scholarly journals Use of a fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia.

1997 ◽  
Vol 41 (2) ◽  
pp. 337-344 ◽  
Author(s):  
G J Leitch ◽  
M Scanlon ◽  
A Shaw ◽  
G S Visvesvara ◽  
S Wallace
Keyword(s):  
Host Cell ◽  
Fluorescent Probe ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Parasitophorous Vacuole ◽  
Detectable Effect ◽  
Drug Induced ◽  
Encephalitozoon Intestinalis ◽  
Infected Cells

Microsporidia are obligate intracellular protozoan parasites. Three species of the genus Encephalitozoon are among the microsporidia that infect immunodeficient humans. These species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, all develop in a parasitophorous vacuole within a host cell. The present study describes a method that uses the fluorescent probe calcein and confocal microscopy to detect drug-induced effects in Encephalitozoon-infected green monkey kidney cells. The effects were as follows: (i) changes in parasite organization within the parasitophorous vacuole; (ii) swelling and gross morphological changes of parasite developing stages in situ; (iii) killing of developing parasite stages in situ, detected by their uptake of the fluorescent probe; and (iv) reduction in the viability of the host cell population, assessed by the loss of the probe. Verapamil and itraconazole were used to increase the vital dye loading by both uninfected and infected cells. Agents with known antimicrosporidial activity, albendazole and fumagillin, caused all three types of parasite changes at concentrations that had no detectable effect on host cell viability. The effective doses of albendazole and fumagillin that caused swelling and disorganization of parasite developing stages were 5 x 10(-7) and 10(-6) M respectively. Killing of developing stages was detected at 10-fold-higher concentrations for these agents and at 10(-5) M for metronidazole. This method can be used to screen candidate antimicrosporidial agents in infected cultured cells.

scholarly journals Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

2006 ◽  
Vol 17 (8) ◽  
pp. 3557-3568 ◽  
Author(s):  
James C. Warren ◽  
Adam Rutkowski ◽  
Lynne Cassimeris
Keyword(s):  
Host Cell ◽  
Dynamic Instability ◽  
Infection Process ◽  
Microtubule Dynamics ◽  
Cell Periphery ◽  
Adenovirus Infection ◽  
Drug Induced ◽  
Infected Cells ◽  
The Stability ◽  
Induced Changes

Adenovirus translocation to the nucleus occurs through a well characterized minus end-directed transport along microtubules. Here, we show that the adenovirus infection process has a significant impact on the stability and dynamic behavior of host cell microtubules. Adenovirus-infected cells had elevated levels of acetylated and detyrosinated microtubules compared with uninfected cells. The accumulation of modified microtubules within adenovirus-infected cells required active RhoA. Adenovirus-induced changes in microtubule dynamics were characterized at the centrosome and at the cell periphery in living cells. Adenovirus infection resulted in a transient enhancement of centrosomal microtubule nucleation frequency. At the periphery of adenovirus-infected cells, the dynamic instability of microtubules plus ends shifted toward net growth, compared with the nearly balanced growth and shortening observed in uninfected cells. In infected cells, microtubules spent more time in growth, less time in shortening, and underwent catastrophes less frequently compared with those in uninfected cells. Drug-induced inhibition of Rac1 prevented most of these virus-induced shifts in microtubule dynamic instability. These results demonstrate that adenovirus infection induces a significant stabilizing effect on host cell microtubule dynamics, which involve, but are not limited to, the activation of the RhoGTPases RhoA and Rac1.

scholarly journals Evidence for Host Cells as the Major Contributor of Lipids in the Intravacuolar Network of Toxoplasma-Infected Cells

2011 ◽  
Vol 10 (8) ◽  
pp. 1095-1099 ◽  
Author(s):  
Carolina E. Caffaro ◽  
John C. Boothroyd
Keyword(s):  
Host Cell ◽  
Fluorescence Recovery After Photobleaching ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Host Cells ◽  
Cell Plasma Membrane ◽  
Content Type ◽  
Cell Plasma ◽  
Continuous Stream ◽  
Infected Cells

ABSTRACT The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.

Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell

1987 ◽  
Vol 88 (2) ◽  
pp. 231-239
Author(s):  
I. Kimata ◽  
K. Tanabe
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Anterior Part ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Immunoperoxidase Staining ◽  
Cell Plasma ◽  
Sds Page ◽  
Infected Cells ◽  
Triton X

