, two of which were vegetated with a host plant, Alnus glutinosa, and two with a non-host plant, Betula nigra. Analyses of DAPI-stained cells at three locations, i.e., at a distance of less than 1 m (near stem), 2.5 m (middle crown), and 3-5 m (crown edge) from the stems of both tree species revealed no statistically significant differences in abundance. Frankiae generally accounted for 0.01 to 0.04 % of these cells, with values between 4 and 36 × 10(5) cells (g soil)(-1). In three out of four soils, abundance of frankiae was significantly higher at locations "near stem" and/or "middle crown" compared to "crown edge," while numbers at these locations were not different in the fourth soil. Frankiae of the Alnus host infection group were dominant in all samples accounting for about 75 % and more of the cells, with no obvious differences with distance to stem. In three of the soils, all of these cells were represented by strain Ag45/Mut15. In the fourth soil that was vegetated with older A. glutinosa trees, about half of these cells belonged to a different subgroup represented by strain ArI3. In all soils, the remaining cells belonged to the Elaeagnus host infection group represented by strain EAN1pec. Casuarina-infective frankiae were not found. Abundance and relative distribution of Frankia host infection groups were similar in soils under the host plant A. glutinosa and the non-host plant B. nigra. Results did thus not reveal any specific effects of plant species on soil Frankia populations.

Medaka embryonic stem cells are capable of generating entire organs and embryo-like miniatures.

PubMed

Hong, Ni; He, Bei Ping; Schartl, Manfred; Hong, Yunhan

2013-03-01

Embryonic stem (ES) cells have the potency to produce many cell types of the embryo and adult body. Upon transplantation into early host embryos, ES cells are able to differentiate into various specialized cells and contribute to host tissues and organs of all germ layers. Here we present data in the fish medaka (Oryzias latipes) that ES cells have a novel ability to form extra organs and even embryo-like miniatures. Upon transplantation as individual cells according to the standard procedure, ES cells distributed widely to various organ systems of 3 germ layers. Upon transplantation as aggregates, ES cells were able to form extra organs, including the hematopoietic organ and contracting heart. We show that localized ES cell transplantation often led to the formation of extra axes that comprised essentially of either host cells or donor ES cells. These extra axes were associated with the head region of the embryo proper or formed at ectopic sites on the yolk sac. Surprisingly, certain ectopic axes were even capable of forming embryo-like miniatures. We conclude that ES cells have the ability to form entire organs and even embryo-like miniatures under proper environmental conditions. This finding points to a new possibility to generate ES cell-derived axes and organs.

The effect of inhibition of host MreB on the infection of thermophilic phage GVE2 in high temperature environment.

PubMed

Jin, Min; Chen, Yanjiang; Xu, Chenxi; Zhang, Xiaobo

2014-04-28

In eukaryotes, the manipulation of the host actin cytoskeleton is a necessary strategy for viral pathogens to invade host cells. Increasing evidence indicates that the actin homolog MreB of bacteria plays key roles in cell shape formation, cell polarity, cell wall biosynthesis, and chromosome segregation. However, the role of bacterial MreB in the bacteriophage infection is not extensively investigated. To address this issue, in this study, the MreB of thermophilic Geobacillus sp. E263 from a deep-sea hydrothermal field was characterized by inhibiting the MreB polymerization and subsequently evaluating the bacteriophage GVE2 infection. The results showed that the host MreB played important roles in the bacteriophage infection at high temperature. After the host cells were treated with small molecule drug A22 or MP265, the specific inhibitors of MreB polymerization, the adsorption of GVE2 and the replication of GVE2 genome were significantly repressed. The confocal microscopy data revealed that MreB facilitated the GVE2 infection by inducing the polar distribution of virions during the phage infection. Our study contributed novel information to understand the molecular events of the host in response to bacteriophage challenge and extended our knowledge about the host-virus interaction in deep-sea vent ecosystems.

Yersinia vs. host Immunity: how a pathogen evades or triggers a protective response

PubMed Central

Chung, Lawton K.; Bliska, James B.

2015-01-01

The human pathogenic Yersinia species cause diseases that represent a significant source of morbidity and mortality. Despite this, specific mechanisms underlying Yersinia pathogenesis and protective host responses remain poorly understood. Recent studies have shown that Yersinia disrupt cell death pathways, perturb inflammatory processes and exploit immune cells to promote disease. The ensuing host responses following Yersinia infection include coordination of innate and adaptive immune responses in an attempt to control bacterial replication. Here, we highlight current advances in our understanding of the interactions between the pathogenic yersiniae and host cells, as well as the protective host responses mobilized to counteract these pathogens. Together, these studies enhance our understanding of Yersinia pathogenesis and highlight the ongoing battle between host and microbe. PMID:26638030

The hidden life of integrative and conjugative elements

PubMed Central

Delavat, François; Miyazaki, Ryo; Carraro, Nicolas; Pradervand, Nicolas

2017-01-01

Abstract Integrative and conjugative elements (ICEs) are widespread mobile DNA that transmit both vertically, in a host-integrated state, and horizontally, through excision and transfer to new recipients. Different families of ICEs have been discovered with more or less restricted host ranges, which operate by similar mechanisms but differ in regulatory networks, evolutionary origin and the types of variable genes they contribute to the host. Based on reviewing recent experimental data, we propose a general model of ICE life style that explains the transition between vertical and horizontal transmission as a result of a bistable decision in the ICE–host partnership. In the large majority of cells, the ICE remains silent and integrated, but hidden at low to very low frequencies in the population specialized host cells appear in which the ICE starts its process of horizontal transmission. This bistable process leads to host cell differentiation, ICE excision and transfer, when suitable recipients are present. The ratio of ICE bistability (i.e. ratio of horizontal to vertical transmission) is the outcome of a balance between fitness costs imposed by the ICE horizontal transmission process on the host cell, and selection for ICE distribution (i.e. ICE ‘fitness’). From this emerges a picture of ICEs as elements that have adapted to a mostly confined life style within their host, but with a very effective and dynamic transfer from a subpopulation of dedicated cells. PMID:28369623

Strategic targeting of essential host-pathogen interactions in chlamydial disease.

PubMed

Coombes, B K; Johnson, D L; Mahony, J B

2002-09-01

The chlamydiae are obligate intracellular gram-negative bacteria that are exquisitely adapted for exploitation of their hosts and contribute to a wide range of acute and chronic human diseases. Acute infections treated with non-cidal antibiotics can lead to the development of persistent, non-replicating bacteria with the corollary that these persistent (yet viable) chlamydiae can resist eradication by further antimicrobial treatment and cause chronic disease. These findings highlight an urgent need for therapeutics that are effective against persistent infections and call for creative approaches to identify potential drug targets. The C. pneumoniae and C. trachomatis genome projects have greatly expanded our knowledge of chlamydial pathogenesis and have provided an enormous potential for the identification and characterization of unknown genes and potential virulence factors in these bacteria. As intracellular pathogens, chlamydiae rely on host cells for all aspects of their survival, from the initial attachment with host cell membranes, to cellular invasion, acquisition of host cell metabolites and intracellular replication. As such, the molecules participating in interactions with the host could be attractive targets for therapeutic intervention. This review describes recent advances in chlamydial genomics, proteomics and cell biology that have cast light on host-pathogen relations that are essential for chlamydial survival. Using this knowledge, we discuss how strategically interfering with essential interactions between chlamydiae and the host cell could be exploited to develop an innovative, and potentially more relevant arsenal of therapeutic compounds.

Impairment of T Cell Function in Parasitic Infections

PubMed Central

Rodrigues, Vasco; Cordeiro-da-Silva, Anabela; Laforge, Mireille; Ouaissi, Ali; Akharid, Khadija; Silvestre, Ricardo; Estaquier, Jérôme

2014-01-01

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence. PMID:24551250

Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines.

PubMed

Maghodia, Ajay B; Jarvis, Donald L

2017-12-01

Sf-rhabdovirus was only recently identified as an adventitious agent of Spodoptera frugiperda (Sf) cell lines used as hosts for baculovirus vectors. As such, we still know little about its genetic variation, infectivity, and the potential impact of variation on the Sf-rhabdovirus-host interaction. Here, we characterized Sf-rhabdoviruses from two widely used Sf cell lines to confirm and extend information on Sf-rhabdovirus variation. We then used our novel Sf-rhabdovirus-negative (Sf-RVN) Sf cell line to assess the infectivity of variants with and without a 320bp X/L deletion and found both established productive persistent infections in Sf-RVN cells. We also assessed their infectivity using heterologous insect and mammalian cell lines and found neither established productive persistent infections in these cells. These results are the first to directly demonstrate Sf-rhabdoviruses are infectious for Sf cells, irrespective of the X/L deletion. They also confirm and extend previous results indicating Sf-rhabdoviruses have a narrow host range. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines.

PubMed

Offerdahl, Danielle K; Dorward, David W; Hansen, Bryan T; Bloom, Marshall E

2017-01-15

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60-100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20-30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. Published by Elsevier Inc.

Pathogen trafficking pathways and host phosphoinositide metabolism.

PubMed

Weber, Stefan S; Ragaz, Curdin; Hilbi, Hubert

2009-03-01

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.

Fungal Strategies to Evade the Host Immune Recognition.

PubMed

Hernández-Chávez, Marco J; Pérez-García, Luis A; Niño-Vega, Gustavo A; Mora-Montes, Héctor M

2017-09-23

The recognition of fungal cells by the host immune system is key during the establishment of a protective anti-fungal response. Even though the immune system has evolved a vast number of processes to control these organisms, they have developed strategies to fight back, avoiding the proper recognition by immune components and thus interfering with the host protective mechanisms. Therefore, the strategies to evade the immune system are as important as the virulence factors and attributes that damage the host tissues and cells. Here, we performed a thorough revision of the main fungal tactics to escape from the host immunosurveillance processes. These include the composition and organization of the cell wall, the fungal capsule, the formation of titan cells, biofilms, and asteroid bodies; the ability to undergo dimorphism; and the escape from nutritional immunity, extracellular traps, phagocytosis, and the action of humoral immune effectors.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

PubMed

Kortebi, Mounia; Milohanic, Eliane; Mitchell, Gabriel; Péchoux, Christine; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

2017-11-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

PubMed Central

Mitchell, Gabriel

2017-01-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle*

PubMed Central

Bruening, Janina; Weigel, Bettina; Pietschmann, Thomas

2017-01-01

Protein–protein interactions govern biological functions in cells, in the extracellular milieu, and at the border between cells and extracellular space. Viruses are small intracellular parasites and thus rely on protein interactions to produce progeny inside host cells and to spread from cell to cell. Usage of host proteins by viruses can have severe consequences e.g. apoptosis, metabolic disequilibria, or altered cell proliferation and mobility. Understanding protein interactions during virus infection can thus educate us on viral infection and pathogenesis mechanisms. Moreover, it has led to important clinical translations, including the development of new therapeutic and vaccination strategies. Here, we will discuss protein interactions of members of the Flaviviridae family, which are small enveloped RNA viruses. Dengue virus, Zika virus and hepatitis C virus belong to the most prominent human pathogenic Flaviviridae. With a genome of roughly ten kilobases encoding only ten viral proteins, Flaviviridae display intricate mechanisms to engage the host cell machinery for their purpose. In this review, we will highlight how dengue virus, hepatitis C virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, yellow fever virus, and Zika virus proteins engage host proteins and how this knowledge helps elucidate Flaviviridae infection. We will specifically address the protein composition of the virus particle as well as the protein interactions during virus entry, replication, particle assembly, and release from the host cell. Finally, we will give a perspective on future challenges in Flaviviridae interaction proteomics and why we believe these challenges should be met. PMID:28077444

Recombinant protein production and insect cell culture and process

NASA Technical Reports Server (NTRS)

Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

1993-01-01

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

Inhibitors of the entry of HIV into host cells.

PubMed

Meanwell, Nicholas A; Kadow, John F

2003-07-01

The development of mechanistic insight into the process by which HIV enters host cells has revealed a panoply of targets that offer considerable potential as sites for pharmacological intervention. The gp120/gp41 protein complex, expressed on the virion surface, mediates HIV entry by a process initiated by the engagement of the host cell receptor CD4. Subtle conformational changes triggered by this interaction expose elements of gp120 to the seven-transmembrane, G protein-coupled chemokine receptors CCR5 or CXCR4 expressed on host cells, a contact that relieves constraints imposed on gp41 by gp120. This leads to a major conformational rearrangement of gp41, which results in the insertion of the fusion peptide into the host cell membrane and the assembly of the amino terminus heptad repeat into a trimeric form that is subsequently recognized by the carboxy terminal heptad repeat. The latter process leads to juxtaposition of the viral and host cell membranes, a prelude to fusion. The most prominent strategies and targets that are actively being exploited as drug discovery opportunities are inhibition of the attachment of HIV to host cells, blockade of chemokine receptors and interference with the function of gp41. Inhibitors of each of these steps in the HIV entry process with potential clinical relevance are reviewed in the context of their status in the drug development process. The most significant entity to emerge from this area of research to date is enfuvirtide, a 36-amino acid derivative that interferes with the function of gp41. Enfuvirtide is the first HIV entry inhibitor to be granted a license for marketing (it was approved in the US and Europe in March 2003), and its introduction portends the beginning of what promises to be an exciting new era of HIV therapy.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

1998-10-13

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

1998-01-01

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous gene

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2007-03-20

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2000-08-22

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

A universal mammalian vaccine cell line substrate.

PubMed

Murray, Jackelyn; Todd, Kyle V; Bakre, Abhijeet; Orr-Burks, Nichole; Jones, Les; Wu, Weilin; Tripp, Ralph A

2017-01-01

Using genome-wide small interfering RNA (siRNA) screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD) enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences) across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205) showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

Enhanced production of enveloped viruses in BST-2-deficient cell lines.

PubMed

Yi, Eunbi; Oh, Jinsoo; Giao, Ngoc Q; Oh, Soohwan; Park, Se-Ho

2017-10-01

Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The role of rare innate immune cells in Type 2 immune activation against parasitic helminths.

PubMed

Webb, Lauren M; Tait Wojno, Elia D

2017-09-01

The complexity of helminth macroparasites is reflected in the intricate network of host cell types that participate in the Type 2 immune response needed to battle these organisms. In this context, adaptive T helper 2 cells and the Type 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13 have been the focus of research for years, but recent work has demonstrated that the innate immune system plays an essential role. Some innate immune cells that promote Type 2 immunity are relatively abundant, such as macrophages and eosinophils. However, we now appreciate that more rare cell types including group 2 innate lymphoid cells, basophils, mast cells and dendritic cells make significant contributions to these responses. These cells are found at low frequency but they are specialized to their roles - located at sites such as the skin, lung and gut, where the host combats helminth parasites. These cells respond rapidly and robustly to worm antigens and worm-induced damage to produce essential cytokines, chemokines, eicosanoids and histamine to activate damaged epithelium and to recruit other effectors. Thus, a greater understanding of how these cells operate is essential to understand how the host protects itself during helminth infection.

A strain-cue hypothesis for biological network formation

PubMed Central

Cox, Brian N.

2011-01-01

The direction of migration of a cell invading a host population is assumed to be controlled by the magnitude of the strains in the host medium (cells plus extracellular matrix) that arise as the host medium deforms to accommodate the invader. The single assumption that invaders are cued by strains external to themselves is sufficient to generate network structures. The strain induced by a line of invaders is greatest at the extremity of the line and thus the strain field breaks symmetry, stabilizing branch formation. The strain cue also triggers sprouting from existing branches, with no further model assumption. Network characteristics depend primarily on the ratio of the rate of advance of the invaders to the rate of relaxation of the host cells after their initial deformation. Intra-cell mechanisms that govern these two rates control network morphology. The strain field that cues an individual invader is a collective response of the combined cell populations, involving the nearest 100 cells, to order of magnitude, to any invader. The mechanism does not rely on the pre-existence of the entire host medium prior to invasion; the host cells need only maintain a layer several cells thick around each invader. Consistent with recent experiments, networks result only from a strain cue that is based on strain magnitudes. Spatial strain gradients do not break symmetry and therefore cannot stabilize branch formation. The theory recreates most of the geometrical features of the nervous network in the mouse gut when the most influential adjustable parameter takes a value consistent with one inferred from human and mouse amelogenesis. Because of similarity in the guiding local strain fields, strain cues could also be a participating factor in the formation of vascular networks. PMID:20671068

Fibroblasts—a key host cell type in tumor initiation, progression, and metastasis

PubMed Central

Strell, Carina; Rundqvist, Helene

2012-01-01

Tumor initiation, growth, invasion, and metastasis occur as a consequence of a complex interplay between the host environment and cancer cells. Fibroblasts are now recognized as a key host cell type involved in host–cancer signaling. This review discusses some recent studies that highlight the roles of fibroblasts in tumor initiation, early progression, invasion, and metastasis. Some clinical studies describing the prognostic significance of fibroblast-derived markers and signatures are also discussed. PMID:22509805

Translational Control of Viral Gene Expression in Eukaryotes

PubMed Central

Gale, Michael; Tan, Seng-Lai; Katze, Michael G.

2000-01-01

As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell. PMID:10839817

A Novel Sphingomyelinase-Like Enzyme in Ixodes scapularis Tick Saliva Drives Host CD4+ T cells to Express IL-4

PubMed Central

Alarcon-Chaidez, F. J.; Boppana, V. D.; Hagymasi, A.T.; Adler, A. J.; Wikel, S. K.

2009-01-01

Tick feeding modulates host immune responses. Tick-induced skewing of host CD4+ T cells towards a Th2 cytokine profile facilitates transmission of tick-borne pathogens that would otherwise be neutralized by Th1 cytokines. Tick-derived factors that drive this Th2 response have not previously been characterized. In the current study, we examined an I. scapularis cDNA library prepared at 18-24 hours of feeding and identified and expressed a tick gene with homology to Loxosceles spider venom proteins with sphingomyelinase activity. This I. scapularis sphingomyelinase-like (IsSMase) protein is a Mg+2-dependent, neutral (pH 7.4) form of sphingomyelinase. Significantly, in an in vivo TCR transgenic adoptive transfer assay IsSMase programmed host CD4+ T cells to express the hallmark Th2 effector cytokine IL-4. IsSMase appears to directly program host CD4 T cell IL-4 expression (as opposed to its metabolic by-products) because induced IL-4 expression was not altered when enzymatic activity was neutralized. TCR transgenic CD4 T cell proliferation (CFSE-dilution) was also significantly increased by IsSMase. Furthermore, a Th2 response is superimposed onto a virally-primed Th1 response by IsSMase. Thus, IsSMase is the first identified tick molecule capable of programming host CD4+ T cells to express IL-4. PMID:19292772

Intracellular survival and vascular cell-to-cell transmission of Porphyromonas gingivalis

PubMed Central

Li, Ling; Michel, Raynald; Cohen, Joshua; DeCarlo, Arthur; Kozarov, Emil

2008-01-01

Background Porphyromonas gingivalis is associated with periodontal disease and invades different cell types including epithelial, endothelial and smooth muscle cells. In addition to P. gingivalis DNA, we have previously identified live invasive bacteria in atheromatous tissue. However, the mechanism of persistence of this organism in vascular tissues remains unclear. Therefore, the objective of this study was to analyze the ability of intracellular P. gingivalis to persist for extended periods of time, transmit to and possibly replicate in different cell types. Results Using antibiotic protection assays, immunofluorescent and laser confocal microscopy, we found that after a prolonged intracellular phase, while P. gingivalis can still be detected by immunostaining, the intracellular organisms lose their ability to be recovered in vitro. Surprisingly however, intracellular P. gingivalis could be recovered in vitro upon co incubation with fresh vascular host cells. We then demonstrated that the organism was able to exit the initially infected host cells, then enter and multiply in new host cells. Further, we found that cell-to-cell contact increased the transmission rate but was not required for transmission. Finally, we found that the invasion of new host cells allowed P. gingivalis to increase its numbers. Conclusion Our results suggest that the persistence of vascular tissue-embedded P. gingivalis is due to its ability to transmit among different cell types. This is the first communication demonstrating the intercellular transmission as a likely mechanism converting latent intracellular bacteria from state of dormancy to a viable state allowing for persistence of an inflammatory pathogen in vascular tissue. PMID:18254977

The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence.

PubMed

Latgé, Jean-Paul; Beauvais, Anne; Chamilos, Georgios

2017-09-08

More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.

Novel Insights into Cell Entry of Emerging Human Pathogenic Arenaviruses.

PubMed

Fedeli, Chiara; Moreno, Héctor; Kunz, Stefan

2018-06-22

Viral hemorrhagic fevers caused by emerging RNA viruses of the Arenavirus family are among the most devastating human diseases. Climate change, global trade, and increasing urbanization promote the emergence and re-emergence of these human pathogenic viruses. Emerging pathogenic arenaviruses are of zoonotic origin and reservoir-to-human transmission is crucial for spillover into human populations. Host cell attachment and entry are the first and most fundamental steps of every virus infection and represent major barriers for zoonotic transmission. During host cell invasion, viruses critically depend on cellular factors, including receptors, co-receptors, and regulatory proteins of endocytosis. An in-depth understanding of the complex interaction of a virus with cellular factors implicated in host cell entry is therefore crucial to predict the risk of zoonotic transmission, define the tissue tropism, and assess disease potential. Over the past years, investigation of the molecular and cellular mechanisms underlying host cell invasion of human pathogenic arenaviruses uncovered remarkable viral strategies and provided novel insights into viral adaptation and virus-host co-evolution that will be covered in the present review. Copyright © 2018. Published by Elsevier Ltd.

Bacterial immunostat: Mycobacterium tuberculosis lipids and their role in the host immune response.

PubMed

Queiroz, Adriano; Riley, Lee W

2017-01-01

The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans) trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose) appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

The activation and suppression of plant innate immunity by parasitic nematodes.

PubMed

Goverse, Aska; Smant, Geert

2014-01-01

Plant-parasitic nematodes engage in prolonged and intimate relationships with their host plants, often involving complex alterations in host cell morphology and function. It is puzzling how nematodes can achieve this, seemingly without activating the innate immune system of their hosts. Secretions released by infective juvenile nematodes are thought to be crucial for host invasion, for nematode migration inside plants, and for feeding on host cells. In the past, much of the research focused on the manipulation of developmental pathways in host plants by plant-parasitic nematodes. However, recent findings demonstrate that plant-parasitic nematodes also deliver effectors into the apoplast and cytoplasm of host cells to suppress plant defense responses. In this review, we describe the current insights in the molecular and cellular mechanisms underlying the activation and suppression of host innate immunity by plant-parasitic nematodes along seven critical evolutionary and developmental transitions in plant parasitism.

Structural Insights into Helicobacter pylori Cag Protein Interactions with Host Cell Factors.

PubMed

Bergé, Célia; Terradot, Laurent

2017-01-01

The most virulent strains of Helicobacter pylori carry a genomic island (cagPAI) containing a set of 27-31 genes. The encoded proteins assemble a syringe-like apparatus to inject the cytotoxin-associated gene A (CagA) protein into gastric cells. This molecular device belongs to the type IV secretion system (T4SS) family albeit with unique characteristics. The cagPAI-encoded T4SS and its effector protein CagA have an intricate relationship with the host cell, with multiple interactions that only start to be deciphered from a structural point of view. On the one hand, the major roles of the interactions between CagL and CagA (and perhaps CagI and CagY) and host cell factors are to facilitate H. pylori adhesion and to mediate the injection of the CagA oncoprotein. On the other hand, CagA interactions with host cell partners interfere with cellular pathways to subvert cell defences and to promote H. pylori infection. Although a clear mechanism for CagA translocation is still lacking, the structural definition of CagA and CagL domains involved in interactions with signalling proteins are progressively coming to light. In this chapter, we will focus on the structural aspects of Cag protein interactions with host cell molecules, critical molecular events precluding H. pylori-mediated gastric cancer development.

Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging

NASA Astrophysics Data System (ADS)

Naemat, Abida; Elsheikha, Hany M.; Boitor, Radu A.; Notingher, Ioan

2016-02-01

This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of L-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting L-Phe(D8) from host cells as soon as it invades the cell. L-Phe(D8) from the host cell completely replaces the L-Phe within T. gondii tachyzoites 7-9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5-1.6 × 104 molecules/s. On the other hand, extracellular tachyzoites were not able to consume L-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell.

Protective role of host aquaporin 6 against Hazara virus, a model for Crimean-Congo hemorrhagic fever virus infection.

PubMed

Molinas, Andrea; Mirazimi, Ali; Holm, Angelika; Loitto, Vesa M; Magnusson, Karl-Eric; Vikström, Elena

2016-04-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus-host interactions, which may lead to less severe outcomes of an infection. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Studies of intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes (PMNS). I. Role of contact inhibition of locomotion

PubMed Central

1975-01-01

Intercellular invasion is the active migration of cells on one type into the interiors of tissues composed of cells of dissimilar cell types. Contact paralysis of locomotion is the cessation of forward extension of the pseudopods of a cell as a result of its collision with another cell. One hypothesis to account for intercellular invasion proposes that a necessary condition for a cell type to be invasive to a given host tissue is that it lack contact paralysis of locomotion during collision with cells of that host tissue. The hypothesis has been tested using rabbit peritoneal neutrophil granulocytes (PMNs) as the invasive cell type and chick embryo fibroblasts as the host tissue. In organ culture, PMNs rapidly invade aggregates of fibroblasts. The behavior of the pseudopods of PMNs during collision with fibroblasts was analyzed for contact paralysis by a study of time-lapse films of cells in mixed monolayer culture. In monolayer culture, PMNs show little sign of paralysis of the pseudopods upon collision with fibroblasts and thus conform in their behavior to that predicted by the hypothesis. PMID:1092702

Helminth Infections: Recognition and Modulation of the Immune Response by Innate Immune Cells

PubMed Central

Motran, Claudia Cristina; Silvane, Leonardo; Chiapello, Laura Silvina; Theumer, Martin Gustavo; Ambrosio, Laura Fernanda; Volpini, Ximena; Celias, Daiana Pamela; Cervi, Laura

2018-01-01

The survival of helminths in the host over long periods of time is the result of a process of adaptation or dynamic co-evolution between the host and the parasite. However, infection with helminth parasites causes damage to the host tissues producing the release of danger signals that induce the recruitment of various cells, including innate immune cells such as macrophages (Mo), dendritic cells (DCs), eosinophils, basophils, and mast cells. In this scenario, these cells are able to secrete soluble factors, which orchestrate immune effector mechanisms that depend on the different niches these parasites inhabit. Here, we focus on recent advances in the knowledge of excretory-secretory products (ESP), resulting from helminth recognition by DCs and Mo. Phagocytes and other cells types such as innate lymphocyte T cells 2 (ILC2), when activated by ESP, participate in an intricate cytokine network to generate innate and adaptive Th2 responses. In this review, we also discuss the mechanisms of innate immune cell-induced parasite killing and the tissue repair necessary to assure helminth survival over long periods of time. PMID:29670630

Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen

PubMed Central

Ene, Iuliana V; Adya, Ashok K; Wehmeier, Silvia; Brand, Alexandra C; MacCallum, Donna M; Gow, Neil A R; Brown, Alistair J P

2012-01-01

The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C. albicans stress adaptation have generally been performed on glucose-grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C. albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C. albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose- and lactate-grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C. albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C. albicans infections. PMID:22587014

Evasion of Early Antiviral Responses by Herpes Simplex Viruses

PubMed Central

Suazo, Paula A.; Ibañez, Francisco J.; Retamal-Díaz, Angello R.; Paz-Fiblas, Marysol V.; Bueno, Susan M.; Kalergis, Alexis M.; González, Pablo A.

2015-01-01

Besides overcoming physical constraints, such as extreme temperatures, reduced humidity, elevated pressure, and natural predators, human pathogens further need to overcome an arsenal of antimicrobial components evolved by the host to limit infection, replication and optimally, reinfection. Herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) infect humans at a high frequency and persist within the host for life by establishing latency in neurons. To gain access to these cells, herpes simplex viruses (HSVs) must replicate and block immediate host antiviral responses elicited by epithelial cells and innate immune components early after infection. During these processes, infected and noninfected neighboring cells, as well as tissue-resident and patrolling immune cells, will sense viral components and cell-associated danger signals and secrete soluble mediators. While type-I interferons aim at limiting virus spread, cytokines and chemokines will modulate resident and incoming immune cells. In this paper, we discuss recent findings relative to the early steps taking place during HSV infection and replication. Further, we discuss how HSVs evade detection by host cells and the molecular mechanisms evolved by these viruses to circumvent early antiviral mechanisms, ultimately leading to neuron infection and the establishment of latency. PMID:25918478

Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture.

PubMed

Boxell, Annika; Hijjawi, Nawal; Monis, Paul; Ryan, Una

2008-09-01

The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptosporidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture; antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes; Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6' diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages.

Obtaining control of cell surface functionalizations via Pre-targeting and Supramolecular host guest interactions

NASA Astrophysics Data System (ADS)

Rood, Mark T. M.; Spa, Silvia J.; Welling, Mick M.; Ten Hove, Jan Bart; van Willigen, Danny M.; Buckle, Tessa; Velders, Aldrik H.; van Leeuwen, Fijs W. B.

2017-01-01

The use of mammalian cells for therapeutic applications is finding its way into modern medicine. However, modification or “training” of cells to make them suitable for a specific application remains complex. By envisioning a chemical toolbox that enables specific, but straight-forward and generic cellular functionalization, we investigated how membrane-receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β-cyclodextrin (CD) and adamantane (Ad). The feasibility of this approach was studied in cells with membranous overexpression of the chemokine receptor 4 (CXCR4). By combining specific targeting of CXCR4, using an adamantane (Ad)-functionalized Ac-TZ14011 peptide (guest; KD = 56 nM), with multivalent host molecules that entailed fluorescent β-CD-Poly(isobutylene-alt-maleic-anhydride)-polymers with different fluorescent colors and number of functionalities, host-guest cell-surface modifications could be studied in detail. A second set of Ad-functionalized entities enabled introduction of additional surface functionalities. In addition, the attraction between CD and Ad could be used to drive cell-cell interactions. Combined we have shown that supramolecular interactions, that are based on specific targeting of an overexpressed membrane-receptor, allow specific and stable, yet reversible, surface functionalization of viable cells and how this approach can be used to influence the interaction between cells and their surroundings.

Adjustment of Host Cells for Accommodation of Symbiotic Bacteria: Vacuole Defunctionalization, HOPS Suppression, and TIP1g Retargeting in Medicago[C][W][OPEN

PubMed Central

Gavrin, Aleksandr; Kaiser, Brent N.; Geiger, Dietmar; Tyerman, Stephen D.; Wen, Zhengyu; Bisseling, Ton; Fedorova, Elena E.

2014-01-01

In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria. PMID:25217511

Innate Immune Responses of Bat and Human Cells to Filoviruses: Commonalities and Distinctions

PubMed Central

Kuzmin, Ivan V.; Schwarz, Toni M.; Ilinykh, Philipp A.; Jordan, Ingo; Ksiazek, Thomas G.; Sachidanandam, Ravi; Basler, Christopher F.

2017-01-01

ABSTRACT Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (Rousettus aegyptiacus); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFN-α and IFN-β, IFN-γ was demonstrated to control filovirus infection in bat cells but not in human cells, suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which were detected in human cells ectopically expressing bat IFN-α and IFN-β. As bat IFN-γ induced the type I IFN pathway, its antiviral effect is likely to be partially induced via cross talk. IMPORTANCE Bats serve as reservoirs for multiple emerging viruses, including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg or Ebola viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution. PMID:28122983

Innate Immune Responses of Bat and Human Cells to Filoviruses: Commonalities and Distinctions.

PubMed

Kuzmin, Ivan V; Schwarz, Toni M; Ilinykh, Philipp A; Jordan, Ingo; Ksiazek, Thomas G; Sachidanandam, Ravi; Basler, Christopher F; Bukreyev, Alexander

2017-04-15

Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat ( Rousettus aegyptiacus ); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFN-α and IFN-β, IFN-γ was demonstrated to control filovirus infection in bat cells but not in human cells, suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which were detected in human cells ectopically expressing bat IFN-α and IFN-β. As bat IFN-γ induced the type I IFN pathway, its antiviral effect is likely to be partially induced via cross talk. IMPORTANCE Bats serve as reservoirs for multiple emerging viruses, including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg or Ebola viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution. Copyright © 2017 American Society for Microbiology.

Host cells and methods for producing 3-methyl-2-buten-1-ol, 3-methyl-3-buten-1-ol, and 3-methyl-butan-1-ol

DOEpatents

Chou, Howard H [Berkeley, CA; Keasling, Jay D [Berkeley, CA

2011-07-26

The invention provides for a method for producing a 5-carbon alcohol in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses a first enzyme capable of catalyzing the dephosphorylation of an isopentenyl pyrophosphate (IPP) or dimethylallyl diphosphate (DMAPP), such as a Bacillus subtilis phosphatase (YhfR), under a suitable condition so that 5-carbon alcohol is 3-methyl-2-buten-1-ol and/or 3-methyl-3-buten-1-ol is produced. Optionally, the host cell may further comprise a second enzyme capable of reducing a 3-methyl-2-buten-1-ol to 3-methyl-butan-1-ol, such as a reductase.

Host Cell Responses to Persistent Mycoplasmas - Different Stages in Infection of HeLa Cells with Mycoplasma hominis

PubMed Central

Hopfe, Miriam; Deenen, René; Degrandi, Daniel; Köhrer, Karl; Henrich, Birgit

2013-01-01

Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen. PMID:23326599

Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ.

PubMed

Jeong, Kwang Cheol; Sexton, Jessica A; Vogel, Joseph P

2015-03-01

Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.

Differential Effect of MyD88 Signal in Donor T Cells on Graft-versus-Leukemia Effect and Graft-versus-Host Disease after Experimental Allogeneic Stem Cell Transplantation.

PubMed

Lim, Ji-Young; Ryu, Da-Bin; Lee, Sung-Eun; Park, Gyeongsin; Choi, Eun Young; Min, Chang-Ki

2015-11-01

Despite the presence of toll like receptor (TLR) expression in conventional TCRαβ T cells, the direct role of TLR signaling via myeloid differentiation factor 88 (MyD88) within T lymphocytes on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT) remains unknown. In the allo-SCT model of C57BL/6 (H-2(b)) → B6D2F1 (H-2(b/d)), recipients received transplants of wild type (WT) T-cell-depleted (TCD) bone marrow (BM) and splenic T cells from either WT or MyD88 deficient (MyD88KO) donors. Host-type (H-2(d)) P815 mastocytoma or L1210 leukemia cells were injected either subcutaneously or intravenously to generate a GVHD/GVL model. Allogeneic recipients of MyD88KO T cells demonstrated a greater tumor growth without attenuation of GVHD severity. Moreover, GVHD-induced GVL effect, caused by increasing the conditioning intensity was also not observed in the recipients of MyD88KO T cells. In vitro, the absence of MyD88 in T cells resulted in defective cytolytic activity to tumor targets with reduced ability to produce IFN-γ or granzyme B, which are known to critical for the GVL effect. However, donor T cell expansion with effector and memory T-cell differentiation were more enhanced in GVHD hosts of MyD88KO T cells. Recipients of MyD88KO T cells experienced greater expansion of Foxp3- and IL4-expressing T cells with reduced INF-γ producing T cells in the spleen and tumor-draining lymph nodes early after transplantation. Taken together, these results highlight a differential role for MyD88 deficiency on donor T-cells, with decreased GVL effect without attenuation of the GVHD severity after experimental allo-SCT.

Dualtropic CXCR6/CCR5 Simian Immunodeficiency Virus (SIV) Infection of Sooty Mangabey Primary Lymphocytes: Distinct Coreceptor Use in Natural versus Pathogenic Hosts of SIV.

PubMed

Elliott, Sarah T C; Wetzel, Katherine S; Francella, Nicholas; Bryan, Steven; Romero, Dino C; Riddick, Nadeene E; Shaheen, Farida; Vanderford, Thomas; Derdeyn, Cynthia A; Silvestri, Guido; Paiardini, Mirko; Collman, Ronald G

2015-09-01

Natural-host sooty mangabeys (SM) infected with simian immunodeficiency virus (SIV) exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4(+) T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which have been linked to low expression of the canonical SIV entry receptor CCR5 on CD4(+) T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by SIV in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIVsmm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIV infection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4(+) T cell subset targeting compared with that in nonnatural hosts, enabling SIV to maintain high-level replication without leading to widespread CD4(+) T cell loss. Natural hosts of SIV, such as sooty mangabeys, sustain high viral loads but do not develop disease, while nonnatural hosts, like rhesus macaques, develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5, suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may redirect the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of nonessential cells and limit infection of critical cells in natural hosts, thus contributing to benign outcome of infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dualtropic CXCR6/CCR5 Simian Immunodeficiency Virus (SIV) Infection of Sooty Mangabey Primary Lymphocytes: Distinct Coreceptor Use in Natural versus Pathogenic Hosts of SIV

PubMed Central

Elliott, Sarah T. C.; Wetzel, Katherine S.; Francella, Nicholas; Bryan, Steven; Romero, Dino C.; Riddick, Nadeene E.; Shaheen, Farida; Vanderford, Thomas; Derdeyn, Cynthia A.; Silvestri, Guido; Paiardini, Mirko

2015-01-01

ABSTRACT Natural-host sooty mangabeys (SM) infected with simian immunodeficiency virus (SIV) exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4+ T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which have been linked to low expression of the canonical SIV entry receptor CCR5 on CD4+ T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by SIV in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIVsmm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIV infection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4+ T cell subset targeting compared with that in nonnatural hosts, enabling SIV to maintain high-level replication without leading to widespread CD4+ T cell loss. IMPORTANCE Natural hosts of SIV, such as sooty mangabeys, sustain high viral loads but do not develop disease, while nonnatural hosts, like rhesus macaques, develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5, suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may redirect the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of nonessential cells and limit infection of critical cells in natural hosts, thus contributing to benign outcome of infection. PMID:26109719

Origins of endothelial and osteogenic cells in the subcutaneous collagen gel implant.

PubMed

Bilic-Curcic, I; Kalajzic, Z; Wang, L; Rowe, D W

2005-11-01

The interdependent relationship between vascular endothelial cells and osteoblasts during bone formation and fracture healing has been long appreciated. This paper reports a heterotopic implant model using FGF-2-expanded bone marrow stromal cells (BMSC) derived from Tie2eGFP (endothelial marker) and pOBCol3.6GFPcyan or topaz (early osteoblast marker) transgenic mice to appreciate the host/donor relationships of cells participating in the process of heterotopic bone formation. The study included various combinations of Tie2eGFP and pOBCol3.6GFPcyan and topaz transgenics as BMSC or whole bone marrow (WBM) donors and also as recipients. Rat tail collagen was used as a carrier of donor cells and implantation was done in lethally irradiated mice rescued with WBM injection. Development of ossicles in the implants was followed weekly during the 4- to 5-week long post-implantation period. By 4-5 weeks after total body irradiation (TBI) and implantation, a well-formed bone spicule had developed that was invested with bone marrow. Experiments showed absolute dominance of donor-derived cells in the formation of endothelial-lined vessels inside the implants as well as the marrow stromal-derived osteogenic cells. Host-derived fibroblasts and osteogenic cells were confined to the fibrous capsule surrounding the implant. In addition, cells lining the endosteal surface of newly formed marrow space carrying a pOBCol3.6GFP marker were observed that were contributed by WBM donor cells and the host. Thus, FGF-2-expanded BMSC appear to be a source of endothelial and osteogenic progenitor cells capable of eliciting heterotopic bone formation independent of cells from the host. This model should be useful for understanding the interactions between these two cell types that control osteogenic differentiation in vivo.

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells

PubMed Central

Bacchetta, Rosa; Lucarelli, Barbarella; Sartirana, Claudia; Gregori, Silvia; Lupo Stanghellini, Maria T.; Miqueu, Patrick; Tomiuk, Stefan; Hernandez-Fuentes, Maria; Gianolini, Monica E.; Greco, Raffaella; Bernardi, Massimo; Zappone, Elisabetta; Rossini, Silvano; Janssen, Uwe; Ambrosi, Alessandro; Salomoni, Monica; Peccatori, Jacopo; Ciceri, Fabio; Roncarolo, Maria-Grazia

2013-01-01

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders. PMID:24550909

Tacrolimus and Methotrexate With or Without Sirolimus in Preventing Graft-Versus-Host Disease in Young Patients Undergoing Donor Stem Cell Transplant for Acute Lymphoblastic Leukemia in Complete Remission

ClinicalTrials.gov

2016-12-16

B-cell Childhood Acute Lymphoblastic Leukemia; Childhood Acute Lymphoblastic Leukemia in Remission; Graft Versus Host Disease; L1 Childhood Acute Lymphoblastic Leukemia; L2 Childhood Acute Lymphoblastic Leukemia; T-cell Childhood Acute Lymphoblastic Leukemia

S1P dependent inter organ trafficking of group 2 innate lymphoid cells suppots host defense

USDA-ARS?s Scientific Manuscript database

Innate lymphoid cells (ILCs) are considered to be the innate counterparts of adaptive T lymphocytes and play important roles in host defense, tissue repair, metabolic homeostasis, and inflammatory diseases. ILCs are generally thought of as tissue-resident cells, but whether ILCs strictly behave in a...

Mesenchymal stromal cells in the antimicrobial host response of hematopoietic stem cell recipients with graft-versus-host disease--friends or foes?

PubMed

Balan, A; Lucchini, G; Schmidt, S; Schneider, A; Tramsen, L; Kuçi, S; Meisel, R; Bader, P; Lehrnbecher, T

2014-10-01

Mesenchymal stromal cells (MSCs) are multipotent cells, which exhibit broad immunosuppressive activities. Moreover, they may be administered irrespectively of human leukocyte antigen (HLA) compatibility, without inducing life-threatening immunological reactions, as they express no HLA class II and limited HLA class I antigens under resting conditions. These characteristics have made MSC an appealing candidate for cell therapy after hematopoietic stem cell transplantation (HSCT), for example, for treatment of graft-versus-host disease (GvHD) or for graft rejection prevention/treatment in allogeneic HSCT recipients. Unfortunately, information regarding the effect of MSC infusion on the host response to infectious agents is scarce, and study results on infectious complications in patients receiving MSC are conflicting. The present review focuses on the available data from in vitro studies and animal models regarding the interaction of MSC with bacterial, viral and fungal pathogens. In a clinical part, we present the current information on infectious complications in allogeneic HSCT recipients who had received MSCs as prophylaxis or treatment of GvHD disease.

Cellstat--A continuous culture system of a bacteriophage for the study of the mutation rate and the selection process at the DNA level

NASA Astrophysics Data System (ADS)

Husimi, Yuzuru; Nishigaki, Koichi; Kinoshita, Yasunori; Tanaka, Toyosuke

1982-04-01

A bacteriophage is continuously cultured in the flow of the host bacterial cell under the control of a minicomputer. In the culture, the population of the noninfected cell is kept constant by the endogeneous regulation mechanism, so it is called the ''cellstat'' culture. Due to the high dilution rate of the host cell, the mutant cell cannot be selected in the cellstat. Therefore, the cellstat is suitable for the study of the mutation rate and the selection process of a bacteriophage under well-defined environmental conditions (including physiological condition of the host cell) without being interfered by host-cell mutations. Applications to coliphage fd, a secretion type phage, are shown as a measurement example. A chimera between fd and a plasmid pBR322 is cultured more than 100 h. The process of population changeovers by deletion mutants indicates that the deletion hot spots exist in this cloning vector and that this apparatus can be used also for testing instability of a recombinant DNA.

Persistence of viral infection despite similar killing efficacy of antiviral CD8(+) T cells during acute and chronic phases of infection.

PubMed

Ganusov, Vitaly V; Lukacher, Aron E; Byers, Anthony M

2010-09-15

Why some viruses establish chronic infections while others do not is poorly understood. One possibility is that the host's immune response is impaired during chronic infections and is unable to clear the virus from the host. In this report, we use a recently proposed framework to estimate the per capita killing efficacy of CD8(+) T cells, specific for the polyoma virus (PyV), which establishes a chronic infection in mice. Surprisingly, the estimated per cell killing efficacy of PyV-specific effector CD8(+) T cells during the acute phase of the infection was very similar to the efficacy of effector CD8(+) T cells specific to lymphocytic choriomeningitis virus (LCMV-Armstrong), which is cleared from the host. Our results suggest that persistence of PyV does not result from the generation of an inefficient PyV-specific CD8(+) T cell response, and that other host or viral factors are responsible for the ability of PyV to establish chronic infection. Copyright 2010 Elsevier Inc. All rights reserved.

The evolution of a mechanism of cell suicide.

PubMed

Blackstone, N W; Green, D R

1999-01-01

In the vertebrates, programmed cell death or apoptosis frequently involves the relocalization of mitochondrial cytochrome c to the cytoplasm. This prominent role in the regulation of apoptosis is in addition to the primary function of cytochrome c in the mitochondrial electron transport chain. These seemingly divergent roles become plausible when considering the symbiotic origin of the mitochondrion. Symbiosis involves conflicts between levels of selection, in this case between the primitive host cell and the protomitochondria. In an aerobic environment, selection on the protomitochondria may have favored routine manipulations of the host cell's phenotype using products and by-products of oxidative phosphorylation, in particular reactive oxygen species (ROS). Blocking the mitochondrial electron transport chain by removing cytochrome c enhances the production of ROS; thus cytochrome c release by protomitochondria may have altered the host cell's phenotype via enhanced ROS production. Subsequently, this signaling pathway may have been refined by selection so that cytochrome c itself became the trigger for changes in the host's phenotype. A mechanism of apoptosis in metazoans may thus be a vestige of evolutionary conflicts within the eukaryotic cell.

Imaging enhancement of malignancy by cyclophosphamide: surprising chemotherapy opposite effects

NASA Astrophysics Data System (ADS)

Yamauchi, Kensuke; Yang, Meng; Hayashi, Katsuhiro; Jiang, Ping; Xu, Mingxu; Yamamoto, Norio; Tsuchiya, Hiroyuki; Tomita, Katsuro; Moossa, A. R.; Bouvet, Michael; Hoffman, Robert M.

2008-02-01

Although side effects of cancer chemotherapy are well known, "opposite effects" of chemotherapy which enhance the malignancy of the treated cancer are not well understood. We have observed a number of steps of malignancy that are enhanced by chemotherapy pre-treatment of mice before transplantation of human tumor cells. The induction of intravascular proliferation, extravasation, and colony formation by cancer cells, critical steps of metastasis was enhanced by pretreatment of host mice with the commonly-used chemotherapy drug cyclophosphamide. Cyclophosphamide appears to interfere with a host process that inhibits intravascular proliferation, extravasation, and extravascular colony formation by at least some tumor cells. Cyclophosphamide does not directly affect the cancer cells since cyclophosphamide has been cleared by the time the cancer cells were injected. Without cyclophosphamide pretreatment, human colon cancer cells died quickly after injection in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours. The number of apoptotic cells rapidly increased within the portal vein within 12 hours of injection. However, when the host mice were pretreated with cyclophosphamide, the cancer cells survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the cancer cells. This review describes an important unexpected "opposite effects" of chemotherapy that enhances critical steps in malignancy rather than inhibiting them, suggesting that certain current approaches to cancer chemotherapy should be modified.