Monoclonal antibodies against Toxoplasma gondii were prepared to characterize antigens of the parasite. Immunoperoxidase staining of parasites fixed with paraformaldehyde and glutaraldehyde (PFAGA) followed by Triton X-100 treatment showed that the antibody of clone I-63 recognized an antigen located in the anterior part of the parasite. When analysed by SDS-PAGE and immunoblotting, the antigen migrated in a 66 × 10(3) Mr region. The parasite antigen diminished greatly in parasites after invasion of host cells, but reappeared around a time when intracellular T. gondii multiplied. Immunodetection on PFAGA-fixed T. gondii-infected cells, whose membranes were permeabilized by freeze-thawing in the presence of 5% glycerol, demonstrated that, immediately after parasite invasion, the I-63 antibody-reactive antigen appeared to become associated with the parasitophorous vacuole (PV) membrane, that had been formed mainly by invagination of the host-cell plasma membrane so as to surround an invading parasite. The antigen remained associated with the PV membrane for some time, but disappeared later when the PV increased in size after the parasites had multiplied several times. These results were strengthened by immunoelectron microscopic observations: the antigen that had been localized at the anterior part of the parasite before invasion appeared in an area of the host cell cytoplasm around the tips of penetrating parasites and, thereafter, extended throughout the surface of the PV membrane when parasites completed invasion. Thus, it appears that the I-63-reactive antigen is secreted by T. gondii upon invasion of the host cell and becomes associated with the PV membrane shortly after invasion.

scholarly journals Class I Major Histocompatibility Complex Presentation of Antigens That Escape from the Parasitophorous Vacuole of Toxoplasma gondii

2005 ◽  
Vol 73 (2) ◽  
pp. 703-711 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Boris Striepen ◽  
Nilabh Shastri ◽  
Mustafa Turkoz ◽  
Ellen A. Robey
Keyword(s):  
Major Histocompatibility Complex ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
T Cell Response ◽  
Class I ◽  
Peptide Transporter ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Infected Cells

ABSTRACT The intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, induces a protective CD8 T-cell response in its host; however, the mechanisms by which T. gondii proteins are presented by the class I major histocompatibility complex remain largely unexplored. T. gondii resides within a specialized compartment, the parasitophorous vacuole, that sequesters the parasite and its secreted proteins from the host cell cytoplasm, suggesting that an alternative cross-priming pathway might be necessary for class I presentation of T. gondii antigens. Here we used a strain of T. gondii expressing yellow fluorescent protein and a secreted version of the model antigen ovalbumin to investigate this question. We found that presentation of ovalbumin secreted by the parasite requires the peptide transporter TAP (transporter associated with antigen processing) and occurs primarily in actively infected cells rather than bystander cells. We also found that dendritic cells are a major target of T. gondii infection in vivo and account for much of the antigen-presenting activity in the spleen. Finally, we obtained evidence that Cre protein secreted by T. gondii can mediate recombination in the nucleus of the host cell. Together, these results indicate that Toxoplasma proteins can escape from the parasitophorous vacuole into the host cytoplasm and be presented by the endogenous class I pathway, leading to direct recognition of infected cells by CD8 T cells.

scholarly journals Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2786
Author(s):  
Hong-Mei Li ◽  
Reena Ghildyal ◽  
Mengjie Hu ◽  
Kim C. Tran ◽  
Lora M. Starrs ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Dna Binding ◽  
Host Cell ◽  
Rna Binding ◽  
M Protein ◽  
Free System ◽  
Transcriptional Inhibition ◽  
Infected Cells ◽  
Syncytial Virus

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M’s RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.

scholarly journals Intracellular Tryptophan Pool Sizes May Account for Differences in Gamma Interferon-Mediated Inhibition and Persistence of Chlamydial Growth in Polarized and Nonpolarized Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1666-1671 ◽  
Author(s):  
Colleen D. Kane ◽  
Renee M. Vena ◽  
Scot P. Ouellette ◽  
Gerald I. Byrne
Keyword(s):  
Amino Acid ◽  
Host Cell ◽  
Cultured Cells ◽  
Polarization State ◽  
Cell Polarization ◽  
Gamma Interferon ◽  
Infected Cells ◽  
Ifn Γ ◽  
Pool Sizes

ABSTRACT Gamma interferon (IFN-γ) is an important factor in the modulating inhibition of intracellular chlamydial growth and persistence. In human epithelial cells and macrophages, this inhibition is the result of depletion of the essential amino acid tryptophan via the IFN-γ-induced enzyme indoleamine 2,3-dioxygenase. Under these conditions, chlamydiae must successfully compete with the host cell for limited resources in order to maintain viability. We provide evidence to support the hypothesis that the host cell polarization state influences the host-pathogen interplay and outcome of IFN-γ-mediated inhibition. In polarized cells, intracellular soluble tryptophan pools were larger than those in nonpolarized cells despite only small differences in the initial uptake rate of this amino acid compared to that in nonpolarized cells. Furthermore, in Chlamydia trachomatis-infected cells, the amounts of tryptophan consumed by the organisms were similar for cells grown in either state. We propose that intracellular tryptophan pool sizes can account for differences in IFN-γ-mediated chlamydial persistence and growth inhibition in polarized and nonpolarized cells. Collectively, these results argue that polarized cell models, which more accurately reflect the conditions in vivo, may be more relevant than conventionally cultured cells in the study of intimate intracellular host-parasite interactions.

scholarly journals Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Michael W. Panas ◽  
Abel Ferrel ◽  
Adit Naor ◽  
Elizabeth Tenborg ◽  
Hernan A. Lorenzi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Large Set ◽  
Gene Encoding ◽  
Cell Functions ◽  
Infected Cells ◽  
Rhoptry Protein

ABSTRACT Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued “in trans” by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction. IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

scholarly journals Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii.