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

PubMed

Aye, Racheal; Mwirigi, Martin Kiogora; Frey, Joachim; Pilo, Paola; Jores, Joerg; Naessens, Jan

2015-02-07

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.

Mechanisms of Immune Evasion in Leishmaniasis

PubMed Central

Gupta, Gaurav; Oghumu, Steve; Satoskar, Abhay R.

2013-01-01

Diseases caused by Leishmania present a worldwide problem, and current therapeutic approaches are unable to achieve a sterile cure. Leishmania is able to persist in host cells by evading or exploiting host immune mechanisms. A thorough understanding of these mechanisms could lead to better strategies for effective management of Leishmania infections. Current research has focused on parasite modification of host cell signaling pathways, entry into phagocytic cells, and modulation of cytokine and chemokine profiles that alter immune cell activation and trafficking to sites of infection. Immuno-therapeutic approaches that target these mechanisms of immune evasion by Leishmania offer promising areas for preclinical and clinical research. PMID:23415155

How the study of Listeria monocytogenes has led to new concepts in biology.

PubMed

Rolhion, Nathalie; Cossart, Pascale

2017-06-01

The opportunistic intracellular bacterial pathogen Listeria monocytogenes has in 30 years emerged as an exceptional bacterial model system in infection biology. Research on this bacterium has provided considerable insight into how pathogenic bacteria adapt to mammalian hosts, invade eukaryotic cells, move intracellularly, interfere with host cell functions and disseminate within tissues. It also contributed to unveil features of normal host cell pathways and unsuspected functions of previously known cellular proteins. This review provides an updated overview of our knowledge on this pathogen. In many examples, findings on L. monocytogenes provided the basis for new concepts in bacterial regulation, cell biology and infection processes.

Host-Toxoplasma gondii Coadaptation Leads to Fine Tuning of the Immune Response.

PubMed

Brasil, Thaís Rigueti; Freire-de-Lima, Celio Geraldo; Morrot, Alexandre; Vetö Arnholdt, Andrea Cristina

2017-01-01

Toxoplasma gondii has successfully developed strategies to evade host's immune response and reach immune privileged sites, which remains in a controlled environment inside quiescent tissue cysts. In this review, we will approach several known mechanisms used by the parasite to modulate mainly the murine immune system at its favor. In what follows, we review recent findings revealing interference of host's cell autonomous immunity and cell signaling, gene expression, apoptosis, and production of microbicide molecules such as nitric oxide and oxygen reactive species during parasite infection. Modulation of host's metalloproteinases of extracellular matrix is also discussed. These immune evasion strategies are determinant to parasite dissemination throughout the host taking advantage of cells from the immune system to reach brain and retina, crossing crucial hosts' barriers.

Yersinia versus host immunity: how a pathogen evades or triggers a protective response.

PubMed

Chung, Lawton K; Bliska, James B

2016-02-01

The human pathogenic Yersinia species cause diseases that represent a significant source of morbidity and mortality. Despite this, specific mechanisms underlying Yersinia pathogenesis and protective host responses remain poorly understood. Recent studies have shown that Yersinia disrupt cell death pathways, perturb inflammatory processes and exploit immune cells to promote disease. The ensuing host responses following Yersinia infection include coordination of innate and adaptive immune responses in an attempt to control bacterial replication. Here, we highlight current advances in our understanding of the interactions between the pathogenic yersiniae and host cells, as well as the protective host responses mobilized to counteract these pathogens. Together, these studies enhance our understanding of Yersinia pathogenesis and highlight the ongoing battle between host and microbe. Copyright © 2015 Elsevier Ltd. All rights reserved.

T-cell and natural killer cell therapies for hematologic malignancies after hematopoietic stem cell transplantation: enhancing the graft-versus-leukemia effect

PubMed Central

Cruz, C. Russell; Bollard, Catherine M.

2015-01-01

Hematopoietic stem cell transplantation has revolutionized the treatment of hematologic malignancies, but infection, graft-versus-host disease and relapse are still important problems. Calcineurin inhibitors, T-cell depletion strategies, and immunomodulators have helped to prevent graft-versus-host disease, but have a negative impact on the graft-versus-leukemia effect. T cells and natural killer cells are both thought to be important in the graft-versus-leukemia effect, and both cell types are amenable to ex vivo manipulation and clinical manufacture, making them versatile immunotherapeutics. We provide an overview of these immunotherapeutic strategies following hematopoietic stem cell transplantation, with discussions centered on natural killer and T-cell biology. We discuss the contributions of each cell type to graft-versus-leukemia effects, as well as the current research directions in the field as related to adoptive cell therapy after hematopoietic stem cell transplantation. PMID:26034113

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

PubMed

McQuate, Sarah E; Young, Alexandra M; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S; Palmer, Amy E

2017-01-01

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. © 2016 John Wiley & Sons Ltd.

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

PubMed Central

McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less

Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

DOE PAGES

Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.; ...

2016-03-25

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less

Host cells and methods for producing diacid compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steen, Eric J.; Fortman, Jeffrey L.; Dietrich, Jeffrey A.

The present invention provides for a method of producing one or more fatty acid derived dicarboxylic acids in a genetically modified host cell which does not naturally produce the one or more derived fatty acid derived dicarboxylic acids. The invention provides for the biosynthesis of dicarboxylic acid ranging in length from C3 to C26. The host cell can be further modified to increase fatty acid production or export of the desired fatty acid derived compound, and/or decrease fatty acid storage or metabolism.

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts.

PubMed

Leiss, Lina; Mutlu, Ercan; Øyan, Anne; Yan, Tao; Tsinkalovsky, Oleg; Sleire, Linda; Petersen, Kjell; Rahman, Mohummad Aminur; Johannessen, Mireille; Mitra, Sidhartha S; Jacobsen, Hege K; Talasila, Krishna M; Miletic, Hrvoje; Jonassen, Inge; Li, Xingang; Brons, Nicolaas H; Kalland, Karl-Henning; Wang, Jian; Enger, Per Øyvind

2017-02-07

Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b + immune and CD31 + endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.

The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts

PubMed Central

Wagner, Daniel S.; Delk, Nikki A.; Lukianova-Hleb, Ekaterina Y.; Hafner, Jason H.; Farach-Carson, Mary C.; Lapotko, Dmitri O.

2010-01-01

Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host. PMID:20630586

The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts.

PubMed

Wagner, Daniel S; Delk, Nikki A; Lukianova-Hleb, Ekaterina Y; Hafner, Jason H; Farach-Carson, Mary C; Lapotko, Dmitri O

2010-10-01

Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host. 2010 Elsevier Ltd. All rights reserved.

Method of forming a package for MEMS-based fuel cell

DOEpatents

Morse, Jeffrey D; Jankowski, Alan F

2013-05-21

A MEMS-based fuel cell package and method thereof is disclosed. The fuel cell package comprises seven layers: (1) a sub-package fuel reservoir interface layer, (2) an anode manifold support layer, (3) a fuel/anode manifold and resistive heater layer, (4) a Thick Film Microporous Flow Host Structure layer containing a fuel cell, (5) an air manifold layer, (6) a cathode manifold support structure layer, and (7) a cap. Fuel cell packages with more than one fuel cell are formed by positioning stacks of these layers in series and/or parallel. The fuel cell package materials such as a molded plastic or a ceramic green tape material can be patterned, aligned and stacked to form three dimensional microfluidic channels that provide electrical feedthroughs from various layers which are bonded together and mechanically support a MEMS-based miniature fuel cell. The package incorporates resistive heating elements to control the temperature of the fuel cell stack. The package is fired to form a bond between the layers and one or more microporous flow host structures containing fuel cells are inserted within the Thick Film Microporous Flow Host Structure layer of the package.

Method of forming a package for mems-based fuel cell

DOEpatents

Morse, Jeffrey D.; Jankowski, Alan F.

2004-11-23

A MEMS-based fuel cell package and method thereof is disclosed. The fuel cell package comprises seven layers: (1) a sub-package fuel reservoir interface layer, (2) an anode manifold support layer, (3) a fuel/anode manifold and resistive heater layer, (4) a Thick Film Microporous Flow Host Structure layer containing a fuel cell, (5) an air manifold layer, (6) a cathode manifold support structure layer, and (7) a cap. Fuel cell packages with more than one fuel cell are formed by positioning stacks of these layers in series and/or parallel. The fuel cell package materials such as a molded plastic or a ceramic green tape material can be patterned, aligned and stacked to form three dimensional microfluidic channels that provide electrical feedthroughs from various layers which are bonded together and mechanically support a MEMOS-based miniature fuel cell. The package incorporates resistive heating elements to control the temperature of the fuel cell stack. The package is fired to form a bond between the layers and one or more microporous flow host structures containing fuel cells are inserted within the Thick Film Microporous Flow Host Structure layer of the package.

Ecological Therapy for Cancer: Defining Tumors Using an Ecosystem Paradigm Suggests New Opportunities for Novel Cancer Treatments1

PubMed Central

Pienta, Kenneth J; McGregor, Natalie; Axelrod, Robert; Axelrod, David E

2008-01-01

We propose that there is an opportunity to devise new cancer therapies based on the recognition that tumors have properties of ecological systems. Traditionally, localized treatment has targeted the cancer cells directly by removing them (surgery) or killing them (chemotherapy and radiation). These modes of therapy have not always been effective because many tumors recur after these therapies, either because not all of the cells are killed (local recurrence) or because the cancer cells had already escaped the primary tumor environment (distant recurrence). There has been an increasing recognition that the tumor microenvironment contains host noncancer cells in addition to cancer cells, interacting in a dynamic fashion over time. The cancer cells compete and/or cooperate with nontumor cells, and the cancer cells may compete and/or cooperate with each other. It has been demonstrated that these interactions can alter the genotype and phenotype of the host cells as well as the cancer cells. The interaction of these cancer and host cells to remodel the normal host organ microenvironment may best be conceptualized as an evolving ecosystem. In classic terms, an ecosystem describes the physical and biological components of an environment in relation to each other as a unit. Here, we review some properties of tumor microenvironments and ecological systems and indicate similarities between them. We propose that describing tumors as ecological systems defines new opportunities for novel cancer therapies and use the development of prostate cancer metastases as an example. We refer to this as “ecological therapy” for cancer. PMID:19043526

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

PubMed Central

Charpentier, Xavier; Gabay, Joëlle E.; Reyes, Moraima; Zhu, Jing W.; Weiss, Arthur; Shuman, Howard A.

2009-01-01

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions. PMID:19578436

Nature's Ballistic Missile.

PubMed

Robb, E J; Barron, G L

1982-12-17

The parasitic fungus Haptoglossa mirabilis infects its rotifer host by means of a gun-shaped attack cell. The anterior end of the cell is elongated to form a barrel; the wall at the mouth is invaginated deep into the cell to form a bore. A walled chamber at the base of the bore houses a complex, missile-like attack apparatus. The projectile is fired from the gun cell at high speed to accomplish initial penetration of the host.

The early interaction of Leishmania with macrophages and dendritic cells and its influence on the host immune response.

PubMed

Liu, Dong; Uzonna, Jude E

2012-01-01

The complicated interactions between Leishmania and the host antigen-presenting cells (APCs) have fundamental effects on the final outcome of the disease. Two major APCs, macrophages and dendritic cells (DCs), play critical roles in mediating resistance and susceptibility during Leishmania infection. Macrophages are the primary resident cell for Leishmania: they phagocytose and permit parasite proliferation. However, these cells are also the major effector cells to eliminate infection. The effective clearance of parasites by macrophages depends on activation of appropriate immune response, which is usually initiated by DCs. Here, we review the early interaction of APCs with Leishmania parasites and how these interactions profoundly impact on the ensuing adaptive immune response. We also discuss how the current knowledge will allow further refinement of our understanding of the interplay between Leishmania and its hosts that leads to resistance or susceptibility.

The early interaction of Leishmania with macrophages and dendritic cells and its influence on the host immune response

PubMed Central

Liu, Dong; Uzonna, Jude E.

2012-01-01

The complicated interactions between Leishmania and the host antigen-presenting cells (APCs) have fundamental effects on the final outcome of the disease. Two major APCs, macrophages and dendritic cells (DCs), play critical roles in mediating resistance and susceptibility during Leishmania infection. Macrophages are the primary resident cell for Leishmania: they phagocytose and permit parasite proliferation. However, these cells are also the major effector cells to eliminate infection. The effective clearance of parasites by macrophages depends on activation of appropriate immune response, which is usually initiated by DCs. Here, we review the early interaction of APCs with Leishmania parasites and how these interactions profoundly impact on the ensuing adaptive immune response. We also discuss how the current knowledge will allow further refinement of our understanding of the interplay between Leishmania and its hosts that leads to resistance or susceptibility. PMID:22919674

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis.

PubMed

Cherubin, Patrick; Quiñones, Beatriz; Teter, Ken

2018-02-06

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

Method and apparatus for calibrating multi-axis load cells in a dexterous robot

NASA Technical Reports Server (NTRS)

Wampler, II, Charles W. (Inventor); Platt, Jr., Robert J. (Inventor)

2012-01-01

A robotic system includes a dexterous robot having robotic joints, angle sensors adapted for measuring joint angles at a corresponding one of the joints, load cells for measuring a set of strain values imparted to a corresponding one of the load cells during a predetermined pose of the robot, and a host machine. The host machine is electrically connected to the load cells and angle sensors, and receives the joint angle values and strain values during the predetermined pose. The robot presses together mating pairs of load cells to form the poses. The host machine executes an algorithm to process the joint angles and strain values, and from the set of all calibration matrices that minimize error in force balance equations, selects the set of calibration matrices that is closest in a value to a pre-specified value. A method for calibrating the load cells via the algorithm is also provided.

Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth.

PubMed

Ishida, Kasumi; Matsuo, Junji; Yamamoto, Yoshimasa; Yamaguchi, Hiroyuki

2014-12-21

Pathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells. We first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A. We discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.

The effect of inhibition of host MreB on the infection of thermophilic phage GVE2 in high temperature environment

PubMed Central

Jin, Min; Chen, Yanjiang; Xu, Chenxi; Zhang, Xiaobo

2014-01-01

In eukaryotes, the manipulation of the host actin cytoskeleton is a necessary strategy for viral pathogens to invade host cells. Increasing evidence indicates that the actin homolog MreB of bacteria plays key roles in cell shape formation, cell polarity, cell wall biosynthesis, and chromosome segregation. However, the role of bacterial MreB in the bacteriophage infection is not extensively investigated. To address this issue, in this study, the MreB of thermophilic Geobacillus sp. E263 from a deep-sea hydrothermal field was characterized by inhibiting the MreB polymerization and subsequently evaluating the bacteriophage GVE2 infection. The results showed that the host MreB played important roles in the bacteriophage infection at high temperature. After the host cells were treated with small molecule drug A22 or MP265, the specific inhibitors of MreB polymerization, the adsorption of GVE2 and the replication of GVE2 genome were significantly repressed. The confocal microscopy data revealed that MreB facilitated the GVE2 infection by inducing the polar distribution of virions during the phage infection. Our study contributed novel information to understand the molecular events of the host in response to bacteriophage challenge and extended our knowledge about the host-virus interaction in deep-sea vent ecosystems. PMID:24769758

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

PubMed Central

Truyen, U; Parrish, C R

1992-01-01

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts. Images PMID:1323703

Do Viruses Exchange Genes across Superkingdoms of Life?

PubMed

Malik, Shahana S; Azem-E-Zahra, Syeda; Kim, Kyung Mo; Caetano-Anollés, Gustavo; Nasir, Arshan

2017-01-01

Viruses can be classified into archaeoviruses, bacterioviruses, and eukaryoviruses according to the taxonomy of the infected host. The host-constrained perception of viruses implies preference of genetic exchange between viruses and cellular organisms of their host superkingdoms and viral origins from host cells either via escape or reduction. However, viruses frequently establish non-lytic interactions with organisms and endogenize into the genomes of bacterial endosymbionts that reside in eukaryotic cells. Such interactions create opportunities for genetic exchange between viruses and organisms of non-host superkingdoms. Here, we take an atypical approach to revisit virus-cell interactions by first identifying protein fold structures in the proteomes of archaeoviruses, bacterioviruses, and eukaryoviruses and second by tracing their spread in the proteomes of superkingdoms Archaea, Bacteria, and Eukarya. The exercise quantified protein structural homologies between viruses and organisms of their host and non-host superkingdoms and revealed likely candidates for virus-to-cell and cell-to-virus gene transfers. Unexpected lifestyle-driven genetic affiliations between bacterioviruses and Eukarya and eukaryoviruses and Bacteria were also predicted in addition to a large cohort of protein folds that were universally shared by viral and cellular proteomes and virus-specific protein folds not detected in cellular proteomes. These protein folds provide unique insights into viral origins and evolution that are generally difficult to recover with traditional sequence alignment-dependent evolutionary analyses owing to the fast mutation rates of viral gene sequences.

Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

PubMed Central

Burns, John A; Zhang, Huanjia; Hill, Elizabeth; Kim, Eunsoo; Kerney, Ryan

2017-01-01

During embryonic development, cells of the green alga Oophila amblystomatis enter cells of the salamander Ambystoma maculatum forming an endosymbiosis. Here, using de novo dual-RNA seq, we compared the host salamander cells that harbored intracellular algae to those without algae and the algae inside the animal cells to those in the egg capsule. This two-by-two-way analysis revealed that intracellular algae exhibit hallmarks of cellular stress and undergo a striking metabolic shift from oxidative metabolism to fermentation. Culturing experiments with the alga showed that host glutamine may be utilized by the algal endosymbiont as a primary nitrogen source. Transcriptional changes in salamander cells suggest an innate immune response to the alga, with potential attenuation of NF-κB, and metabolic alterations indicative of modulation of insulin sensitivity. In stark contrast to its algal endosymbiont, the salamander cells did not exhibit major stress responses, suggesting that the host cell experience is neutral or beneficial. DOI: http://dx.doi.org/10.7554/eLife.22054.001 PMID:28462779

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

2012-01-01

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses.

PubMed

Choi, Dong Hoon; Kim, Kwang Soon; Yang, Se Hwan; Chung, Doo Hyun; Song, Boyeong; Sprent, Jonathan; Cho, Jae Ho; Sung, Young Chul

2011-12-15

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

Characterization and storage of malaria antigens: Fractionation of Plasmodium knowlesi-induced antigens of rhesus monkey erythrocyte membranes*

PubMed Central

Schmidt-Ullrich, R.; Wallach, D. F. H.; Lightholder, J.

1979-01-01

In order to characterize parasite-induced host cell membrane antigens, the plasma membranes of Plasmodium knowlesi-infected rhesus erythrocytes have been compared with those of normal red cells and purified schizonts by immunochemical and biochemical techniques. Host cell membranes and schizonts were separated by differential centrifugation following nitrogen decompression. Isolated schizonts were further fractionated into several subcellular compartments. Crossed-immune electrophoresis, against monkey anti-schizont serum, of Triton X-100-solubilized material identified 7 P. knowlesi-specific antigens, of which 4 could be detected only in the host cell membranes. These membranes also contained 3 proteins, with relative molecular masses of 55 000, 65 000 and 90 000 and isoelectric points at pH 4.5, 4.5 and 5.2, respectively, which are lacking in normal membranes. Pulse-chase experiments with (14C)-glucosamine showed that these parasite-induced host cell membrane components are glycoproteins. ImagesFig. 1Fig. 2 PMID:120762

γδ T cells in homeostasis and host defence of epithelial barrier tissues

PubMed Central

Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

2018-01-01

Epithelial surfaces line the body and provide a critical interface between the body and the external environment which is essential to maintaining the symbiotic relationship between the host and the microbiome. Tissue-resident epithelial γδ T cells represent a major T cell population in epithelia and are ideally positioned to perform barrier surveillance and aid in tissue homeostasis and repair. In this review we focus on the intraepithelial γδ compartment in the two largest epithelial tissues in the body, namely the epidermis and intestine, and provide a comprehensive overview of the crucial contributions of intraepithelial γδ cells at these sites to tissue integrity and repair, host homeostasis and host protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we address epithelia-specific butyrophilin-like molecules and touch upon their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires. PMID:28920588

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

PubMed

Kentner, David; Martano, Giuseppe; Callon, Morgane; Chiquet, Petra; Brodmann, Maj; Burton, Olga; Wahlander, Asa; Nanni, Paolo; Delmotte, Nathanaël; Grossmann, Jonas; Limenitakis, Julien; Schlapbach, Ralph; Kiefer, Patrick; Vorholt, Julia A; Hiller, Sebastian; Bumann, Dirk

2014-07-08

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

PubMed Central

Kentner, David; Martano, Giuseppe; Callon, Morgane; Chiquet, Petra; Brodmann, Maj; Burton, Olga; Wahlander, Asa; Nanni, Paolo; Delmotte, Nathanaël; Grossmann, Jonas; Limenitakis, Julien; Schlapbach, Ralph; Kiefer, Patrick; Vorholt, Julia A.; Hiller, Sebastian; Bumann, Dirk

2014-01-01

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease. PMID:24958876

Lymph Node Cellular and Viral Dynamics in Natural Hosts and Impact for HIV Cure Strategies.

PubMed

Huot, Nicolas; Bosinger, Steven E; Paiardini, Mirko; Reeves, R Keith; Müller-Trutwin, Michaela

2018-01-01

Combined antiretroviral therapies (cARTs) efficiently control HIV replication leading to undetectable viremia and drastic increases in lifespan of people living with HIV. However, cART does not cure HIV infection as virus persists in cellular and anatomical reservoirs, from which the virus generally rebounds soon after cART cessation. One major anatomical reservoir are lymph node (LN) follicles, where HIV persists through replication in follicular helper T cells and is also trapped by follicular dendritic cells. Natural hosts of SIV, such as African green monkeys and sooty mangabeys, generally do not progress to disease although displaying persistently high viremia. Strikingly, these hosts mount a strong control of viral replication in LN follicles shortly after peak viremia that lasts throughout infection. Herein, we discuss the potential interplay between viral control in LNs and the resolution of inflammation, which is characteristic for natural hosts. We furthermore detail the differences that exist between non-pathogenic SIV infection in natural hosts and pathogenic HIV/SIV infection in humans and macaques regarding virus target cells and replication dynamics in LNs. Several mechanisms have been proposed to be implicated in the strong control of viral replication in natural host's LNs, such as NK cell-mediated control, that will be reviewed here, together with lessons and limitations of in vivo cell depletion studies that have been performed in natural hosts. Finally, we discuss the impact that these insights on viral dynamics and host responses in LNs of natural hosts have for the development of strategies toward HIV cure.

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle.

PubMed

Gerold, Gisa; Bruening, Janina; Weigel, Bettina; Pietschmann, Thomas

2017-04-01

Protein-protein interactions govern biological functions in cells, in the extracellular milieu, and at the border between cells and extracellular space. Viruses are small intracellular parasites and thus rely on protein interactions to produce progeny inside host cells and to spread from cell to cell. Usage of host proteins by viruses can have severe consequences e.g. apoptosis, metabolic disequilibria, or altered cell proliferation and mobility. Understanding protein interactions during virus infection can thus educate us on viral infection and pathogenesis mechanisms. Moreover, it has led to important clinical translations, including the development of new therapeutic and vaccination strategies. Here, we will discuss protein interactions of members of the Flaviviridae family, which are small enveloped RNA viruses. Dengue virus, Zika virus and hepatitis C virus belong to the most prominent human pathogenic Flaviviridae With a genome of roughly ten kilobases encoding only ten viral proteins, Flaviviridae display intricate mechanisms to engage the host cell machinery for their purpose. In this review, we will highlight how dengue virus, hepatitis C virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, yellow fever virus, and Zika virus proteins engage host proteins and how this knowledge helps elucidate Flaviviridae infection. We will specifically address the protein composition of the virus particle as well as the protein interactions during virus entry, replication, particle assembly, and release from the host cell. Finally, we will give a perspective on future challenges in Flaviviridae interaction proteomics and why we believe these challenges should be met. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

PubMed

Siddique, Shahid; Radakovic, Zoran S; De La Torre, Carola M; Chronis, Demosthenis; Novák, Ondřej; Ramireddy, Eswarayya; Holbein, Julia; Matera, Christiane; Hütten, Marion; Gutbrod, Philipp; Anjam, Muhammad Shahzad; Rozanska, Elzbieta; Habash, Samer; Elashry, Abdelnaser; Sobczak, Miroslaw; Kakimoto, Tatsuo; Strnad, Miroslav; Schmülling, Thomas; Mitchum, Melissa G; Grundler, Florian M W

2015-10-13

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Mediators involved in the immunomodulatory effects of apoptotic cells.

PubMed

Saas, Philippe; Bonnefoy, Francis; Kury-Paulin, Stephanie; Kleinclauss, François; Perruche, Sylvain

2007-07-15

Immunomodulatory properties are attributed to apoptotic cells. These properties have been used to modulate allogeneic immune responses in experimental transplantation settings. In independent studies, apoptotic cell infusion has been shown to favor hematopoietic cell engraftment, to increase heart graft survival, and to delay the lethal onset of graft-versus-host disease (GVHD). The goal of this review was to discuss how apoptotic cell infusion interferes with graft rejection or host rejection (i.e., GVHD) and to focus on the potential mediators or "perpetuators" involved in apoptotic cell-induced immunomodulation. Particular emphasis on apoptotic cell phagocytosis, transforming growth factor (TGF)-beta secretion, and regulatory T cell induction was performed. Stimulating "naturally" immunosuppressive molecules (i.e., TGF-beta) or immunomodulatory cells ("alternatively-activated" macrophages, certain dendritic cell subsets, or regulatory T cells) in a physiological manner by using apoptotic cell infusion can be a promising way to induce tolerance.

Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity.

PubMed

Ogawa, Mikako; Tomita, Yusuke; Nakamura, Yuko; Lee, Min-Jung; Lee, Sunmin; Tomita, Saori; Nagaya, Tadanobu; Sato, Kazuhide; Yamauchi, Toyohiko; Iwai, Hidenao; Kumar, Abhishek; Haystead, Timothy; Shroff, Hari; Choyke, Peter L; Trepel, Jane B; Kobayashi, Hisataka

2017-02-07

Immunogenic cell death (ICD) is a form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Cancer cells killed via ICD can elicit antitumor immunity. ICD is efficiently induced by near-infrared photo-immunotherapy (NIR-PIT) that selectively kills target-cells on which antibody-photoabsorber conjugates bind and are activated by NIR light exposure. Advanced live cell microscopies showed that NIR-PIT caused rapid and irreversible damage to the cell membrane function leading to swelling and bursting, releasing intracellular components due to the influx of water into the cell. The process also induces relocation of ICD bio markers including calreticulin, Hsp70 and Hsp90 to the cell surface and the rapid release of immunogenic signals including ATP and HMGB1 followed by maturation of immature dendritic cells. Thus, NIR-PIT is a therapy that kills tumor cells by ICD, eliciting a host immune response against tumor.

Motility and more: the flagellum of Trypanosoma brucei

PubMed Central

Langousis, Gerasimos; Hill, Kent L.

2014-01-01

A central feature of trypanosome cell biology and life cycle is the parasite’s single flagellum, which is an essential and multifunctional organelle involved in cell propulsion, morphogenesis and cytokinesis. The flagellar membrane is also a specialized subdomain of the cell surface that harbors multiple parasite virulence factors with roles in signaling and host-parasite interactions. In this review, we discuss the structure, assembly and function of the trypanosome flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host-parasite interaction. PMID:24931043

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

PubMed

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

2016-01-01

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

PubMed Central

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

2016-01-01

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species. PMID:26901159

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1.

PubMed

Kuchipudi, Suresh V; Dunham, Stephen P; Nelli, Rahul; White, Gavin A; Coward, Vivien J; Slomka, Marek J; Brown, Ian H; Chang, Kin Chow

2012-01-01

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

PubMed Central

Gardner, Jameson K.; Herbst-Kralovetz, Melissa M.

2016-01-01

The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies. PMID:27834891

Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

PubMed Central

Liu, Yancheng; Tan, Shumin; Huang, Lu; Abramovitch, Robert B.; Rohde, Kyle H.; Zimmerman, Matthew D.; Chen, Chao; Dartois, Véronique; VanderVen, Brian C.

2016-01-01

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs. PMID:27114608

Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

PubMed

Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

2016-03-03

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Induction of innate immunity and its perturbation by influenza viruses.

PubMed

Goraya, Mohsan Ullah; Wang, Song; Munir, Muhammad; Chen, Ji-Long

2015-10-01

Influenza A viruses (IAV) are highly contagious pathogens causing dreadful losses to human and animal, around the globe. IAVs first interact with the host through epithelial cells, and the viral RNA containing a 5'-triphosphate group is thought to be the critical trigger for activation of effective innate immunity via pattern recognition receptors-dependent signaling pathways. These induced immune responses establish the antiviral state of the host for effective suppression of viral replication and enhancing viral clearance. However, IAVs have evolved a variety of mechanisms by which they can invade host cells, circumvent the host immune responses, and use the machineries of host cells to synthesize and transport their own components, which help them to establish a successful infection and replication. In this review, we will highlight the molecular mechanisms of how IAV infection stimulates the host innate immune system and strategies by which IAV evades host responses.

Struggle for space: viral extinction through competition for cells.

PubMed

Cuesta, José A; Aguirre, Jacobo; Capitán, José A; Manrubia, Susanna C

2011-01-14

The design of protocols to suppress the propagation of viral infections is an enduring enterprise, especially hindered by limited knowledge of the mechanisms leading to viral extinction. Here we report on infection extinction due to intraspecific competition to infect susceptible hosts. Beneficial mutations increase the production of viral progeny, while the host cell may develop defenses against infection. For an unlimited number of host cells, a feedback runaway coevolution between host resistance and progeny production occurs. However, physical space limits the advantage that the virus obtains from increasing offspring numbers; thus, infection clearance may result from an increase in host defenses beyond a finite threshold. Our results might be relevant to devise improved control strategies in environments with mobility constraints or different geometrical properties.

Herpesviruses and Their Host Cells: A Successful Liaison.

PubMed

Adler, Barbara; Sattler, Christine; Adler, Heiko

2017-03-01

During a long history of coevolution, herpesviruses have reached a fine-tuned balance with their hosts, allowing them to successfully persist and spread to new hosts without causing too much damage. Only under certain circumstances, as in neonates or immunocompromised individuals, they may cause serious diseases. The delicate balance between herpesviruses and their hosts results from interactions of a great variety of viral and cellular factors which together shape the tropism for a particular host, tissue, or cell. Understanding these interactions will provide insight into the viral life cycle and cell biology in general. Moreover, it will also facilitate comprehension of herpesvirus pathogenesis, enabling the development of new strategies to combat herpesviruses in cases where they cause disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Noncoding RNPs of viral origin.

PubMed

Steitz, Joan; Borah, Sumit; Cazalla, Demian; Fok, Victor; Lytle, Robin; Mitton-Fry, Rachel; Riley, Kasandra; Samji, Tasleem

2011-03-01

Like their host cells, many viruses produce noncoding (nc)RNAs. These show diversity with respect to time of expression during viral infection, length and structure, protein-binding partners and relative abundance compared with their host-cell counterparts. Viruses, with their limited genomic capacity, presumably evolve or acquire ncRNAs only if they selectively enhance the viral life cycle or assist the virus in combating the host's response to infection. Despite much effort, identifying the functions of viral ncRNAs has been extremely challenging. Recent technical advances and enhanced understanding of host-cell ncRNAs promise accelerated insights into the RNA warfare mounted by this fascinating class of RNPs.

[What makes a parasite "transforming"? Insights into cancer from the agents of an exotic pathology, Theileria spp].

PubMed

Cheeseman, K M; Weitzman, J B

2017-02-01

Theileria are obligate eukaryotic intracellular parasites of cattle. The diseases they cause, Tropical theileriosis and East Coast Fever, cause huge economic loss in East African, Mediterranean and central and South-East Asian countries. These apicomplexan parasites are the only intracellular eukaryotic parasites known to transform their host cell and represent a unique model to study host-parasite interactions and mechanisms of cancer onset.Here, we review how Theileria parasites induce transformation of their leukocyte host cell and discuss similarities with tumorigenesis. We describe how genomic innovation, epigenetic changes and hijacking of signal transductions enable a eukaryotic parasite to transform its host cell.

Acting on Actin: Rac and Rho Played by Yersinia.

PubMed

Aepfelbacher, Martin; Wolters, Manuel

2017-01-01

Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.

Energy transfer in “parasitic” cancer metabolism

PubMed Central

Martinez-Outschoorn, Ubaldo E; Pestell, Richard G; Howell, Anthony; Tykocinski, Mark L; Nagajyothi, Fnu; Machado, Fabiana S; Tanowitz, Herbert B

2011-01-01

It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extra-cellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy and aerobic glycolysis), which are then transferred to cancer cells, to fuel anabolic tumor growth. Stromal cell derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS), to produce ATP efficiently. However, “parasitic” energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via β-oxidation (β-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism via autophagy, mitophagy and lipolysis may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a “parasitic organism.” Hence, we propose the term “parasitic cancer metabolism” to describe this type of metabolic-coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and β-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can confer drug resistance. Importantly, this metabolic chemo-resistance is reversed by blocking OXPHOS in cancer cell mitochondria, with drugs like Metformin, a mitochondrial “poison.” In summary, parasitic cancer metabolism is achieved architecturally by dividing tumor tissue into at least two well-defined opposing “metabolic compartments:” catabolic and anabolic. PMID:22033146

The civRT operon is important for Campylobacter jejuni strain 81-176 host cell interactions through regulation of the formate dehydrogenase operon

USDA-ARS?s Scientific Manuscript database

C. jejuni colonizes the intestinal mucosa, and the severity of disease in different strains is correlated with host cell interaction and invasion. A microarray screen to identify genes differentially regulated during C. jejuni interaction with tissue culture cells revealed the up-regulation of a two...

Mast cell activators as novel immune regulators.

PubMed

Johnson-Weaver, Brandi; Choi, Hae Woong; Abraham, Soman N; Staats, Herman F

2018-05-26

Mast cells are an important cell type of the innate immune system that when activated, play a crucial role in generating protective innate host responses after bacterial and viral infection. Additionally, activated mast cells influence lymph node composition to regulate the induction of adaptive immune responses. The recognition that mast cells play a beneficial role in host responses to microbial infection and induction of adaptive immunity has provided the rationale to evaluate mast cell activators for use as antimicrobials or vaccine adjuvants. This review summarizes the role of mast cell activators in antimicrobial responses while also discussing the use of different classes of mast cell activators as potent vaccine adjuvants that enhance the induction of protective immune responses. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural characterization of the cell division cycle in Strigomonas culicis, an endosymbiont-bearing trypanosomatid.

PubMed

Brum, Felipe Lopes; Catta-Preta, Carolina Moura Costa; de Souza, Wanderley; Schenkman, Sergio; Elias, Maria Carolina; Motta, Maria Cristina Machado

2014-02-01

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

Isotope labeling of proteins in insect cells.

PubMed

Skora, Lukasz; Shrestha, Binesh; Gossert, Alvar D

2015-01-01

Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies. © 2015 Elsevier Inc. All rights reserved.

A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

DOE PAGES

Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

2017-01-27

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

Host cell interactome of PA protein of H5N1 influenza A virus in chicken cells.

PubMed

Wang, Qiao; Li, Qinghe; Liu, Ranran; Zheng, Maiqing; Wen, Jie; Zhao, Guiping

2016-03-16

Influenza A virus (IAV) heavily depends on viral-host protein interactions in order to replicate and spread. Identification of host factors that interact with viral proteins plays crucial roles in understanding the mechanism of IAV infection. Here we report the interaction landscape of H5N1 IAV PA protein in chicken cells through the use of affinity purification and mass spectrometry. PA protein was expressed in chicken cells and PA interacting complexes were captured by co-immunoprecipitation and analyzed by mass spectrometry. A total of 134 proteins were identified as PA-host interacting factors. Protein complexes including the minichromosome maintenance complex (MCM), 26S proteasome and the coat protein I (COPI) complex associated with PA in chicken cells, indicating the essential roles of these functional protein complexes during the course of IAV infection. Gene Ontology and pathway enrichment analysis both showed strong enrichment of PA interacting proteins in the category of DNA replication, covering genes such as PCNA, MCM2, MCM3, MCM4, MCM5 and MCM7. This study has uncovered the comprehensive interactome of H5N1 IAV PA protein in its chicken host and helps to establish the foundation for further investigation into the newly identified viral-host interactions. Influenza A virus (IAV) is a great threat to public health and avian production. However, the manner in which avian IAV recruits the host cellular machinery for replication and how the host antagonizes the IAV infection was previously poorly understood. Here we present the viral-host interactome of the H5N1 IAV PA protein and reveal the comprehensive association of host factors with PA. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanism of eliciting host immunity against cancer cells treated with silica-phthalocyanine-based near infrared photoimmunotherapy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kobayashi, Hisataka

2016-03-01

Near infrared (NIR) photoimmunotherapy (PIT) is a new type of molecularly-targeted cancer photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (MAb) targeting cancer-specific cell-surface molecules. When exposed to NIR light, the conjugate induces a highly-selective necrotic/ immunogenic cell death (ICD) only in receptor-positive, MAb-IR700-bound cancer cells. This cell death occurs as early as 1 minute after exposure to NIR light. Meanwhile, immediately adjacent receptor-negative cells including immune cells are unharmed. Therefore, we hypothesized that NIR-PIT could efficiently elicit host immunity against treated cancer cells. Three-dimensional dynamic quantitative phase contrast microscopy and selective plane illumination microscopy of tumor cells undergoing PIT showed rapid swelling in treated cells immediately after light exposure suggesting rapid water influx into cells, followed by irreversible morphologic changes such as bleb formation, and rupture of vesicles. Furthermore, biological markers of ICD including relocation of HSP70/90 and calreticulin, and release of ATP and High Mobility Group Box 1 (HMGB1), were clearly detected immediately after NIR-PIT. When NIR-PIT was performed in a mixture of cancer cells and immature dendritic cells, maturation of immature dendritic cells was strongly induced rapidly after NIR-PIT. In summary, NIR-PIT can induce necrotic/ immunogenic cell death that promotes rapid maturation of immature dendritic cells adjacent to dying cancer cells. Therefore, NIR-PIT could efficiently initiate host immune response against NIR-PIT treated cancer cells growing in patients.

Gammaherpesvirus Colonization of the Spleen Requires Lytic Replication in B Cells.

PubMed

Lawler, Clara; de Miranda, Marta Pires; May, Janet; Wyer, Orry; Simas, J Pedro; Stevenson, Philip G

2018-04-01

Gammaherpesviruses infect lymphocytes and cause lymphocytic cancers. Murid herpesvirus-4 (MuHV-4), Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus all infect B cells. Latent infection can spread by B cell recirculation and proliferation, but whether this alone achieves systemic infection is unclear. To test the need of MuHV-4 for lytic infection in B cells, we flanked its essential ORF50 lytic transactivator with loxP sites and then infected mice expressing B cell-specific Cre (CD19-Cre). The floxed virus replicated normally in Cre - mice. In CD19-Cre mice, nasal and lymph node infections were maintained; but there was little splenomegaly, and splenic virus loads remained low. Cre-mediated removal of other essential lytic genes gave a similar phenotype. CD19-Cre spleen infection by intraperitoneal virus was also impaired. Therefore, MuHV-4 had to emerge lytically from B cells to colonize the spleen. An important role for B cell lytic infection in host colonization is consistent with the large CD8 + T cell responses made to gammaherpesvirus lytic antigens during infectious mononucleosis and suggests that vaccine-induced immunity capable of suppressing B cell lytic infection might reduce long-term virus loads. IMPORTANCE Gammaherpesviruses cause B cell cancers. Most models of host colonization derive from cell cultures with continuous, virus-driven B cell proliferation. However, vaccines based on these models have worked poorly. To test whether proliferating B cells suffice for host colonization, we inactivated the capacity of MuHV-4, a gammaherpesvirus of mice, to reemerge from B cells. The modified virus was able to colonize a first wave of B cells in lymph nodes but spread poorly to B cells in secondary sites such as the spleen. Consequently, viral loads remained low. These results were consistent with virus-driven B cell proliferation exploiting normal host pathways and thus having to transfer lytically to new B cells for new proliferation. We conclude that viral lytic infection is a potential target to reduce B cell proliferation. Copyright © 2018 American Society for Microbiology.

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

PubMed Central

Silva, Ricardo Jorge; Cruz, Ana Rita; Mano, Miguel

2017-01-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. PMID:28394930

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

PubMed

Sunkavalli, Ushasree; Aguilar, Carmen; Silva, Ricardo Jorge; Sharan, Malvika; Cruz, Ana Rita; Tawk, Caroline; Maudet, Claire; Mano, Miguel; Eulalio, Ana

2017-04-01

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

Genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status.

PubMed

Verrier, Eloi R; Langevin, Christelle; Tohry, Corinne; Houel, Armel; Ducrocq, Vincent; Benmansour, Abdenour; Quillet, Edwige; Boudinot, Pierre

2012-01-01

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction--that was not observed in the susceptible cells--and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

PubMed

Douchkov, Dimitar; Lueck, Stefanie; Hensel, Goetz; Kumlehn, Jochen; Rajaraman, Jeyaraman; Johrde, Annika; Doblin, Monika S; Beahan, Cherie T; Kopischke, Michaela; Fuchs, René; Lipka, Volker; Niks, Rients E; Bulone, Vincent; Chowdhury, Jamil; Little, Alan; Burton, Rachel A; Bacic, Antony; Fincher, Geoffrey B; Schweizer, Patrick

2016-10-01

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Estimation of the size of genetic bottlenecks in cell-to-cell movement of soil-borne wheat mosaic virus and the possible role of the bottlenecks in speeding up selection of variations in trans-acting genes or elements.

PubMed

Miyashita, Shuhei; Kishino, Hirohisa

2010-02-01

Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil-borne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 +/- 0.22 on average and 5.02 +/- 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans-acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.

Aspergillosis and stem cell transplantation: An overview of experimental pathogenesis studies.

PubMed

Al-Bader, Nadia; Sheppard, Donald C

2016-11-16

Invasive aspergillosis is a life-threatening infection caused by the opportunistic filamentous fungus Aspergillus fumigatus. Patients undergoing haematopoietic stem cell transplant (HSCT) for the treatment of hematological malignancy are at particularly high risk of developing this fatal infection. The susceptibility of HSCT patients to infection with A. fumigatus is a consequence of a complex interplay of both fungal and host factors. Here we review our understanding of the host-pathogen interactions underlying the susceptibility of the immunocompromised host to infection with A. fumigatus with a focus on the experimental validation of fungal and host factors relevant to HSCT patients. These include fungal factors such as secondary metabolites, cell wall constituents, and metabolic adaptations that facilitate immune evasion and survival within the host microenvironment, as well as the innate and adaptive immune responses involved in host defense against A. fumigatus.

Therapeutic cloning in individual parkinsonian mice

PubMed Central

Tabar, Viviane; Tomishima, Mark; Panagiotakos, Georgia; Wakayama, Sayaka; Menon, Jayanthi; Chan, Bill; Mizutani, Eiji; Al-Shamy, George; Ohta, Hiroshi; Wakayama, Teruhiko; Studer, Lorenz

2009-01-01

Cell transplantation with embryonic stem (ES) cell progeny requires immunological compatibility with host tissue. ‘Therapeutic cloning’ is a strategy to overcome this limitation by generating nuclear transfer (nt)ES cells that are genetically matched to an individual. Here we establish the feasibility of treating individual mice via therapeutic cloning. Derivation of 187 ntES cell lines from 24 parkinsonian mice, dopaminergic differentiation, and transplantation into individually matched host mice showed therapeutic efficacy and lack of immunological response. PMID:18376409

Industrial production of clotting factors: Challenges of expression, and choice of host cells.

PubMed

Kumar, Sampath R

2015-07-01

The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional engraftment of the medial ganglionic eminence cells in experimental stroke model.

PubMed

Daadi, Marcel M; Lee, Sang Hyung; Arac, Ahmet; Grueter, Brad A; Bhatnagar, Rishi; Maag, Anne-Lise; Schaar, Bruce; Malenka, Robert C; Palmer, Theo D; Steinberg, Gary K

2009-01-01

Currently there are no effective treatments targeting residual anatomical and behavioral deficits resulting from stroke. Evidence suggests that cell transplantation therapy may enhance functional recovery after stroke through multiple mechanisms. We used a syngeneic model of neural transplantation to explore graft-host communications that enhance cellular engraftment.The medial ganglionic eminence (MGE) cells were derived from 15-day-old transgenic rat embryos carrying green fluorescent protein (GFP), a marker, to easily track the transplanted cells. Adult rats were subjected to transient intraluminal occlusion of the medial cerebral artery. Two weeks after stroke, the grafts were deposited into four sites, along the rostro-caudal axis and medially to the stroke in the penumbra zone. Control groups included vehicle and fibroblast transplants. Animals were subjected to motor behavioral tests at 4 week posttransplant survival time. Morphological analysis demonstrated that the grafted MGE cells differentiated into multiple neuronal subtypes, established synaptic contact with host cells, increased the expression of synaptic markers, and enhanced axonal reorganization in the injured area. Initial patch-clamp recording demonstrated that the MGE cells received postsynaptic currents from host cells. Behavioral analysis showed reduced motor deficits in the rotarod and elevated body swing tests. These findings suggest that graft-host interactions influence the fate of grafted neural precursors and that functional recovery could be mediated by neurotrophic support, new synaptic circuit elaboration, and enhancement of the stroke-induced neuroplasticity.

Molecular Biology of Prune Dwarf Virus—A Lesser Known Member of the Bromoviridae but a Vital Component in the Dynamic Virus–Host Cell Interaction Network

PubMed Central

Bujarski, Józef J.

2017-01-01

Prune dwarf virus (PDV) is one of the members of Bromoviridae family, genus Ilarvirus. Host components that participate in the regulation of viral replication or cell-to-cell movement via plasmodesmata are still unknown. In contrast, viral infections caused by some other Bromoviridae members are well characterized. Bromoviridae can be distinguished based on localization of their replication process in infected cells, cell-to-cell movement mechanisms, and plant-specific response reactions. Depending upon the genus, “genome activation” and viral replication are linked to various membranous structures ranging from endoplasmic reticulum, to tonoplast. In the case of PDV, there is still no evidence of natural resistance sources in the host plants susceptible to virus infection. Apparently, PDV has a great ability to overcome the natural defense responses in a wide spectrum of plant hosts. The first manifestations of PDV infection are specific cell membrane alterations, and the formation of replicase complexes that support PDV RNA replication inside the spherules. During each stage of its life cycle, the virus uses cell components to replicate and to spread in whole plants, within the largely suppressed cellular immunity environment. This work presents the above stages of the PDV life cycle in the context of current knowledge about other Bromoviridae members. PMID:29258199

Membrane traffic and synaptic cross-talk during host cell entry by Trypanosoma cruzi.