1996 ◽  
Vol 44 (10) ◽  
pp. 1123-1129 ◽  
Author(s):  
L Pingret ◽  
J M Millot ◽  
S Sharonov ◽  
A Bonhomme ◽  
M Manfait ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Calcium Ionophore ◽  
Living Cells ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Subcellular Compartment ◽  
Subcellular Compartments ◽  
Infected Cells

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.

scholarly journals The Apicomplexan Pathogen Neospora caninum Inhibits Host Cell Apoptosis in the Absence of Discernible NF-κB Activation

2007 ◽  
Vol 75 (9) ◽  
pp. 4255-4262 ◽  
Author(s):  
Rebecca K. Herman ◽  
Robert E. Molestina ◽  
Anthony P. Sinai ◽  
Daniel K. Howe
Keyword(s):  
Host Cell ◽  
Cell Apoptosis ◽  
Nuclear Translocation ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Death Receptor ◽  
Host Cells ◽  
Iκb Kinase ◽  
Host Cell Apoptosis ◽  
Infected Cells

ABSTRACT Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-κB subunit p65 was not detected in N. caninum-infected cells, although this host transcription factor has been shown to upregulate prosurvival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated IκB that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative IκB kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-κB translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-κB pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.

scholarly journals Accumulation and Intranuclear Distribution of Unintegrated Human Immunodeficiency Virus Type 1 DNA

2001 ◽  
Vol 75 (16) ◽  
pp. 7683-7691 ◽  
Author(s):  
Peter Bell ◽  
Luis J. Montaner ◽  
Gerd G. Maul
Keyword(s):  
Human Immunodeficiency Virus ◽  
Host Cell ◽  
Human Immunodeficiency Virus Type ◽  
Cell Nucleus ◽  
Viral Dna ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The RNA genome of human immunodeficiency virus type 1 (HIV-1) is converted into DNA after infection in order to integrate into the host cell DNA. However, a large number of these reverse-transcribed genomes remain unintegrated in the nucleus of infected cells. Currently, there are no data available about the intranuclear distribution pattern of unintegrated HIV-1 DNA in relation to nuclear structures as observed on the single-cell level. In the present study, we investigated the intranuclear fate of unintegrated viral DNA in cell lines expressing CD4 and coreceptors (HOS-CD4.CCR5 and U373-MAGI-CXCR4CEM) infected with HIV-1 (strain 89.6). We used a novel approach to distinguish in situ unintegrated from integrated viral DNA by performing fluorescent in situ hybridization on cells in which stress-induced chromosome condensation had been induced, a procedure that contracts chromosomes independent of the cell cycle. Cells infected for 15 h accumulated large amounts of HIV-1 DNA which was located between the condensed chromosome strands, allowing the identification of this viral DNA as unintegrated. In contrast, in HeLa/LAV, a cell line carrying integrated HIV-1 genomes, the great majority of viral DNA colocalized with the cellular DNA. We show that unintegrated HIV-1 DNA does not evenly distribute within the host cell nucleus but tends to aggregate into clusters containing many copies of the viral genomes. The formation of these DNA clusters was independent of viral DNA replication and thus appeared to result solely from multiple infections. The DNA aggregates remained in the nuclei of infected cells for at least 25 h after the infection was stopped. The emergence of transcription sites, which most likely denote sites of the integrated provirus, lagged clearly behind the accumulation of viral DNA. These transcription foci could not be linked to unintegrated DNA molecules, suggesting that this DNA type is unable to transcribe, at least at levels comparable to those of integrated DNA. Neither unintegrated HIV-1 DNA nor transcription foci nor integrated DNA was observed to associate with nuclear domain 10 (ND10), a nuclear structure known to represent the site where several DNA viruses replicate and transcribe. Also, HIV-1 does not modify ND10 at early or late times of infection. There was no specific association of HIV-1 transcripts with splicing factor SC35 domains, in contrast to what has been reported for a number of both cellular and viral genes. Surprisingly, unintegrated HIV-1 DNA was found to accumulate within or in close association with SC35 domains, demonstrating a specific distribution of the viral DNA within the host cell nucleus. Taken together, our results demonstrate that unintegrated proviral HIV-1 DNA does not randomly localize within infected cells but preferentially aggregates in the nucleus within SC35 domains.

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