PubMed

Butler, Claire E; Tyler, Kevin M

2012-09-01

It is widely accepted that Trypanosoma cruzi can exploit the natural exocytic response of the host to cell damage, utilizing host cell lysosomes as important effectors. It is, though, increasingly clear that the parasite also exploits endocytic mechanisms which allow for incorporation of plasma membrane into the parasitophorous vacuole. Further, that these endocytic mechanisms are involved in cross-talk with the exocytic machinery, in the recycling of vesicles and in the manipulation of the cytoskeleton. Here we review the mechanisms by which T. cruzi exploits features of the exocytic and endocytic pathways in epithelial and endothelial cells and the evidence for cross-talk between these pathways. © 2012 Blackwell Publishing Ltd.

Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts

PubMed Central

de Souza, Wanderley; de Carvalho, Tecia M. Ulisses

2013-01-01

In the present short review, we analyze past experiments that addressed the interactions of intracellular pathogenic protozoa (Trypanosoma cruzi, Toxoplasma gondii, and Plasmodium) with host cells and the initial use of the term active penetration to indicate that a protozoan “crossed the host cell membrane, penetrating into the cytoplasm.” However, the subsequent use of transmission electron microscopy showed that, for all of the protozoans and cell types examined, endocytosis, classically defined as involving the formation of a membrane-bound vacuole, took place during the interaction process. As a consequence, the recently penetrated parasites are always within a vacuole, designated the parasitophorous vacuole (PV). PMID:23355838

Cell biology and immunology lessons taught by Legionella pneumophila.

PubMed

Zhu, Wenhan; Luo, Zhao-Qing

2016-01-01

Legionella pneumophila is a facultative intracellular pathogen capable of replicating within a broad range of hosts. One unique feature of this pathogen is the cohort of ca. 300 virulence factors (effectors) delivered into host cells via its Dot/Icm type IV secretion system. Study of these proteins has produced novel insights into the mechanisms of host function modulation by pathogens, the regulation of essential processes of eukaryotic cells and of immunosurveillance. In this review, we will briefly discuss the roles of some of these effectors in the creation of a niche permissive for bacterial replication in phagocytes and recent advancements in the dissection of the innate immune detection mechanisms by challenging immune cells with L. pneumophila.

Endophytic fungi specifically introduce novel metabolites into grape flesh cells in vitro

PubMed Central

Ao, Xiu-Jin; Ren, An-Yun; Zhang, Han-Bo

2018-01-01

Since endophytes can affect metabolism of host plants, they are expected to be used to improve crop quality, especially for crops with organoleptic sensitive products such as wine grape. However, details of metabolic interactions between endophytes and host plants were less understood. In this work, we used high pressure liquid chromatography (HPLC) to analyze the metabolites of fruit flesh cells of grape treated with dual culture of different endophytic fungal strains (EFS). We observed that the dual-culture with different fungal strains show different metabolites composition in grape cells. In response to different EFS, quantities of detected metabolites in grape cells varied from 6 to 17 in this assay, and 1 to 11 novel metabolites were introduced into metabolome of grape cells. Dual-culture with fungal strains CS2, RH16 and RH5 introduced the highest quantities (10 or 11) of novel metabolites in grape cells. More importantly, the modification of metabolic profiles in grape cells via fungal endophytes appeared to be fungal strain/genus-specificity. Overall, this work revealed that introduction of specific metabolites in host plants may be one consequence during the process of endophytes-host metabolic interactions, which raise the possibility to shape grape qualities and characteristics using tool of fungal endophytes. PMID:29734364

Herpesvirus microRNAs for use in gene therapy immune-evasion strategies.

PubMed

Bots, S T F; Hoeben, R C

2017-07-01

Transplantation of allogeneic cells as well as of genetically corrected autologous cells are potent approaches to restore cellular functions in patients suffering from genetic diseases. The recipient's immune responses against non-self-antigens may compromise the survival of the grafted cells. Recipients of the graft may therefore require lifelong treatment with immunosuppressive drugs. An alternative approach to reduce graft rejection could involve the use of immune-evasion molecules. Expression of such molecules in cells of the graft may subvert recognition by the host's immune system. Viruses in particular are masters of exploitation and modulation of their hosts immune response. The Herpesviridae family provides a proof of concept for this as these viruses are capable to establish latency and a lifelong persistence in the infected hosts. While several viral proteins involved in immune evasion have been characterized, the Herpesviridae also encode a multitude of viral microRNA (miRNAs). Several of these miRNAs have been demonstrated to reduce the sensitivity of the infected cells to the destructive action of the host's immune cells. In this review, the miRNAs of some common herpesviruses that are associated with immune modulation will be discussed with a focus on their potential use in strategies aiming at generating non-immunogenic cells for transplantation.

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors.

PubMed

Kushwaha, Ambuj K; Apolis, Liana; Ito, Daisuke; Desai, Sanjay A

2018-05-03

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca ++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca ++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca ++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca ++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca ++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca ++ ] is consistent with parasite killing specifically via action on one or more Ca ++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca ++ transport and may be starting points for new antimalarial drugs. © 2018 John Wiley & Sons Ltd.

Mediators involved in the immunomodulatory effects of apoptotic cells

PubMed Central

Saas, Philippe; Bonnefoy, Francis; Kury-Paulin, Stephanie; Kleinclauss, François M.; Perruche, Sylvain

2007-01-01

Immunomodulatory properties are attributed to apoptotic cells. These properties have been used to modulate allogeneic immune responses in experimental transplantation settings. In independent studies, apoptotic cell infusion has been shown to favor hematopoietic cell engraftment, to increase heart graft survival and to delay the lethal onset of graft-versus-host disease (GVHD). The goal of this review was to discuss how apoptotic cell infusion interferes with graft rejection or host rejection (i.e., GVHD) and to focus on the potential mediators or “perpetuators” involved in apoptotic cell-induced immunomodulation. Particular emphasis on apoptotic cell phagocytosis, TGF-β secretion and regulatory T cell induction was performed. Stimulating “naturally” immunosuppressive molecules (i.e., TGF-β) or immunomodulatory cells (“alternatively-activated” macrophages, certain DC subsets or regulatory T cells) in a physiological manner by using apoptotic cell infusion can be a promising way to induce tolerance. PMID:17632410

Graft-versus-host disease after radiation therapy in patients who have undergone allogeneic stem cell transplantation: two case reports.

PubMed

Milgrom, Sarah A; Nieto, Yago; Pinnix, Chelsea C; Smith, Grace L; Wogan, Christine F; Rondon, Gabriela; Medeiros, L Jeffrey; Kebriaei, Partow; Dabaja, Bouthaina S

2016-07-28

Patients who undergo allogeneic stem cell transplantation and subsequent radiation therapy uncommonly develop graft-versus-host disease within the irradiated area. We quantified the incidence of this complication, which is a novel contribution to the field. From 2010 to 2014, 1849 patients underwent allogeneic stem cell transplantation, and 41 (2 %) received radiation therapy afterward. Of these, two patients (5 %) developed graft-versus-host disease within the irradiated tissues during or immediately after radiation therapy. The first patient is a 37-year-old white man who had Hodgkin lymphoma; he underwent allogeneic stem cell transplantation from a matched unrelated donor and received radiation therapy for an abdominal and pelvic nodal recurrence. After 28.8 Gy, he developed grade 4 gastrointestinal graft-versus-host disease, refractory to tacrolimus and steroids, but responsive to pentostatin and photopheresis. The other patient is a 24-year-old white man who had acute leukemia; he underwent allogeneic stem cell transplantation from a matched related donor and received craniospinal irradiation for a central nervous system relapse. After 24 cobalt Gy equivalent, he developed severe cutaneous graft-versus-host disease, sharply delineated within the radiation therapy field, which was responsive to tacrolimus and methylprednisolone. We conclude that graft-versus-host disease within irradiated tissues is an uncommon but potentially serious complication that may follow radiation therapy in patients who have undergone allogeneic stem cell transplantation. Clinicians must be aware of this complication and prepared with strategies to mitigate risk. Patients who have undergone allogeneic stem cell transplantation represent a unique population that may offer novel insight into the pathways involved in radiation-related inflammation.

Function of Macrophage and Parasite Phosphatases in Leishmaniasis

PubMed Central

Soulat, Didier; Bogdan, Christian

2017-01-01

The kinetoplastid protozoan parasites belonging to the genus Leishmania are the causative agents of different clinical forms of leishmaniasis, a vector-borne infectious disease with worldwide prevalence. The protective host immune response against Leishmania parasites relies on myeloid cells such as dendritic cells and macrophages in which upon stimulation by cytokines (e.g., interferon-γ) a complex network of signaling pathways is switched on leading to strong antimicrobial activities directed against the intracellular parasite stage. The regulation of these pathways classically depends on post-translational modifications of proteins, with phosphorylation events playing a cardinal role. Leishmania parasites deactivate their phagocytic host cells by inducing specific mammalian phosphatases that are capable to impede signaling. On the other hand, there is now also evidence that Leishmania spp. themselves express phosphatases that might target host cell molecules and thereby facilitate the intracellular survival of the parasite. This review will present an overview on the modulation of host phosphatases by Leishmania parasites as well as on the known families of Leishmania phosphatases and their possible function as virulence factors. A more detailed understanding of the role of phosphatases in Leishmania–host cell interactions might open new avenues for the treatment of non-healing, progressive forms of leishmaniasis. PMID:29312331

Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

PubMed

Pentland, Ieisha; Parish, Joanna L

2015-07-06

All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

Engineering host-derived resistance against plant parasites through RNA interference: challenges and opportunities.

PubMed

Runo, Steven

2011-01-01

RNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral - worms and insects, viral infections, viruses - or through a vascular connections - parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.

Archaeal viruses at the cell envelope: entry and egress

PubMed Central

Quemin, Emmanuelle R. J.; Quax, Tessa E. F.

2015-01-01

The cell envelope represents the main line of host defense that viruses encounter on their way from one cell to another. The cytoplasmic membrane in general is a physical barrier that needs to be crossed both upon viral entry and exit. Therefore, viruses from the three domains of life employ a wide range of strategies for perforation of the cell membrane, each adapted to the cell surface environment of their host. Here, we review recent insights on entry and egress mechanisms of viruses infecting archaea. Due to the unique nature of the archaeal cell envelope, these particular viruses exhibit novel and unexpected mechanisms to traverse the cellular membrane. PMID:26097469

Transforming growth factor β: a master regulator of the gut microbiota and immune cell interactions.

PubMed

Bauché, David; Marie, Julien C

2017-04-01

The relationship between host organisms and their microbiota has co-evolved towards an inter-dependent network of mutualistic interactions. This interplay is particularly well studied in the gastrointestinal tract, where microbiota and host immune cells can modulate each other directly, as well as indirectly, through the production and release of chemical molecules and signals. In this review, we define the functional impact of transforming growth factor-beta (TGF-β) on this complex interplay, especially through its modulation of the activity of local regulatory T cells (Tregs), type 17 helper (Th17) cells, innate lymphoid cells (ILCs) and B cells.

Investigation of the open-circuit voltage in wide-bandgap InGaP-host InP quantum dot intermediate-band solar cells

NASA Astrophysics Data System (ADS)

Aihara, Taketo; Tayagaki, Takeshi; Nagato, Yuki; Okano, Yoshinobu; Sugaya, Takeyoshi

2018-04-01

To analyze the open-circuit voltage (V oc) in intermediate-band solar cells, we investigated the current-voltage characteristics in wide-bandgap InGaP-based InP quantum dot (QD) solar cells. From the temperature dependence of the current-voltage curves, we show that the V oc in InP QD solar cells increases with decreasing temperature. We use a simple diode model to extract V oc at the zero-temperature limit, V 0, and the temperature coefficient C of the solar cells. Our results show that, while the C of InP QD solar cells is slightly larger than that of the reference InGaP solar cells, V 0 significantly decreases and coincides with the bandgap energy of the InP QDs rather than that of the InGaP host. This V 0 indicates that the V oc reduction in the InP QD solar cells is primarily caused by the breaking of the Fermi energy separation between the QDs and the host semiconductor in intermediate-band solar cells, rather than by enhanced carrier recombination.

Intrinsic properties of limb bud cells can be differentially reset.

PubMed

Saiz-Lopez, Patricia; Chinnaiya, Kavitha; Towers, Matthew; Ros, Maria A

2017-02-01

An intrinsic timing mechanism specifies the positional values of the zeugopod (i.e. radius/ulna) and then autopod (i.e. wrist/digits) segments during limb development. Here, we have addressed whether this timing mechanism ensures that patterning events occur only once by grafting GFP-expressing autopod progenitor cells to the earlier host signalling environment of zeugopod progenitor cells. We show by detecting Hoxa13 expression that early and late autopod progenitors fated for the wrist and phalanges, respectively, both contribute to the entire host autopod, indicating that the autopod positional value is irreversibly determined. We provide evidence that Hoxa13 provides an autopod-specific positional value that correctly allocates cells into the autopod, most likely through the control of cell-surface properties as shown by cell-cell sorting analyses. However, we demonstrate that only the earlier autopod cells can adopt the host proliferation rate to permit normal morphogenesis. Therefore, our findings reveal that the ability of embryonic cells to differentially reset their intrinsic behaviours confers robustness to limb morphogenesis. We speculate that this plasticity could be maintained beyond embryogenesis in limbs with regenerative capacity. © 2017. Published by The Company of Biologists Ltd.

Cryptosporidium parvum Induces an Endoplasmic Stress Response in the Intestinal Adenocarcinoma HCT-8 Cell Line*

PubMed Central

Morada, Mary; Pendyala, Lakhsmi; Wu, Gang; Merali, Salim; Yarlett, Nigel

2013-01-01

Invasion of human intestinal epithelial cells (HCT-8) by Cryptosporidium parvum resulted in a rapid induction of host cell spermidine/spermine N1-acetyltransferase 1 (hSSAT-1) mRNA, causing a 4-fold increase in SSAT-1 enzyme activity after 24 h of infection. In contrast, host cell SSAT-2, spermine oxidase, and acetylpolyamine oxidase (hAPAO) remained unchanged during this period. Intracellular polyamine levels of C. parvum-infected human epithelial cells were determined, and it was found that spermidine remained unchanged and putrescine increased by 2.5-fold after 15 h and then decreased after 24 h, whereas spermine decreased by 3.9-fold after 15 h. Concomitant with these changes, N1-acetylspermine and N1-acetylspermidine both increased by 115- and 24-fold, respectively. Increased SSAT-1 has previously been shown to be involved in the endoplasmic reticulum (ER) stress response leading to apoptosis. Several stress response proteins were increased in HCT-8 cells infected with C. parvum, including calreticulin, a major calcium-binding chaperone in the ER; GRP78/BiP, a prosurvival ER chaperone; and Nrf2, a transcription factor that binds to antioxidant response elements, thus activating them. However, poly(ADP-ribose) polymerase, a protein involved in DNA repair and programmed cell death, was decreased. Cumulatively, these results suggest that the invasion of HCT-8 cells by C. parvum results in an ER stress response by the host cell that culminates in overexpression of host cell SSAT-1 and elevated N1-acetylpolyamines, which can be used by a parasite that lacks ornithine decarboxylase. PMID:23986438

A new model for myxosporean (Myxozoa) development explains the endogenous budding phenomenon, the nature of cell within cell life stages and evolution of parasitism from a cnidarian ancestor.

PubMed

Morris, D J

2012-08-01

The phylum Myxozoa is composed of endoparasitic species that have predominately been recorded within aquatic vertebrates. The simple body form of a trophic cell containing other cells within it, as observed within these hosts, has provided few clues to relationships with other organisms. In addition, the placement of the group using molecular phylogenies has proved very difficult, although the majority of analyses now suggest that they are cnidarians. There have been relatively few studies of myxozoan stages within invertebrate hosts, even though these exhibit multicellular and sexual stages that may provide clues to myxozoan evolution. Therefore an ultrastructural examination of a myxozoan infection of a freshwater oligochaete was conducted, to reassess and formulate a model for myxozoan development in these hosts. This deemed that meiosis occurs within the oligochaete, but that fertilisation is not immediate. Rather, the resultant haploid germ cell (oocyte) is engulfed by a diploid sporogonic cell (nurse cell) to form a sporoplasm. It is this sporoplasm that infects the fish, resulting in the multicellular stages observed. Fertilisation occurs after the parasites leave the fish and enter the oligochaete host. The nurse cell/oocyte model explains previously conflicting evidence in the literature regarding myxosporean biology, and aligns phenomena considered distinctive to the Myxozoa, such as endogenous budding and cell within cell development, with processes recorded in cnidarians. Finally, the evolutionary origin of the Myxozoa as cnidarian parasites of ova is hypothesised. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses▿ †

PubMed Central

Caro, Audrey; Gros, Olivier; Got, Patrice; De Wit, Rutger; Troussellier, Marc

2007-01-01

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host. PMID:17259363

The membrane as the gatekeeper of infection: Cholesterol in host-pathogen interaction.

PubMed

Kumar, G Aditya; Jafurulla, Md; Chattopadhyay, Amitabha

2016-09-01

The cellular plasma membrane serves as a portal for the entry of intracellular pathogens. An essential step for an intracellular pathogen to gain entry into a host cell therefore is to be able to cross the cell membrane. In this review, we highlight the role of host membrane cholesterol in regulating the entry of intracellular pathogens using insights obtained from work on the interaction of Leishmania and Mycobacterium with host cells. The entry of these pathogens is known to be dependent on host membrane cholesterol. Importantly, pathogen entry is inhibited either upon depletion (or complexation), or enrichment of membrane cholesterol. In other words, an optimum level of host membrane cholesterol is necessary for efficient infection by pathogens. In this overall context, we propose a general mechanism, based on cholesterol-induced conformational changes, involving cholesterol binding sites in host cell surface receptors that are implicated in this process. A therapeutic strategy targeting modulation of membrane cholesterol would have the advantage of avoiding the commonly encountered problem of drug resistance in tackling infection by intracellular pathogens. Insights into the role of host membrane cholesterol in pathogen entry would be instrumental in the development of novel therapeutic strategies to effectively tackle intracellular pathogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The plant host pathogen interface: cell wall and membrane dynamics of pathogen-induced responses.

PubMed

Day, Brad; Graham, Terry

2007-10-01

Perception of pathogens by their hosts is the outcome of a highly coordinated and sophisticated surveillance network, tightly regulated by both host and pathogen elicitors, effectors, and signaling processes. In this article, we focus on two relatively well-studied host-pathogens systems, one involving a bacterial-plant interaction (Pseudomonas syringae-Arabidopsis) and the other involving an oomycete-plant interaction (Phytophthora sojae-soybean). We discuss the status of current research related to events occurring at the host-pathogen interface in these two systems, and how these events influence the organization and activation of resistance responses in the respective hosts. This recent research has revealed that in addition to the previously identified resistance machinery (R-proteins, molecular chaperones, etc.), the dynamics of the cell wall, membrane trafficking, and the actin cytoskeleton are intimately associated with the activation of resistance in plants. Specifically, in Arabidopsis, a possible connection between the actin machinery and R-protein- mediated induction of disease resistance is described. In the case of the P. sojae-soybean interaction, we describe the fact that a classical basal resistance elicitor, the cell wall glucan elicitor from the pathogen, can directly activate host hypersensitive cell death, which is apparently modulated in a race-specific manner by the presence of R genes in the host.

Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts.

PubMed

Bhattacharyya, Sankar; Md Sakib Hossain, Dewan; Mohanty, Suchismita; Sankar Sen, Gouri; Chattopadhyay, Sreya; Banerjee, Shuvomoy; Chakraborty, Juni; Das, Kaushik; Sarkar, Diptendra; Das, Tanya; Sa, Gaurisankar

2010-07-01

Immune dysfunction is well documented during tumor progression and likely contributes to tumor immune evasion. CD8(+) cytotoxic T lymphocytes (CTLs) are involved in antigen-specific tumor destruction and CD4(+) T cells are essential for helping this CD8(+) T cell-dependent tumor eradication. Tumors often target and inhibit T-cell function to escape from immune surveillance. This dysfunction includes loss of effector and memory T cells, bias towards type 2 cytokines and expansion of T regulatory (Treg) cells. Curcumin has previously been shown to have antitumor activity and some research has addressed the immunoprotective potential of this plant-derived polyphenol in tumor-bearing hosts. Here we examined the role of curcumin in the prevention of tumor-induced dysfunction of T cell-based immune responses. We observed severe loss of both effector and memory T-cell populations, downregulation of type 1 and upregulation of type 2 immune responses and decreased proliferation of effector T cells in the presence of tumors. Curcumin, in turn, prevented this loss of T cells, expanded central memory T cell (T(CM))/effector memory T cell (T(EM)) populations, reversed the type 2 immune bias and attenuated the tumor-induced inhibition of T-cell proliferation in tumor-bearing hosts. Further investigation revealed that tumor burden upregulated Treg cell populations and stimulated the production of the immunosuppressive cytokines transforming growth factor (TGF)-beta and IL-10 in these cells. Curcumin, however, inhibited the suppressive activity of Treg cells by downregulating the production of TGF-beta and IL-10 in these cells. More importantly, curcumin treatment enhanced the ability of effector T cells to kill cancer cells. Overall, our observations suggest that the unique properties of curcumin may be exploited for successful attenuation of tumor-induced suppression of cell-mediated immune responses.

Induction of virulence factors in Giardia duodenalis independent of host attachment

PubMed Central

Emery, Samantha J.; Mirzaei, Mehdi; Vuong, Daniel; Pascovici, Dana; Chick, Joel M.; Lacey, Ernest; Haynes, Paul A.

2016-01-01

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response. PMID:26867958

Mycobacterium tuberculosis Infection and Innate Responses in a New Model of Lung Alveolar Macrophages.

PubMed

Woo, Minjeong; Wood, Connor; Kwon, Doyoon; Park, Kyu-Ho Paul; Fejer, György; Delorme, Vincent

2018-01-01

Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis ( Mtb ) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hamper the detailed molecular understanding of host- Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung AMs, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular, and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell lines. MPI cells were also suitable for the determination of anti-tuberculosis (TB) drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, interleukin 6 (IL-6), IL-1α, and IL-1β, as compared to stimulation with heat-killed (HK) bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including HK Mtb . By contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using light chain 3B immunostaining, as well as LysoTracker labeling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for TB research, allowing a deeper understanding of AMs functions in this pathology.

Identification of legionella effectors using bioinformatic approaches.

PubMed

Segal, Gil

2013-01-01

Legionella pneumophila the causative agent of Legionnaires' disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which translocates about 300 effector proteins into host cells during infection. Many of these effectors were first identified as effector candidates by several bioinformatic approaches, and these predicted effectors were later examined experimentally for translocation and a large number of which were validated as effector proteins. Here, I summarized the bioinformatic approaches that were used to identify these effectors.

Producing a trimethylpentanoic acid using hybrid polyketide synthases

DOEpatents

Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

2014-10-07

The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

Nanotechnology as an adjunct tool for transplanting engineered cells and tissues.

PubMed

Borlongan, Cesar V; Masuda, Tadashi; Walker, Tiffany A; Maki, Mina; Hara, Koichi; Yasuhara, Takao; Matsukawa, Noriyuki; Emerich, Dwaine F

2007-11-01

Laboratory and clinical studies have provided evidence of feasibility, safety and efficacy of cell transplantation to treat a wide variety of diseases characterized by tissue and cell dysfunction ranging from diabetes to spinal cord injury. However, major hurdles remain and limit pursuing large clinical trials, including the availability of a universal cell source that can be differentiated into specific cellular phenotypes, methods to protect the transplanted allogeneic or xenogeneic cells from rejection by the host immune system, techniques to enhance cellular integration of the transplant within the host tissue, strategies for in vivo detection and monitoring of the cellular implants, and new techniques to deliver genes to cells without eliciting a host immune response. Finding ways to circumvent these obstacles will benefit considerably from being able to understand, visualize, and control cellular interactions at a sub-micron level. Cutting-edge discoveries in the multidisciplinary field of nanotechnology have provided us a platform to manipulate materials, tissues, cells, and DNA at the level of and within the individual cell. Clearly, the scientific innovations achieved with nanotechnology are a welcome strategy for enhancing the generally encouraging results already achieved in cell transplantation. This review article discusses recent progress in the field of nanotechnology as a tool for tissue engineering, gene therapy, cell immunoisolation, and cell imaging, highlighting its direct applications in cell transplantation therapy.

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

PubMed

Anastasia, Luigi; Holguera, Javier; Bianchi, Anna; D'Avila, Francesca; Papini, Nadia; Tringali, Cristina; Monti, Eugenio; Villar, Enrique; Venerando, Bruno; Muñoz-Barroso, Isabel; Tettamanti, Guido

2008-03-01

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

PubMed

Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

2017-01-03

The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017 Loh et al.

Host-microbiota interactions in the intestine.

PubMed

Elson, Charles O; Alexander, Katie L

2015-01-01

The comprehensive collection of bacterial species, termed microbiota, within human and other mammalian hosts has profound effects on both innate and adaptive immunity. Multiple host innate mechanisms contribute to intestinal homeostasis, including epithelial production of protective mucin layers maintaining spatial segregation in the intestine as well as epithelial cell secretion of a broad range of antimicrobial peptides. Additionally, epithelial cells employ autophagy to contain and eliminate invading bacteria; interestingly, genetic variants in specific autophagy genes are linked to susceptibility to Crohn's disease. Innate lymphoid cells, which rapidly respond to cytokine and microbial signals, have emerged as important regulators of the intestinal immune response to the microbiota. With regard to adaptive immunity, specific microbial species stimulate induction of regulatory T cells while others induce effector T cells within the gut. Such stimulation is subject to dysregulation during inflammation and disease, contributing to 'dysbiosis' or an abnormal microbiota composition that has been associated with a variety of immune-mediated inflammatory disorders, including celiac disease. The microbiota communicates with the immune system and vice versa; thus, an abnormal microbiota composition likely translates into an altered host immune response, though the exact mechanisms of such are not yet clear. Immunoglobulin A plays a critical role in limiting bacterial access to the host and in maintaining mutualism with the microbiota. Perturbation of the mucosal barrier via infection or other means can induce effector T cells reactive to the intestinal microbiota, and these cells can persist as memory cells for extended periods of time and potentially serve as pathogenic effector cells upon re-encounter with antigen. Health is associated with a diverse microbiota that functions to maintain the balance between T effector and T regulatory cells in the intestine. Whether dysbiosis can be reversed in immune-mediated disease, thus restoring health, is a question of intense interest for this active area of research. © 2015 S. Karger AG, Basel.

Roles of Pro-viral Host Factors in Mosquito-Borne Flavivirus Infections.

PubMed

Campos, Rafael K; Garcia-Blanco, Mariano A; Bradrick, Shelton S

2017-07-09

Identification and analysis of viral host factors is a growing area of research which aims to understand the how viruses molecularly interface with the host cell. Investigations into flavivirus-host interactions has led to new discoveries in viral and cell biology, and will potentially bolster strategies to control the important diseases caused by these pathogens. Here, we address the current knowledge of prominent host factors required for the flavivirus life-cycle and mechanisms by which they promote infection.

Neem leaf glycoprotein generates superior tumor specific central memory CD8+ T cells than cyclophosphamide that averts post-surgery solid sarcoma recurrence.

PubMed

Ghosh, Sarbari; Sarkar, Madhurima; Ghosh, Tithi; Guha, Ipsita; Bhuniya, Avishek; Saha, Akata; Dasgupta, Shayani; Barik, Subhasis; Bose, Anamika; Baral, Rathindranath

2017-08-03

The success of cancer vaccines is limited as most of them induce corrupted CD8 + T cell memory populations. We reported earlier that a natural immunomodulator, neem leaf glycoprotein (NLGP), therapeutically restricts tumor growth in a CD8 + T cell-dependent manner. Here, our objective is to study whether memory CD8 + T cell population is generated in sarcoma hosts after therapeutic NLGP treatment and their role in prevention of post-surgery tumor recurrence, in comparison to the immunostimulatory metronomic cyclophosphamide (CTX) treatment. We found that therapeutic NLGP and CTX treatment generates central memory CD8 + T (TCM) cells with characteristic CD44 + CD62L high CCR7 high IL-2 high phenotypes. But these TCM cells are functionally impaired to prevent re-appearance of tumors along with compromised proliferative, IL-2 secretive and cytotoxic status. This might be due to the presence of tumor load, even a small one in the host, which serves as a persistent source of tumor antigens thereby corrupting the TCM cells so generated. Surgical removal of the persisting tumors from the host restored the functional characteristics of memory CD8 + T cells, preventing tumor recurrence after surgery till end of the experiment. Moreover, we observed that generation of superior TCM cells in NLGP treated surgically removed tumor hosts is related to the activation of Wnt signalling in memory CD8 + T cells with concomitant inhibition of GSK-3β and stabilisation of β-catenin, which ultimately activates transcription of Wnt target genes, like, eomesodermin, a signature molecule of CD8 + TCM cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Complex Cell Wall Composition of Syncytia Induced by Plant Parasitic Cyst Nematodes Reflects Both Function and Host Plant.

PubMed

Zhang, Li; Lilley, Catherine J; Imren, Mustafa; Knox, J Paul; Urwin, Peter E

2017-01-01

Plant-parasitic cyst nematodes induce the formation of specialized feeding structures, syncytia, within their host roots. These unique plant organs serve as the sole nutrient resource for development and reproduction throughout the biotrophic interaction. The multinucleate syncytium, which arises through local dissolution of cell walls and protoplast fusion of multiple adjacent cells, has dense cytoplasm containing numerous organelles, surrounded by thickened outer cell walls that must withstand high turgor pressure. However, little is known about how the constituents of the syncytial cell wall and their conformation support its role during nematode parasitism. We used a set of monoclonal antibodies, targeted to a range of plant cell wall components, to reveal the microstructures of syncytial cell walls induced by four of the most economically important cyst nematode species, Globodera pallida , Heterodera glycines , Heterodera avenae and Heterodera filipjevi , in their respective potato, soybean, and spring wheat host roots. In situ fluorescence analysis revealed highly similar cell wall composition of syncytia induced by G. pallida and H. glycines . Both consisted of abundant xyloglucan, methyl-esterified homogalacturonan and pectic arabinan. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain little xyloglucan but are rich in feruloylated xylan and arabinan residues, with variable levels of mixed-linkage glucan. The overall chemical composition of syncytial cell walls reflected the general features of root cell walls of the different host plants. We relate specific components of syncytial cell walls, such as abundant arabinan, methyl-esterification status of pectic homogalacturonan and feruloylation of xylan, to their potential roles in forming a network to support both the strength and flexibility required for syncytium function.

Cell replacement and visual restoration by retinal sheet transplants

PubMed Central

Seiler, Magdalene J.; Aramant, Robert B.

2012-01-01

Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a ‘nursing’ role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance – they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity. In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy. PMID:22771454

Actin polymerization drives septation of Listeria monocytogenes namA hydrolase mutants, demonstrating host correction of a bacterial defect.

PubMed

Alonzo, Francis; McMullen, P David; Freitag, Nancy E

2011-04-01

The Gram-positive bacterial cell wall presents a structural barrier that requires modification for protein secretion and large-molecule transport as well as for bacterial growth and cell division. The Gram-positive bacterium Listeria monocytogenes adjusts cell wall architecture to promote its survival in diverse environments that include soil and the cytosol of mammalian cells. Here we provide evidence for the enzymatic flexibility of the murein hydrolase NamA and demonstrate that bacterial septation defects associated with a loss of NamA are functionally complemented by physical forces associated with actin polymerization within the host cell cytosol. L. monocytogenes ΔnamA mutants formed long bacterial chains during exponential growth in broth culture; however, normal septation could be restored if mutant cells were cocultured with wild-type L. monocytogenes bacteria or by the addition of exogenous NamA. Surprisingly, ΔnamA mutants were not significantly attenuated for virulence in mice despite the pronounced exponential growth septation defect. The physical force of L. monocytogenes-mediated actin polymerization within the cytosol was sufficient to sever ΔnamA mutant intracellular chains and thereby enable the process of bacterial cell-to-cell spread so critical for L. monocytogenes virulence. The inhibition of actin polymerization by cytochalasin D resulted in extended intracellular bacterial chains for which septation was restored following drug removal. Thus, despite the requirement for NamA for the normal septation of exponentially growing L. monocytogenes cells, the hydrolase is essentially dispensable once L. monocytogenes gains access to the host cell cytosol. This phenomenon represents a notable example of eukaryotic host cell complementation of a bacterial defect.

The Complex Cell Wall Composition of Syncytia Induced by Plant Parasitic Cyst Nematodes Reflects Both Function and Host Plant

PubMed Central

Zhang, Li; Lilley, Catherine J.; Imren, Mustafa; Knox, J. Paul; Urwin, Peter E.

2017-01-01

Plant–parasitic cyst nematodes induce the formation of specialized feeding structures, syncytia, within their host roots. These unique plant organs serve as the sole nutrient resource for development and reproduction throughout the biotrophic interaction. The multinucleate syncytium, which arises through local dissolution of cell walls and protoplast fusion of multiple adjacent cells, has dense cytoplasm containing numerous organelles, surrounded by thickened outer cell walls that must withstand high turgor pressure. However, little is known about how the constituents of the syncytial cell wall and their conformation support its role during nematode parasitism. We used a set of monoclonal antibodies, targeted to a range of plant cell wall components, to reveal the microstructures of syncytial cell walls induced by four of the most economically important cyst nematode species, Globodera pallida, Heterodera glycines, Heterodera avenae and Heterodera filipjevi, in their respective potato, soybean, and spring wheat host roots. In situ fluorescence analysis revealed highly similar cell wall composition of syncytia induced by G. pallida and H. glycines. Both consisted of abundant xyloglucan, methyl-esterified homogalacturonan and pectic arabinan. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain little xyloglucan but are rich in feruloylated xylan and arabinan residues, with variable levels of mixed-linkage glucan. The overall chemical composition of syncytial cell walls reflected the general features of root cell walls of the different host plants. We relate specific components of syncytial cell walls, such as abundant arabinan, methyl-esterification status of pectic homogalacturonan and feruloylation of xylan, to their potential roles in forming a network to support both the strength and flexibility required for syncytium function. PMID:28680436

Progression from productive infection to integration and oncogenic transformation in human papillomavirus type 59-immortalized foreskin keratinocytes.

PubMed

Spartz, Helena; Lehr, Elizabeth; Zhang, Benyue; Roman, Ann; Brown, Darron R

2005-05-25

Studies of changes in the virus and host cell upon progression from human papillomavirus (HPV) episomal infection to integration are critical to understanding HPV-related malignant transformation. However, there exist only a few in vitro models of both productive HPV infection and neoplastic progression on the same host background. We recently described a unique foreskin keratinocyte cell line (ERIN 59) that contains HPV 59 (a close relative of HPV 18). Early passages of ERIN 59 cells (passages 9-13) contained approximately 50 copies of episomes/cell, were feeder cell-dependent, and could be induced to differentiate and produce infectious virus in a simple culture system. We now report that late passage cells (passages greater than 50) were morphologically different from early passage cells, were feeder cell independent, and did not differentiate or produce virus. These late passage cells contained HPV in an integrated form. An integration-derived oncogene transcript was expressed in late passage cells. The E2 open reading frame was interrupted in this transcript at nucleotide 3351. Despite a lower viral genome copy number in late passage ERIN 59 cells, expression of E6/E7 oncogene transcripts was similar to early passage cells. We conclude that ERIN 59 cells are a valuable cell line representing a model of progression from HPV 59 episomal infection and virus production to HPV 59 integration and associated oncogenic transformation on the same host background.

Transcriptional Responses of Candida albicans to Epithelial and Endothelial Cells▿ †

PubMed Central

Park, Hyunsook; Liu, Yaoping; Solis, Norma; Spotkov, Joshua; Hamaker, Jessica; Blankenship, Jill R.; Yeaman, Michael R.; Mitchell, Aaron P.; Liu, Haoping; Filler, Scott G.

2009-01-01

Candida albicans interacts with oral epithelial cells during oropharyngeal candidiasis and with vascular endothelial cells when it disseminates hematogenously. We set out to identify C. albicans genes that govern interactions with these host cells in vitro. The transcriptional response of C. albicans to the FaDu oral epithelial cell line and primary endothelial cells was determined by microarray analysis. Contact with epithelial cells caused a decrease in transcript levels of genes related to protein synthesis and adhesion, whereas contact with endothelial cells did not significantly influence any specific functional category of genes. Many genes whose transcripts were increased in response to either host cell had not been previously characterized. We constructed mutants with homozygous insertions in 22 of these uncharacterized genes to investigate their function during host-pathogen interaction. By this approach, we found that YCK2, VPS51, and UEC1 are required for C. albicans to cause normal damage to epithelial cells and resist antimicrobial peptides. YCK2 is also necessary for maintenance of cell polarity. VPS51 is necessary for normal vacuole formation, resistance to multiple stressors, and induction of maximal endothelial cell damage. UEC1 encodes a unique protein that is required for resistance to cell membrane stress. Therefore, some C. albicans genes whose transcripts are increased upon contact with epithelial or endothelial cells are required for the organism to damage these cells and withstand the stresses that it likely encounters during growth in the oropharynx and bloodstream. PMID:19700637

Productive Lifecycle of Human Papillomaviruses that Depends Upon Squamous Epithelial Differentiation

PubMed Central

Kajitani, Naoko; Satsuka, Ayano; Kawate, Akifumi; Sakai, Hiroyuki

2012-01-01

Human papillomaviruses (HPVs) target the stratified epidermis, and can causes diseases ranging from benign condylomas to malignant tumors. Infections of HPVs in the genital tract are among the most common sexually transmitted diseases, and a major risk factor for cervical cancer. The virus targets epithelial cells in the basal layer of the epithelium, while progeny virions egress from terminally differentiated cells in the cornified layer, the surface layer of the epithelium. In infected basal cells, the virus maintains its genomic DNA at low-copy numbers, at which the viral productive lifecycle cannot proceed. Progression of the productive lifecycle requires differentiation of the host cell, indicating that there is tight crosstalk between viral replication and host differentiation programs. In this review, we discuss the regulation of the HPV lifecycle controlled by the differentiation program of the host cells. PMID:22536200

Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects.

PubMed

Bill, Roslyn M; von der Haar, Tobias

2015-06-01

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

From microbiology to cell biology: when an intracellular bacterium becomes part of its host cell.

PubMed

McCutcheon, John P

2016-08-01

Mitochondria and chloroplasts are now called organelles, but they used to be bacteria. As they transitioned from endosymbionts to organelles, they became more and more integrated into the biochemistry and cell biology of their hosts. Work over the last 15 years has shown that other symbioses show striking similarities to mitochondria and chloroplasts. In particular, many sap-feeding insects house intracellular bacteria that have genomes that overlap mitochondria and chloroplasts in terms of size and coding capacity. The massive levels of gene loss in some of these bacteria suggest that they, too, are becoming highly integrated with their host cells. Understanding these bacteria will require inspiration from eukaryotic cell biology, because a traditional microbiological framework is insufficient for understanding how they work. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunohistochemical investigation of the necrotropic phase of the fungus Colletotrichum gloeosporioides in the biocontrol of Hemp sesbania (Sesbania exaltata; Papilionaceae)

USDA-ARS?s Scientific Manuscript database

Fungal plant pathogens exert much of their effect on plant cells through alterations in the host cell walls. However, biochemical proof for this change is difficult because of the relatively small number of cells that are affected by the pathogen in the bulk of host tissue. In this study, we examine...

Coronavirus Attachment and Replication

DTIC Science & Technology

1988-03-28

is also inhibited by protease treatment (Richter, 1976). Rhabdovirus binding to host cells was inhibited by phosholipase and neuraminadase, but not...protease treatment of host cells. Therefore the rhabdovirus receptor was postulated to be a glyco- or phospholipid (Wunner et &,. 1984). More...Rabies virus and VSV competed for binding on cultured neural and non-neural cells, suggesting a common receptor for rhabdoviruses (Wunner et g1, 1984

Just a Spoonful of Sugar, HTLV-1 Style.

PubMed

Taylor, Naomi

2017-11-16

Host cell metabolism regulates viral infection. In this issue of Cell Chemical Biology, Kulkarni et al. (2017) reveal the importance of oxygen concentrations and glycolysis in the reactivation of human T cell leukemia virus (HTLV-1). Identifying the host metabolic networks that regulate infection will foster our understanding of HTLV-1-associated pathologies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Marrow transplantation in the treatment of a murine heritable hemolytic anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barker, J.E.; McFarland-Starr, E.C.

1989-05-15

Mice with hemolytic anemia, sphha/sphha, have extremely fragile RBCs with a lifespan of approximately one day. Neither splenectomy nor simple transplantation of normal marrow after lethal irradiation cures the anemia but instead causes rapid deterioration and death of the mutant unless additional prophylactic procedures are used. In this report, we show that normal marrow transplantation preceded by sublethal irradiation increases but does not normalize RBC count. The mutant RBCs but not all the WBCs are replaced by donor cells. Splenectomy of the improved recipient causes a dramatic decrease in RBC count, indicating that the mutant spleen is a site ofmore » donor-origin erythropoiesis as well as of RBC destruction. Injections of iron dextran did not improve RBC counts. Transplantation of primary recipient marrow cells into a secondary host with a heritable stem cell deficiency (W/Wv) corrects the defect caused by residence of the normal cells in the sphha/sphha host. The original +/+ donor cells replace the RBCs of the secondary host, and the RBC count is normalized. Results indicate that the environment in the sphha/sphha host is detrimental to normal (as well as mutant) erythroid cells but the restriction is not transmitted.« less

Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry.

PubMed

Ofir-Birin, Yifat; Abou Karam, Paula; Rudik, Ariel; Giladi, Tal; Porat, Ziv; Regev-Rudzki, Neta

2018-01-01

Extracellular vesicles are essential for long distance cell-cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites ( Plasmodium falciparum, Pf ), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo's internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf -DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host-pathogen and pathogen-pathogen systems.

The Role of Oligosaccharides in Host-Microbial Interactions for Human Health.

PubMed

Ross, Sarah A; Lane, Jonathan A; Marotta, Mariarosaria; Kavanaugh, Devon; Ryan, Joseph Thomas; Joshi, Lokesh; Hickey, Rita M

Milk oligosaccharides have many associated bioactivities which can contribute to human health and offer protective properties to the host. Such bioactivities include anti-infective properties whereby oligosaccharides interact with bacterial cells and prevent adhesion to the host and subsequent colonization. Milk oligosaccharides have also been shown to alter the glycosylation of intestinal cells, leading to a reduction in pathogenic colonization. In addition, these sugars promote adhesion of commensal bacterial strains to host cells as well as possessing the ability to alter mucin expression in intestinal cells and improve barrier function. The ability of milk oligosaccharides to alter the transcriptome of both commensal bacterial strains and intestinal epithelial cells has also been revealed, indicating the potential of many cell types to detect the presence of milk oligosaccharides and respond accordingly at the genetic level. Interestingly, domestic animal milk may provide a bioactive source of oligosaccharides for formula supplementation with the aim of emulating the gold standard that is human milk. Overall, this review highlights the ability of milk oligosaccharides to promote health in a variety of ways, for example, through direct bacterial interactions, immunomodulatory activities, promotion of gut barrier function, and induction of protective transcriptional responses.

Necroptotic debris including damaged mitochondria elicits sepsis-like syndrome during late-phase tularemia.

PubMed

Singh, Anju; Periasamy, Sivakumar; Malik, Meenakshi; Bakshi, Chandra Shekhar; Stephen, Laurie; Ault, Jeffrey G; Mannella, Carmen A; Sellati, Timothy J

2017-01-01

Infection with Francisella tularensis ssp. tularensis ( Ft ) strain SchuS4 causes an often lethal disease known as tularemia in rodents, non-human primates, and humans. Ft subverts host cell death programs to facilitate their exponential replication within macrophages and other cell types during early respiratory infection (⩽72 h). The mechanism(s) by which cell death is triggered remains incompletely defined, as does the impact of Ft on mitochondria, the host cell's organellar 'canary in a coal mine'. Herein, we reveal that Ft infection of host cells, particularly macrophages and polymorphonuclear leukocytes, drives necroptosis via a receptor-interacting protein kinase 1/3-mediated mechanism. During necroptosis mitochondria and other organelles become damaged. Ft -induced mitochondrial damage is characterized by: (i) a decrease in membrane potential and consequent mitochondrial oncosis or swelling, (ii) increased generation of superoxide radicals, and (iii) release of intact or damaged mitochondria into the lung parenchyma. Host cell recognition of and response to released mitochondria and other damage-associated molecular patterns engenders a sepsis-like syndrome typified by production of TNF, IL-1 β , IL-6, IL-12p70, and IFN- γ during late-phase tularemia (⩾72 h), but are absent early during infection.

Measles virus: Background and oncolytic virotherapy.

PubMed

Bhattacharjee, Sankhajit; Yadava, Pramod Kumar

2018-03-01

Measles is a highly transmissible disease caused by measles virus and remains a major cause of child mortality in developing countries. Measles virus nucleoprotein (N) encapsidates the RNA genome of the virus for providing protection from host cell endonucleases and for specific recognition of viral RNA as template for transcription and replication. This protein is over-expressed at the time of viral replication. The C-terminal of N protein is intrinsically disordered, which enables this protein to interact with several host cell proteins. It was previously proved in our laboratory that N expressing human cancerous cells undergo programmed cell death because of reactive oxygen species (ROS) generation as well as Caspase 3 activation. The phosphoprotein (P) along with N protein enclosed viral genomic RNA forming a ribonucleoprotein complex (RNP). It also establishes interaction with the large protein (L) i.e. viral RNA dependent RNA polymerase to ensure viral replication within host cells. The host cell receptors of this virus are CD46, SLAM/CD150 and PVRL4. Measles virus is latently oncotropic in nature and possesses oncolytic property by syncytia formation. We try to highlight the application of this property in developing a virotherapeutic vehicle.

Pre-existing malignancy results in increased prevalence of distinct populations of CD4+ T cells during sepsis.

PubMed

Xie, Jianfeng; Robertson, Jennifer M; Chen, Ching-Wen; Zhang, Wenxiao; Coopersmith, Craig M; Ford, Mandy L

2018-01-01

The presence of pre-existing malignancy in murine hosts results in increased immune dysregulation and risk of mortality following a septic insult. Based on the known systemic immunologic changes that occur in cancer hosts, we hypothesized that the presence of pre-existing malignancy would result in phenotypic and functional changes in CD4+ T cell responses following sepsis. In order to conduct a non-biased, unsupervised analysis of phenotypic differences between CD4+ T cell compartments, cohorts of mice were injected with LLC1 tumor cells and tumors were allowed to grow for 3 weeks. These cancer hosts and age-matched non-cancer controls were then subjected to CLP. Splenocytes were harvested at 24h post CLP and flow cytometry and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) were used to analyze populations of CD4+ cells most different between the two groups. Results indicated that relative to non-cancer controls, cancer mice contained more resting memory CD4+ T cells, more activated CD4+ effectors, and fewer naïve CD4+ T cells during sepsis, suggesting that the CD4+ T cell compartment in cancer septic hosts is one of increased activation and differentiation. Moreover, cancer septic animals exhibited expansion of two distinct subsets of CD4+ T cells relative to previously healthy septic controls. Specifically, we identified increases in both a PD-1hi population and a distinct 2B4hi BTLAhi LAG-3hi population in cancer septic animals. By combining phenotypic analysis of exhaustion markers with functional analysis of cytokine production, we found that PD-1+ CD4+ cells in cancer hosts failed to make any cytokines following CLP, while the 2B4+ PD-1lo cells in cancer mice secreted increased TNF during sepsis. In sum, the immunophenotypic landscape of cancer septic animals is characterized by both increased CD4+ T cell activation and exhaustion, findings that may underlie the observed increased mortality in mice with pre-existing malignancy following sepsis.

Pre-existing malignancy results in increased prevalence of distinct populations of CD4+ T cells during sepsis

PubMed Central

Xie, Jianfeng; Robertson, Jennifer M.; Chen, Ching-wen; Zhang, Wenxiao

2018-01-01

The presence of pre-existing malignancy in murine hosts results in increased immune dysregulation and risk of mortality following a septic insult. Based on the known systemic immunologic changes that occur in cancer hosts, we hypothesized that the presence of pre-existing malignancy would result in phenotypic and functional changes in CD4+ T cell responses following sepsis. In order to conduct a non-biased, unsupervised analysis of phenotypic differences between CD4+ T cell compartments, cohorts of mice were injected with LLC1 tumor cells and tumors were allowed to grow for 3 weeks. These cancer hosts and age-matched non-cancer controls were then subjected to CLP. Splenocytes were harvested at 24h post CLP and flow cytometry and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) were used to analyze populations of CD4+ cells most different between the two groups. Results indicated that relative to non-cancer controls, cancer mice contained more resting memory CD4+ T cells, more activated CD4+ effectors, and fewer naïve CD4+ T cells during sepsis, suggesting that the CD4+ T cell compartment in cancer septic hosts is one of increased activation and differentiation. Moreover, cancer septic animals exhibited expansion of two distinct subsets of CD4+ T cells relative to previously healthy septic controls. Specifically, we identified increases in both a PD-1hi population and a distinct 2B4hi BTLAhi LAG-3hi population in cancer septic animals. By combining phenotypic analysis of exhaustion markers with functional analysis of cytokine production, we found that PD-1+ CD4+ cells in cancer hosts failed to make any cytokines following CLP, while the 2B4+ PD-1lo cells in cancer mice secreted increased TNF during sepsis. In sum, the immunophenotypic landscape of cancer septic animals is characterized by both increased CD4+ T cell activation and exhaustion, findings that may underlie the observed increased mortality in mice with pre-existing malignancy following sepsis. PMID:29338031

Transcriptional Profiling Confirms the Therapeutic Effects of Mast Cell Stabilization in a Dengue Disease Model.

PubMed

Morrison, Juliet; Rathore, Abhay P S; Mantri, Chinmay K; Aman, Siti A B; Nishida, Andrew; St John, Ashley L

2017-09-15

There are no approved therapeutics for the treatment of dengue disease despite the global prevalence of dengue virus (DENV) and its mosquito vectors. DENV infections can lead to vascular complications, hemorrhage, and shock due to the ability of DENV to infect a variety of immune and nonimmune cell populations. Increasingly, studies have implicated the host response as a major contributor to severe disease. Inflammatory products of various cell types, including responding T cells, mast cells (MCs), and infected monocytes, can contribute to immune pathology. In this study, we show that the host response to DENV infection in immunocompetent mice recapitulates transcriptional changes that have been described in human studies. We found that DENV infection strongly induced metabolic dysregulation, complement signaling, and inflammation. DENV also affected the immune cell content of the spleen and liver, enhancing NK, NKT, and CD8 + T cell activation. The MC-stabilizing drug ketotifen reversed many of these responses without suppressing memory T cell formation and induced additional changes in the transcriptome and immune cell composition of the spleen, consistent with reduced inflammation. This study provides a global transcriptional map of immune activation in DENV target organs of an immunocompetent host and supports the further development of targeted immunomodulatory strategies to treat DENV disease. IMPORTANCE Dengue virus (DENV), which causes febrile illness, is transmitted by mosquito vectors throughout tropical and subtropical regions of the world. Symptoms of DENV infection involve damage to blood vessels and, in rare cases, hemorrhage and shock. Currently, there are no targeted therapies to treat DENV infection, but it is thought that drugs that target the host immune response may be effective in limiting symptoms that result from excessive inflammation. In this study, we measured the host transcriptional response to infection in multiple DENV target organs using a mouse model of disease. We found that DENV infection induced metabolic dysregulation and inflammatory responses and affected the immune cell content of the spleen and liver. The use of the mast cell stabilization drug ketotifen reversed many of these responses and induced additional changes in the transcriptome and immune cell repertoire that contribute to decreased dengue disease. Copyright © 2017 Morrison et al.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

PubMed

Amatngalim, Gimano D; Schrumpf, Jasmijn A; Dishchekenian, Fernanda; Mertens, Tinne C J; Ninaber, Dennis K; van der Linden, Abraham C; Pilette, Charles; Taube, Christian; Hiemstra, Pieter S; van der Does, Anne M

2018-04-01

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections. Copyright ©ERS 2018.

Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

PubMed

Lin, Yi-Han; Machner, Matthias P

2017-06-15

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.

Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

PubMed Central

Krishnamurthy, Shruthi; Deng, Bin; del Rio, Roxana; Buchholz, Kerry R.; Treeck, Moritz; Urban, Siniša; Boothroyd, John; Lam, Ying-Wai

2016-01-01

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T. gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. PMID:27624124

A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

PubMed

Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J

2015-02-01

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

Interaction of Staphylococcus aureus persister cells with the host when in a persister state and following awakening.

PubMed

Mina, Elin G; Marques, Cláudia N H

2016-08-10

Persister cells, a tolerant cell sub-population, are commonly associated with chronic and recurrent infections. However, little is known about their ability to actually initiate or establish an infection, become virulent and cause pathogenicity within a host. Here we investigated whether Staphylococcus aureus persister cells initiate an infection and are recognized by macrophages, while in a persister cell status, and upon awakening due to exposure to cis-2-decenoic acid (cis-DA). Our results show that S. aureus persister cells are not able to initiate infections in A. thaliana and present significantly reduced virulence towards C. elegans compared to total populations. In contrast, awakened S. aureus persister cells are able to initiate infections in A. thaliana and in C. elegans albeit, with lower mortality than total population. Furthermore, exposure of S. aureus persister cells to cis-DA led to a loss of tolerance to ciprofloxacin, and an increase of the bacterial fluorescence to levels found in total population. In addition, macrophage engulfment of persister cells was significantly lower than engulfment of total population, both before and following awakening. Overall our findings indicate that upon awakening of a persister population the cells regain their ability to infect hosts despite the absence of an increased immune response.

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

PubMed Central

2015-01-01

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals. PMID:26064970

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection.

PubMed

Ufimtseva, Elena

2015-01-01

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals.

Localization of azithromycin in Toxoplasma gondii-infected cells.

PubMed Central

Schwab, J C; Cao, Y; Slowik, M R; Joiner, K A

1994-01-01

Agents effective against intracellular pathogens must enter infected cells, crossing vacuolar membranes surrounding the organisms and then penetrating into the microbe and localizing to the microbial target site. We have characterized these parameters for azithromycin entry into Toxoplasma gondii-infected Chinese hamster ovary cells and murine macrophage-like J774 cells. Azithromycin uptake into infected host cells was concentrative and was dependent upon proton gradients. Subcellular fractionation of azithromycin-loaded infected CHO cells demonstrated > 95% intracellular drug in host cell lysosomes and cytosol, with < 5% associated with the parasite. Uptake of azithromycin into the T. gondii vacuole increased if parasites were coated with antibody prior to internalization by murine J774 cells, conditions which result in the formation of acidified phagolysosomes. No redistribution or retention of azithromycin in the parasite was observed when drug efflux from antibiotic-loaded infected CHO cells was monitored. Azithromycin entry into extracellular T. gondii was concentrative, was temperature and pH dependent, and was not different when azithromycin-sensitive and -resistant parasites were compared. These results demonstrate that azithromycin concentrates primarily in acidified compartments in parasites and host cells. The high concentration of azithromycin within these compartments may not be biologically relevant to inhibition of intracellular parasite growth by this agent. PMID:7979295

IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection.

PubMed

Booty, Matthew G; Barreira-Silva, Palmira; Carpenter, Stephen M; Nunes-Alves, Cláudio; Jacques, Miye K; Stowell, Britni L; Jayaraman, Pushpa; Beamer, Gillian; Behar, Samuel M

2016-11-07

IL-21 is produced predominantly by activated CD4 + T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (γ c ) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8 + T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4 + and CD8 + T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R -/- mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R -/- T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis.

IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection

PubMed Central

Booty, Matthew G.; Barreira-Silva, Palmira; Carpenter, Stephen M.; Nunes-Alves, Cláudio; Jacques, Miye K.; Stowell, Britni L.; Jayaraman, Pushpa; Beamer, Gillian; Behar, Samuel M.

2016-01-01

IL-21 is produced predominantly by activated CD4+ T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (γc) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8+ T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4+ and CD8+ T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R−/− mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R−/− T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis. PMID:27819295

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Offerdahl, Danielle K.

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranesmore » to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60–100 nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20–30 nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. - Highlights: •First electron tomography of Zika virus cytoarchitecture. •Comparison of Zika virus infection in human neuroblastoma and mosquito cells. •Ultrastructure of Zika virus infection in human neuroblastoma and mosquito cells.« less

Memory T cells: A helpful guard for allogeneic hematopoietic stem cell transplantation without causing graft-versus-host disease.

PubMed

Huang, Wei; Chao, Nelson J

2017-12-01

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (AHSCT) and the major cause of nonrelapse morbidity and mortality of AHSCT. In AHSCT, donor T cells facilitate hematopoietic stem cell (HSC) engraftment, contribute to anti-infection immunity, and mediate graft-versus-leukemia (GVL) responses. However, activated alloreactive T cells also attack recipient cells in vital organs, leading to GVHD. Different T-cell subsets, including naïve T (T N ) cells, memory T (T M ) cells, and regulatory T (T reg ) cells mediate different forms of GVHD and GVL; T N cells mediate severe GVHD, whereas T M cells do not cause GVHD, but preserve T-cell function including GVL. In addition, metabolic reprogramming controls T-cell differentiation and activation in these disease states. This minireview focuses on the role and the related mechanisms of T M cells in AHSCT, and the potential manipulation of T cells in AHSCT. Copyright © 2017 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

PubMed

Blomström, Anne-Lie; Gu, Quan; Barry, Gerald; Wilkie, Gavin; Skelton, Jessica K; Baird, Margaret; McFarlane, Melanie; Schnettler, Esther; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

2015-04-19

Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material.

PubMed

Bonfante-Fasolo, P; Vian, B; Perotto, S; Faccio, A; Knox, J P

1990-03-01

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

PubMed Central

Prado, Monica; Eickel, Nina; De Niz, Mariana; Heitmann, Anna; Agop-Nersesian, Carolina; Wacker, Rahel; Schmuckli-Maurer, Jacqueline; Caldelari, Reto; Janse, Chris J; Khan, Shahid M; May, Jürgen; Meyer, Christian G; Heussler, Volker T

2015-01-01

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition. PMID:26208778

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruegg, Thomas Lawrence; Thelen, Michael P.

The present invention provides for a method of genetically modifying microorganisms to enhance resistance to ionic liquids, host cells genetically modified in accordance with the methods, and methods of using the host cells in a reaction comprising biomass that has been pretreated with ionic liquids.

A Novel Secreted Protein, MYR1, Is Central to Toxoplasma ’s Manipulation of Host Cells

DOE PAGES

Franco, Magdalena; Panas, Michael W.; Marino, Nicole D.; ...

2016-02-02

ABSTRACT The intracellular protozoanToxoplasma gondiidramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc–GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification ofMYR1(Mycregulation1;TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion ofMYR1revealed that in additionmore » to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability ofToxoplasmatachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocatesToxoplasmaeffectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient inMYR1were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in howToxoplasmadelivers effector proteins to the infected host cell and that this is crucial to virulence. IMPORTANCEToxoplasma gondiiis an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell withToxoplasmatachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.« less

Failure in generating hemopoietic stem cells is the primary cause of death from cytomegalovirus disease in the immunocompromised host

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mutter, W.; Reddehase, M.J.; Busch, F.W.

1988-05-01

We have shown in a murine model system for cytomegalovirus (CMV) disease in the immunocompromised host that CMV infection interferes with the earliest detectable step in hemopoiesis, the generation of the stem cell CFU-S-I, and thereby prevents the autoreconstitution of bone marrow after sublethal irradiation. The antihemopoietic effect could not be ascribed to a direct infection of stem cells. The failure in hemopoiesis was prevented by adoptive transfer of antiviral CD8+ T lymphocytes and could be overcome by syngeneic bone marrow transplantation. CD8+ T lymphocytes and bone marrow cells both mediated survival, although only CD8+ T lymphocytes were able tomore » limit virus multiplication in host tissues. We concluded that not the cytopathic effect of virus replication in host tissues, but the failure in hemopoiesis, is the primary cause of death in murine CMV disease.« less

Murine Leukemia Viruses: Objects and Organisms

PubMed Central

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera. PMID:22312342

Murine leukemia viruses: objects and organisms.

PubMed

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes-protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.

IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals.

PubMed

Kanda, Takehiro; Ozawa, Makoto; Tsukiyama-Kohara, Kyoko

2016-03-31

Foot-and-mouth disease virus (FMDV) possess a positive sense, single stranded RNA genome. Internal ribosomal entry site (IRES) element exists within its 5' untranslated region (5'UTR) of the viral RNA. Translation of the viral RNA is initiated by internal entry of the 40S ribosome within the IRES element. This process is facilitated by cellular factors known as IRES trans-acting factors (ITAFs). Foot-and-mouth disease (FMD) is host-restricted disease for cloven-hoofed animals such as cattle and pigs, but the factors determining the host range have not been identified yet. Although, ITAFs are known to promote IRES-mediated translation, these findings were confirmed only in cells derived from FMDV-insusceptible animals so far. We evaluated and compared the IRES-mediated translation activities among cell lines derived from four different animal species using bicistronic luciferase reporter plasmid, which possesses an FMDV-IRES element between Renilla and Firefly luciferase genes. Furthermore, we analyzed the effect of the cellular factors on IRES-mediated translation by silencing the cellular factors using siRNA in both FMDV-susceptible and -insusceptible animal cells. Our data indicated that IRES-mediated translational activity was not linked to FMDV host range. ITAF45 promoted IRES-mediated translation in all cell lines, and the effects of poly-pyrimidine tract binding protein (PTB) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were observed only in FMDV-susceptible cells. Thus, PTB and 4E-BP1 may influence the host range of FMDV. IRES-mediated translation activity of FMDV was not predictive of its host range. ITAF45 promoted IRES-mediated translation in all cells, and the effects of PTB and 4E-BP1 were observed only in FMDV-susceptible cells.

Leptospira interrogans Binds to Cadherins

PubMed Central

Evangelista, Karen; Franco, Ricardo; Schwab, Andrew; Coburn, Jenifer

2014-01-01

Leptospirosis, caused by pathogenic species of Leptospira, is the most widespread zoonosis and has emerged as a major public health problem worldwide. The adhesion of pathogenic Leptospira to host cells, and to extracellular matrix (ECM) components, is likely to be necessary for the ability of leptospires to penetrate, disseminate and persist in mammalian host tissues. Previous work demonstrated that pathogenic L. interrogans binds to host cells more efficiently than to ECM. Using two independent screening methods, mass spectrometry and protein arrays, members of the cadherin family were identified as potential L. interrogans receptors on mammalian host surfaces. We focused our investigation on vascular endothelial (VE)-cadherin, which is widely expressed on endothelia and is primarily responsible for endothelial cell-cell adhesion. Monolayers of EA.hy926 and HMEC-1 endothelial cells produce VE-cadherin, bind L. interrogans in vitro, and are disrupted upon incubation with the bacteria, which may reflect the endothelial damage seen in vivo. Dose-dependent and saturable binding of L. interrogans to the purified VE-cadherin receptor was demonstrated and pretreatment of purified receptor or endothelial cells with function-blocking antibody against VE-cadherin significantly inhibited bacterial attachment. The contribution of VE-cadherin to leptospiral adherence to host endothelial cell surfaces is biologically significant because VE-cadherin plays an important role in maintaining the barrier properties of the vasculature. Attachment of L. interrogans to the vasculature via VE-cadherin may result in vascular damage, facilitating the escape of the pathogen from the bloodstream into different tissues during disseminated infection, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to describe a mammalian cell surface protein as a receptor for L. interrogans. PMID:24498454

Cellular transfer of magnetic nanoparticles via cell microvesicles: impact on cell tracking by magnetic resonance imaging.

PubMed

Silva, Amanda K Andriola; Wilhelm, Claire; Kolosnjaj-Tabi, Jelena; Luciani, Nathalie; Gazeau, Florence

2012-05-01

Cell labeling with magnetic nanoparticles can be used to monitor the fate of transplanted cells in vivo by magnetic resonance imaging. However, nanoparticles initially internalized in administered cells might end up in other cells of the host organism. We investigated a mechanism of intercellular cross-transfer of magnetic nanoparticles to different types of recipient cells via cell microvesicles released under cellular stress. Three cell types (mesenchymal stem cells, endothelial cells and macrophages) were labeled with 8-nm iron oxide nanoparticles. Then cells underwent starvation stress, during which they produced microvesicles that were subsequently transferred to unlabeled recipient cells. The analysis of the magnetophoretic mobility of donor cells indicated that magnetic load was partially lost under cell stress. Microvesicles shed by stressed cells participated in the release of magnetic label. Moreover, such microvesicles were uptaken by naïve cells, resulting in cellular redistribution of nanoparticles. Iron load of recipient cells allowed their detection by MRI. Cell microvesicles released under stress may be disseminated throughout the organism, where they can be uptaken by host cells. The transferred cargo may be sufficient to allow MRI detection of these secondarily labeled cells, leading to misinterpretations of the effectiveness of transplanted cells.

Smuggling across the border: how arthropod-borne pathogens evade and exploit the host defense system of the skin.

PubMed

Bernard, Quentin; Jaulhac, Benoit; Boulanger, Nathalie

2014-05-01

The skin is a critical barrier between hosts and pathogens in arthropod-borne diseases. It harbors many resident cells and specific immune cells to arrest or limit infections by secreting inflammatory molecules or by directly killing pathogens. However, some pathogens are able to use specific skin cells and arthropod saliva for their initial development, to hide from the host immune system, and to establish persistent infection in the vertebrate host. A better understanding of the initial mechanisms taking place in the skin should allow the development of new strategies to fight these vector-borne pathogens that are spread worldwide and are of major medical importance.

Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation.

PubMed

Ost, Kyla S; Esher, Shannon K; Leopold Wager, Chrissy M; Walker, Louise; Wagener, Jeanette; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

2017-01-31

Compared to other fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune cells. To explore fungal cellular features involved in immune avoidance, we characterized cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ cell wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both cell wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of cell wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans cell wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune cells detect C. neoformans and other fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan. Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their cell surfaces to mask immunogenic epitopes. We have created a fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its cell wall, resulting in a dramatic immune reaction during infection. This mutant cell wall is defective in hiding important cell wall components, such as the chito-oligomers chitin and chitosan. By creating a series of cell wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection. Copyright © 2017 Ost et al.

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins.

PubMed

Hewezi, Tarek

2015-10-01

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. © 2015 American Society of Plant Biologists. All Rights Reserved.

The immunological contribution of NF-κB within the tumor microenvironment: A potential protective role of zinc as an anti-tumor agent

PubMed Central

Bao, Bin; Thakur, Archana; Li, Yiwei; Ahmad, Aamir; Azmi, Asfar S.; Banerjee, Sanjeev; Kong, Dejuan; Ali, Shadan; Lum, Lawrence G.; Sarkar, Fazlul H.

2013-01-01

Over decades, cancer treatment has been mainly focused on targeting cancer cells and not much attention to host tumor microenvironment. Recent advances suggest that the tumor microenvironment requires in-depth investigation for understanding the interactions between tumor cell biology and immunobiology in order to optimize therapeutic approaches. Tumor microenvironment consists of cancer cells and tumor associated reactive fibroblasts, infiltrating non-cancer cells, secreted soluble factors or molecules, and non-cellular support materials. Tumor associated host immune cells such as Th1, Th2, Th17, regulatory cells, dendritic cells, macrophages, and myeloid-derived suppressor cells are major components of the tumor microenvironment. Accumulating evidence suggests that these tumor associated immune cells may play important roles in cancer development and progression. However, the exact functions of these cells in the tumor microenvironment are poorly understood. In the tumor microenvironment, NF-κB plays an important role in cancer development and progression because this is a major transcription factor which regulates immune functions within the tumor microenvironment. In this review, we will focus our discussion on the immunological contribution of NF-κB in tumor associated host immune cells within the tumor microenvironment. We will also discuss the potential protective role of zinc, a well-known immune response mediator, in the regulation of these immune cells and cancer cells in the tumor microenvironment especially because zinc could be useful for conditioning the tumor microenvironment toward innovative cancer therapy. PMID:22155217

HIV-1 and hijacking of the host immune system: the current scenario.

PubMed

Imran, Muhammad; Manzoor, Sobia; Saalim, Muhammad; Resham, Saleha; Ashraf, Javed; Javed, Aneela; Waqar, Ahmed Bilal

2016-10-01

Human immunodeficiency virus (HIV) infection is a major health burden across the world which leads to the development of acquired immune deficiency syndrome (AIDS). This review article discusses the prevalence of HIV, its major routes of transmission, natural immunity, and evasion from the host immune system. HIV is mostly prevalent in Sub-Saharan Africa and low income countries. It is mostly transmitted by sharing syringe needles, blood transfusion, and sexual routes. The host immune system is categorized into three main types; the innate, the adaptive, and the intrinsic immune system. Regarding the innate immune system against HIV, the key players are mucosal membrane, dendritic cells (DCs), complement system, interferon, and host Micro RNAs. The major components of the adaptive immune system exploited by HIV are T cells mainly CD4+ T cells and B cells. The intrinsic immune system confronted by HIV involves (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) APOBEC3G, tripartite motif 5-α (TRIM5a), terherin, and (SAM-domain HD-domain containing protein) SAMHD1. HIV-1 efficiently interacts with the host immune system, exploits the host machinery, successfully replicates and transmits from one cell to another. Further research is required to explore evasion strategies of HIV to develop novel therapeutic approaches against HIV. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Theileria induces oxidative stress and HIF1α activation that are essential for host leukocyte transformation.

PubMed

Medjkane, S; Perichon, M; Marsolier, J; Dairou, J; Weitzman, J B

2014-04-03

Complex links between infection and cancer suggest that we still can learn much about tumorigenesis by studying how infectious agents hijack the host cell machinery. We studied the effects of an intracellular parasite called Theileria that infects bovine leukocytes and turns them into invasive cancer-like cells. We investigated the host cells pathways that are deregulated in infected leukocytes and might link infection and lymphoproliferative disease. We show that intracellular Theileria parasites drive a Warburg-like phenotype in infected host leukocytes, characterized by increased expression of metabolic regulators, increased glucose uptake and elevated lactate production, which were lost when the parasite was eliminated. The cohabitation of the parasites within the host cells leads to disruption of the redox balance (as measured by reduced/oxidized glutathione ratio) and elevated ROS (reactive oxygen species) levels, associated with chronic stabilization of the hypoxia-inducible factor 1 alpha (HIF1α). Inhibition of HIF1α (pharmacologically or genetically), or treatment with antioxidants, led to a marked reduction in expression of aerobic glycolytic genes and inhibited the transformed phenotype. These data show that stabilization of HIF1α, following increased ROS production, modulates host glucose metabolism and is critical for parasite-induced transformation. Our study expands knowledge about the molecular strategy used by the parasite Theileria to induce the transformed phenotypes of infected cells via reprogramming of glucose metabolism and redox signaling.

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

PubMed Central

Zhou, Yan; Dong, Na; Hu, Liyan; Shao, Feng

2013-01-01

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells. PMID:23469023

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

PubMed

Bouklas, Tejas; Alonso-Crisóstomo, Luz; Székely, Tamás; Diago-Navarro, Elizabeth; Orner, Erika P; Smith, Kalie; Munshi, Mansa A; Del Poeta, Maurizio; Balázsi, Gábor; Fries, Bettina C

2017-05-01

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2016-04-01

The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes.

PubMed

Florentino, Pilar T V; Real, Fernando; Orikaza, Cristina M; da Cunha, Julia P C; Vitorino, Francisca N L; Cordero, Esteban M; Sobreira, Tiago J P; Mortara, Renato A

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas' disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo . Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas' disease.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

PubMed Central

Florentino, Pilar T. V.; Real, Fernando; Orikaza, Cristina M.; da Cunha, Julia P. C.; Vitorino, Francisca N. L.; Cordero, Esteban M.; Sobreira, Tiago J. P.; Mortara, Renato A.

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. PMID:29692765

A self-lysis pathway that enhances the virulence of a pathogenic bacterium.

PubMed

McFarland, Kirsty A; Dolben, Emily L; LeRoux, Michele; Kambara, Tracy K; Ramsey, Kathryn M; Kirkpatrick, Robin L; Mougous, Joseph D; Hogan, Deborah A; Dove, Simon L

2015-07-07

In mammalian cells, programmed cell death (PCD) plays important roles in development, in the removal of damaged cells, and in fighting bacterial infections. Although widespread among multicellular organisms, there are relatively few documented instances of PCD in bacteria. Here we describe a potential PCD pathway in Pseudomonas aeruginosa that enhances the ability of the bacterium to cause disease in a lung infection model. Activation of the system can occur in a subset of cells in response to DNA damage through cleavage of an essential transcription regulator we call AlpR. Cleavage of AlpR triggers a cell lysis program through de-repression of the alpA gene, which encodes a positive regulator that activates expression of the alpBCDE lysis cassette. Although this is lethal to the individual cell in which it occurs, we find it benefits the population as a whole during infection of a mammalian host. Thus, host and pathogen each may use PCD as a survival-promoting strategy. We suggest that activation of the Alp cell lysis pathway is a disease-enhancing response to bacterial DNA damage inflicted by the host immune system.

Candida albicans Iff11, a secreted protein required for cell wall structure and virulence.

PubMed

Bates, Steven; de la Rosa, José M; MacCallum, Donna M; Brown, Alistair J P; Gow, Neil A R; Odds, Frank C

2007-06-01

The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be O glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence.

Magnetofection™ of NMDA Receptor Subunits GluN1 and GluN2A Expression Vectors in Non-Neuronal Host Cells.

PubMed

Bruneau, Nadine; Szepetowski, Pierre

2017-01-01

The functional study of reconstituted NMDA receptors (NMDARs) in host cells requires that the corresponding vectors for the expression of the NMDAR subunits are co-transfected with high efficiency. Magnetofection™ is a technology used to deliver nucleic acids to cells. It is driven and site-specifically guided by the attractive forces of magnetic fields acting on magnetic nanoparticles that are associated with nucleic acid vectors. In magnetofection™, cationic lipids form self-assembled complexes with the nucleic acid vectors of interest. Those complexes are then associated with magnetic nanoparticles that are concentrated at the surface of cultured cells by applying a permanent magnetic field. Magnetofection™ is a simple method to transfect cultured cells with high transfection rates. Satisfactory expression levels are obtained with very low amounts of nucleic acid vector. Moreover, incubation time with host cells is less than 1 h, as compared with the several hours needed with standard transfection assays.

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner

PubMed Central

Hughes, K. R.; Harnisch, L. C.; Alcon-Giner, C.; Mitra, S.; Wright, C. J.; Ketskemety, J.

2017-01-01

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi—a process termed ‘cell shedding’. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. PMID:28123052

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

PubMed

Hughes, K R; Harnisch, L C; Alcon-Giner, C; Mitra, S; Wright, C J; Ketskemety, J; van Sinderen, D; Watson, A J M; Hall, L J

2017-01-01

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. © 2017 The Authors.

Protozoa lectins and their role in host-pathogen interactions.

PubMed

Singh, Ram Sarup; Walia, Amandeep Kaur; Kanwar, Jagat Rakesh

2016-01-01

Lectins are proteins/glycoproteins of non-immune origin that agglutinate red blood cells, lymphocytes, fibroblasts, etc., and bind reversibly to carbohydrates present on the apposing cells. They have at least two carbohydrate binding sites and their binding can be inhibited by one or more carbohydrates. Owing to carbohydrate binding specificity of lectins, they mediate cell-cell interactions and play role in protozoan adhesion and host cell cytotoxicity, thus are central to the pathogenic property of the parasite. Several parasitic protozoa possess lectins which mediate parasite adherence to host cells based on their carbohydrate specificities. These interactions could be exploited for development of novel therapeutics, targeting the adherence and thus helpful in eradicating wide spread of protozoan diseases. The current review highlights the present state knowledge with regard to protozoal lectins with an emphasis on their haemagglutination activity, carbohydrate specificity, characteristics and also their role in pathogenesis notably as adhesion molecules, thereby aiding the pathogen in disease establishment. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamic Electrochemical Control of Cell Capture-and-Release Based on Redox-Controlled Host-Guest Interactions.

PubMed

Gao, Tao; Li, Liudi; Wang, Bei; Zhi, Jun; Xiang, Yang; Li, Genxi

2016-10-18

Artificial control of cell adhesion on smart surface is an on-demand technique in areas ranging from tissue engineering, stem cell differentiation, to the design of cell-based diagnostic system. In this paper, we report an electrochemical system for dynamic control of cell catch-and-release, which is based on the redox-controlled host-guest interaction. Experimental results reveal that the interaction between guest molecule (ferrocene, Fc) and host molecule (β-cyclodextrin, β-CD) is highly sensitive to electrochemical stimulus. By applying a reduction voltage, the uncharged Fc can bind to β-CD that is immobilized at the electrode surface. Otherwise, it is disassociated from the surface as a result of electrochemical oxidation, thus releasing the captured cells. The catch-and-release process on this voltage-responsive surface is noninvasive with the cell viability over 86%. Moreover, because Fc can act as an electrochemical probe for signal readout, the integration of this property has further extended the ability of this system to cell detection. Electrochemical signal has been greatly enhanced for cell detection by introducing branched polymer scaffold that are carrying large quantities of Fc moieties. Therefore, a minimum of 10 cells can be analyzed. It is anticipated that such redox-controlled system can be an important tool in biological and biomedical research, especially for electrochemical stimulated tissue engineering and cell-based clinical diagnosis.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease.

PubMed

Kordelas, Lambros; Steckel, Nina-Kristin; Horn, Peter A; Beelen, Dietrich W; Rebmann, Vera

2016-10-27

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease

PubMed Central

Kordelas, Lambros; Steckel, Nina-Kristin; Horn, Peter A.; Beelen, Dietrich W.; Rebmann, Vera

2016-01-01

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention. PMID:27801784

Mutation in the Fas Pathway Impairs CD8+ T Cell Memory1

PubMed Central

Dudani, Renu; Russell, Marsha; van Faassen, Henk; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-γ and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections. PMID:18292515

Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

PubMed Central

Barretto, Naina; Sainz, Bruno; Hussain, Snawar

2014-01-01

ABSTRACT Hepatitis C virus (HCV) infects 180 million people worldwide and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. It has been shown that HCV can spread to naive cells using two distinct entry mechanisms, “cell-free” entry of infectious extracellular virions that have been released by infected cells and direct “cell-to-cell” transmission. Here, we examined host cell requirements for HCV spread and found that the cholesterol uptake receptor NPC1L1, which we recently identified as being an antiviral target involved in HCV cell-free entry/spread, is also required for the cell-to-cell spread. In contrast, the very low density lipoprotein (VLDL) pathway, which is required for the secretion of cell-free infectious virus and thus has been identified as an antiviral target for blocking cell-free virus secretion/spread, is not required for cell-to-cell spread. Noting that HCV cell-free and cell-to-cell spread share some common factors but not others, we tested the therapeutic implications of these observations and demonstrate that inhibitors that target cell factors required for both forms of HCV spread exhibit synergy when used in combination with interferon (a representative inhibitor of intracellular HCV production), while inhibitors that block only cell-free spread do not. This provides insight into the mechanistic basis of synergy between interferon and HCV entry inhibitors and highlights the broader, previously unappreciated impact blocking HCV cell-to-cell spread can have on the efficacy of HCV combination therapies. IMPORTANCE HCV can spread to naive cells using distinct mechanisms: “cell-free” entry of extracellular virus and direct “cell-to-cell” transmission. Herein, we identify the host cell HCV entry factor NPC1L1 as also being required for HCV cell-to-cell spread, while showing that the VLDL pathway, which is required for the secretion of cell-free infectious virus, is not required for cell-to-cell spread. While both these host factors are considered viable antiviral targets, we demonstrate that only inhibitors that block factors required for both forms of HCV entry/spread (i.e., NPC1L1) exhibit synergy when used in combination with interferon, while inhibitors that block factors required only for cell-free spread (i.e., VLDL pathway components) do not. Thus, this study advances our understanding of HCV cell-to-cell spread, provides mechanistic insight into the basis of drug synergy, and highlights inhibition of HCV spread as a previously unappreciated consideration in HCV therapy design. PMID:24554660

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

PubMed

Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

2009-09-01

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Chemerin regulation and role in host defense.

PubMed

Zabel, Brian A; Kwitniewski, Mateusz; Banas, Magdalena; Zabieglo, Katarzyna; Murzyn, Krzysztof; Cichy, Joanna

2014-01-01

Chemerin is a widely distributed multifunctional secreted protein implicated in immune cell migration, adipogenesis, osteoblastogenesis, angiogenesis, myogenesis, and glucose homeostasis. Chemerin message is regulated by nuclear receptor agonists, metabolic signaling proteins and intermediates, and proinflammatory cytokines. Following translation chemerin is secreted as an inactive pro-protein, and its secretion can be regulated depending on cell type. Chemerin bioactivity is largely dependent on carboxyl-terminal proteolytic processing and removal of inhibitory residues. Chemerin is abundant in human epidermis where it is well-placed to provide barrier protection. In host defense, chemerin plays dual roles as a broad spectrum antimicrobial protein and as a leukocyte attractant for macrophages, dendritic cells, and NK cells. Here we review the mechanisms underlying chemerin regulation and its function in host defense.

Method for fabrication of electrodes

DOEpatents

Jankowski, Alan F.; Morse, Jeffrey D.; Barksdale, Randy

2004-06-22

Described herein is a method to fabricate porous thin-film electrodes for fuel cells and fuel cell stacks. Furthermore, the method can be used for all fuel cell electrolyte materials which utilize a continuous electrolyte layer. An electrode layer is deposited on a porous host structure by flowing gas (for example, Argon) from the bottomside of the host structure while simultaneously depositing a conductive material onto the topside of the host structure. By controlling the gas flow rate through the pores, along with the process conditions and deposition rate of the thin-film electrode material, a film of a pre-determined thickness can be formed. Once the porous electrode is formed, a continuous electrolyte thin-film is deposited, followed by a second porous electrode to complete the fuel cell structure.

Biological macromolecules based targeted nanodrug delivery systems for the treatment of intracellular infections.

PubMed

Aparna, V; Shiva, M; Biswas, Raja; Jayakumar, R

2018-04-15

Intracellular infections are tricky to treat, the reason being the poor penetration of antibiotics/antimycotics into the microbial niche (host cell). Macrophages are primary targets of facultative and obligate intracellular bacteria/fungi to be abused as host cells. The need for drugs with better intracellular penetration led to the development of endocytosable drug carriers, which can cross the cell membrane of the host cells (macrophages) by imitating the entry path of the pathogens. Therefore, the drugs can be targeted to macrophages ensuring enhanced therapeutic effect. This review discusses the exploitation of various nanocarriers for targeted delivery of drugs to the macrophages in the last two decades. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of inflammation by microbiota interactions with the host.

PubMed

Blander, J Magarian; Longman, Randy S; Iliev, Iliyan D; Sonnenberg, Gregory F; Artis, David

2017-07-19

The study of the intestinal microbiota has begun to shift from cataloging individual members of the commensal community to understanding their contributions to the physiology of the host organism in health and disease. Here, we review the effects of the microbiome on innate and adaptive immunological players from epithelial cells and antigen-presenting cells to innate lymphoid cells and regulatory T cells. We discuss recent studies that have identified diverse microbiota-derived bioactive molecules and their effects on inflammation within the intestine and distally at sites as anatomically remote as the brain. Finally, we highlight new insights into how the microbiome influences the host response to infection, vaccination and cancer, as well as susceptibility to autoimmune and neurodegenerative disorders.

Proteomics reveal energy metabolism and mitogen-activated protein kinase signal transduction perturbation in human Borna disease virus Hu-H1-infected oligodendroglial cells.

PubMed

Liu, X; Yang, Y; Zhao, M; Bode, L; Zhang, L; Pan, J; Lv, L; Zhan, Y; Liu, S; Zhang, L; Wang, X; Huang, R; Zhou, J; Xie, P

2014-05-30

Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK-RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK-RSK complex of the Raf/MEK/ERK signaling cascade, and disturbs host cell proliferation possibly through impaired nuclear translocation of pERK, a finding which warrants further research. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing.

PubMed

Steuerman, Yael; Cohen, Merav; Peshes-Yaloz, Naama; Valadarsky, Liran; Cohn, Ofir; David, Eyal; Frishberg, Amit; Mayo, Lior; Bacharach, Eran; Amit, Ido; Gat-Viks, Irit

2018-06-01

The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model.

PubMed

Sabanero López, Myrna; Flores Villavicencio, Lérida L; Soto Arredondo, Karla; Barbosa Sabanero, Gloria; Villagómez-Castro, Julio César; Cruz Jiménez, Gustavo; Sandoval Bernal, Gerardo; Torres Guerrero, Haydee

Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. To evaluate the proteolytic activity of S. schenckii on epithelial cells. The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

PubMed

Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Macías, David; Gálvez, Victoria; Pastor, Angel M

2014-04-01

Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants. Copyright © 2014 Wiley Periodicals, Inc.

Emergent Behaviors from a Cellular Automaton Model for Invasive Tumor Growth in Heterogeneous Microenvironments

PubMed Central

Jiao, Yang; Torquato, Salvatore

2011-01-01

Understanding tumor invasion and metastasis is of crucial importance for both fundamental cancer research and clinical practice. In vitro experiments have established that the invasive growth of malignant tumors is characterized by the dendritic invasive branches composed of chains of tumor cells emanating from the primary tumor mass. The preponderance of previous tumor simulations focused on non-invasive (or proliferative) growth. The formation of the invasive cell chains and their interactions with the primary tumor mass and host microenvironment are not well understood. Here, we present a novel cellular automaton (CA) model that enables one to efficiently simulate invasive tumor growth in a heterogeneous host microenvironment. By taking into account a variety of microscopic-scale tumor-host interactions, including the short-range mechanical interactions between tumor cells and tumor stroma, degradation of the extracellular matrix by the invasive cells and oxygen/nutrient gradient driven cell motions, our CA model predicts a rich spectrum of growth dynamics and emergent behaviors of invasive tumors. Besides robustly reproducing the salient features of dendritic invasive growth, such as least-resistance paths of cells and intrabranch homotype attraction, we also predict nontrivial coupling between the growth dynamics of the primary tumor mass and the invasive cells. In addition, we show that the properties of the host microenvironment can significantly affect tumor morphology and growth dynamics, emphasizing the importance of understanding the tumor-host interaction. The capability of our CA model suggests that sophisticated in silico tools could eventually be utilized in clinical situations to predict neoplastic progression and propose individualized optimal treatment strategies. PMID:22215996

Genetic Resistance to Rhabdovirus Infection in Teleost Fish Is Paralleled to the Derived Cell Resistance Status

PubMed Central

Verrier, Eloi R.; Langevin, Christelle; Tohry, Corinne; Houel, Armel; Ducrocq, Vincent; Benmansour, Abdenour; Quillet, Edwige; Boudinot, Pierre

2012-01-01

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses. PMID:22514610

Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata.

PubMed

Suzuki, M; Tanaka, T

2007-10-01

The solitary endoparasitoid Meteorus pulchricornis can parasitize many lepidopteran host species successfully. In the case of parasitization of Pseudaletia separata, developmental duration of M. pulchricornis was 8-9 days from egg to larval emergence and 6 days from prepupa to adult emergence. Successful parasitism by M. pulchricornis decreased with host age. Following parasitization of day-0 4th host instar, the parasitoid embryo, whilst still enclosed in serosal cell membrane, hatched out of the egg chorion 2 days after oviposition. Subsequently, the 1st instar parasitoid emerged from the surrounding serosal cell membrane. Serosal cells dissociated and developed as teratocytes 3.5 days after oviposition. One embryo of M. pulchricornis gave rise to approximately 1200 teratocytes, a number that remained constant until 6 days after parasitization, but decreased drastically to 200 at 7 days post-oviposition. The teratocytes of M. pulchricornis were round- or oval-shaped and grew from 65 microm at 4 days to 200 microm in the long axis at 6 days post-parasitization. At 4 days post-parasitization, many cells or cell clusters with lipid particles were observed in the hemocoels of parasitized hosts. In addition, paraffin sections of parasitized hosts revealed that many teratocytes were attached to the host's fat body and contributed to disrupting the fat body tissue. Further, examination of the total hemocyte count (THC) during parasitization revealed that THC was maintained at low levels. Surprisingly, a temporal decrease followed by restoration of THC was observed in hosts injected with virus-like particles of M. pulchricornis (MpVLPs) plus venom, which contrasts with the constant THC suppression seen in parasitized hosts. This indicates that MpVLP function is temporal and is involved in regulation of the host during early parasitism. Therefore, teratocytes, a host regulation factor in late parasitism, could be involved in keeping THC at a low level.

The Leishmania-macrophage interaction: a metabolic perspective.

PubMed

Naderer, Thomas; McConville, Malcolm J

2008-02-01

Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophages although they have the capacity to infect a variety of other phagocytic and non-phagocytic mammalian cells. Unlike most other intramacrophage pathogens, the major proliferative stage of Leishmania resides in the mature phagolysosomes of these host cells. In this review we highlight some of the strategies utilized by the intracellular amastigote stage of Leishmania to survive in this compartment. Remarkably, and in contrast to many other intracellular pathogens, Leishmania amastigotes have a minimalist surface glycocalyx which may facilitate uptake of essential lipids and promote exposure of phospholipids required for phagocytosis via macrophage apoptotic cell receptors. Leishmania amastigotes also differ from many other intracellular pathogens in having complex nutritional requirements which must be scavenged from the host cell. Amino acids and polyamines appear to be important carbon sources and growth-limiting nutrients, respectively, and their availability to intracellular amastigotes may be regulated by the activation state of host macrophages. Metabolic processes in both the parasite and host cell may thus be crucial determinants of disease outcome.

The role of lipids in host microbe interactions.

PubMed

Lang, Roland; Mattner, Jochen

2017-06-01

Lipids are one of the major subcellular constituents and serve as signal molecules, energy sources, metabolic precursors and structural membrane components in various organisms. The function of lipids can be modified by multiple biochemical processes such as (de-)phosphorylation or (de-)glycosylation, and the organization of fatty acids into distinct cellular pools and subcellular compartments plays a pivotal role for the morphology and function of various cell populations. Thus, lipids regulate, for example, phagosome formation and maturation within host cells and thus, are critical for the elimination of microbial pathogens. Vice versa, microbial pathogens can manipulate the lipid composition of phagosomal membranes in host cells, and thus avoid their delivery to phagolysosomes. Lipids of microbial origin belong also to the strongest and most versatile inducers of mammalian immune responses upon engagement of distinct receptors on myeloid and lymphoid cells. Furthermore, microbial lipid toxins can induce membrane injuries and cell death. Thus, we will review here selected examples for mutual host-microbe interactions within the broad and divergent universe of lipids in microbial defense, tissue injury and immune evasion.

Interleukin-22 in Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation

PubMed Central

Lamarthée, Baptiste; Malard, Florent; Saas, Philippe; Mohty, Mohamad; Gaugler, Béatrice

2016-01-01

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative treatment for hematologic malignancies and non-malignant diseases. Because of the lower toxicity of reduced intensity conditioning, the number of transplants is in constant increase. However, allo-HSCT is still limited by complications, such as graft-versus-host disease (GVHD), which is associated with important morbidity and mortality. Acute GVHD is an exacerbated inflammatory response that leads to the destruction of healthy host tissues by donor immune cells. Recently, the contribution of innate immunity in GVHD triggering has been investigated by several groups and resulted in the identification of new cellular and molecular effectors involved in GVHD pathogenesis. Interleukin-22 (IL-22) is produced by both immune and adaptive cells and has both protective and inflammatory properties. Its role in GVHD processes has been investigated, and the data suggest that its effect depends on the timing, the target tissue, and the origin of the producing cells (donor/host). In this review, we discuss the role of IL-22 in allo-HSCT and GVHD. PMID:27148267

Interleukin-22 in Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation.

PubMed

Lamarthée, Baptiste; Malard, Florent; Saas, Philippe; Mohty, Mohamad; Gaugler, Béatrice

2016-01-01

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative treatment for hematologic malignancies and non-malignant diseases. Because of the lower toxicity of reduced intensity conditioning, the number of transplants is in constant increase. However, allo-HSCT is still limited by complications, such as graft-versus-host disease (GVHD), which is associated with important morbidity and mortality. Acute GVHD is an exacerbated inflammatory response that leads to the destruction of healthy host tissues by donor immune cells. Recently, the contribution of innate immunity in GVHD triggering has been investigated by several groups and resulted in the identification of new cellular and molecular effectors involved in GVHD pathogenesis. Interleukin-22 (IL-22) is produced by both immune and adaptive cells and has both protective and inflammatory properties. Its role in GVHD processes has been investigated, and the data suggest that its effect depends on the timing, the target tissue, and the origin of the producing cells (donor/host). In this review, we discuss the role of IL-22 in allo-HSCT and GVHD.