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

2012 ◽  
Vol 12 (2) ◽  
pp. 265-277 ◽  
Author(s):  
Julia D. Romano ◽  
Catherine de Beaumont ◽  
Jose A. Carrasco ◽  
Karen Ehrenman ◽  
Patrik M. Bavoil ◽  
...  
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Productive Infection ◽  
Microtubule Organizing Center ◽  
Nutrient Pools ◽  
Content Type ◽  
Cell Structures ◽  
Organizing Center ◽  
A Cell ◽  
Fierce Competition

ABSTRACTThe prokaryoteChlamydia trachomatisand the protozoanToxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similarmodus operandito 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 byC. trachomatisandT. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and thatToxoplasmaholds a significant competitive advantage overChlamydia. Here we have expanded our coinfection studies by examining the respective abilities ofChlamydiaandToxoplasmato 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,ToxoplasmaoutcompetesChlamydiato the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected withChlamydia, the centrosomes become associated with the chlamydial inclusion, while theToxoplasmaparasitophorous 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 bothChlamydiaandToxoplasmadepends 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.

scholarly journals Protease-Dependent Uncoating of a Complex Retrovirus

2005 ◽  
Vol 79 (14) ◽  
pp. 9244-9253 ◽  
Author(s):  
Jacqueline Lehmann-Che ◽  
Marie-Lou Giron ◽  
Olivier Delelis ◽  
Martin Löchelt ◽  
Patricia Bittoun ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Viral Genome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Microtubule Organizing Center ◽  
Viral Protease ◽  
Virus Life Cycle ◽  
Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

scholarly journals Targeted Disruption of Toxoplasma gondii Serine Protease Inhibitor 1 Increases Bradyzoite Cyst FormationIn Vitroand Parasite Tissue Burden in Mice

2011 ◽  
Vol 80 (3) ◽  
pp. 1156-1165 ◽  
Author(s):  
Viviana Pszenny ◽  
Paul H. Davis ◽  
Xing W. Zhou ◽  
Christopher A. Hunter ◽  
Vern B. Carruthers ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protease Inhibitor ◽  
Host Cell ◽  
Serine Protease ◽  
Parasitophorous Vacuole ◽  
Parasite Burden ◽  
Serine Protease Inhibitor ◽  
Genomic Locus ◽  
Content Type

As an intracellular protozoan parasite,Toxoplasma gondiiis likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities.T. gondiiserine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces twoTgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigateTgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles withDolichos bifloruslectin under conditions promotingin vitrodifferentiation. The differentiation phenotype can be partially complemented by eitherTgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and thisin vivophenotype is also complemented by eitherTgPI1 isoform. These results demonstrate thatTgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.

scholarly journals Microtubules control cellular shape and coherence in amoeboid migrating cells

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Aglaja Kopf ◽  
Jörg Renkawitz ◽  
Robert Hauschild ◽  
Irute Girkontaite ◽  
Kerry Tedford ◽  
...  
Keyword(s):  
Cell Shape ◽  
Shape Control ◽  
Microtubule Depolymerization ◽  
Microtubule Organizing Center ◽  
Amoeboid Cell ◽  
Actomyosin Contractility ◽  
Organizing Center ◽  
A Cell ◽  
And Control ◽  
Rate Limit

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.

scholarly journals Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host Cell

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Gustavo Arrizabalaga
Keyword(s):  
Host Cell ◽  
Protein Translocation ◽  
Parasitophorous Vacuole ◽  
Opportunistic Pathogen ◽  
Dense Granule ◽  
Effector Proteins ◽  
Content Type ◽  
Dense Granules ◽  
Cell Functions ◽  
Host Signaling

ABSTRACT The opportunistic pathogen Toxoplasma gondii is highly adept at manipulating host cell functions. While inside a host cell, Toxoplasma divides within a parasitophorous vacuole from which it secretes numerous effector proteins from its dense granules. Many of these so-called GRA proteins are translocated from the parsitophorous vacuole into the host cell where they directly disrupt host signaling pathways. The machinery that drives the translocation of GRA proteins across the parasitophorous vacuole membrane is being elucidated through both genetic and biochemical approaches. A new mSphere research article (M. W. Panas, A. Ferrel, A. Naor, E. Tenborg, et al., mSphere 4:e00276-19, 2019, https://doi.org/10.1128/mSphere.00276-19) describes how the kinase ROP17, which is secreted from the parasite’s rhoptries into the host cell during invasion, regulates the translocation of GRA effectors.

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells

1997 ◽  
Vol 110 (19) ◽  
pp. 2391-2401 ◽  
Author(s):  
A.M. Yvon ◽  
P. Wadsworth
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Human Kidney ◽  
Light Level ◽  
Pause Duration ◽  
Living Cells ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

scholarly journals The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
William J. Blakely ◽  
Michael J. Holmes ◽  
Gustavo Arrizabalaga
Keyword(s):  
Acid Phosphatase ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Parasitophorous Vacuole ◽  
Severe Disease ◽  
Immune Recognition ◽  
Phosphatase Domain ◽  
Content Type ◽  
Cell Defense

ABSTRACT During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

scholarly journals Toxoplasma Uses GRA16 To Upregulate Host c-Myc

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Michael W. Panas ◽  
John C. Boothroyd
Keyword(s):  
Host Cell ◽  
Protein Phosphatase ◽  
Crucial Role ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Regulatory Subunit ◽  
Close Relative ◽  
Effector Protein ◽  
Animal Cells ◽  
Content Type

ABSTRACT Manipulation of the host cell is a crucial part of life for many intracellular organisms. We have recently come to appreciate the extent to which the intracellular pathogen Toxoplasma gondii reprograms its host cell, and this is illustrated by the marked upregulation of the central regulator c-Myc, an oncogene that coordinates myriad cellular functions. In an effort to identify an effector protein capable of regulating c-Myc, our laboratory constructed a screen for mutant parasites unable to accomplish this upregulation. Interestingly, this screen identified numerous components of a complex located in/on the parasitophorous vacuole membrane necessary to translocate Toxoplasma proteins out into the host cytosol, but it never identified a specific effector protein. Thus, how the parasite upregulates c-Myc has largely been a mystery. Previously, the Toxoplasma dense granule protein GRA16 has been described to bind to one isoform of PP2A-B, a regulatory subunit that coordinates the activity of the catalytic protein phosphatase PP2A. As other PP2A subunits have been reported to target PP2A protein phosphatase activity to c-Myc, subsequently leading to c-Myc destabilization, we examined whether GRA16 has an impact on host c-Myc accumulation. Expression of Toxoplasma’s GRA16 protein in Neospora caninum, a close relative of Toxoplasma that does not naturally upregulate host c-Myc, conferred the ability on Neospora to do this now. Further support was obtained by deleting the GRA16 gene from Toxoplasma and observing a severely diminished ability of Toxoplasma tachyzoites to upregulate host c-Myc. Thus, GRA16 is an effector protein central to Toxoplasma’s ability to upregulate host c-Myc. IMPORTANCE The proto-oncogene c-Myc plays a crucial role in the growth and division of many animal cells. Previous studies have identified an active upregulation of c-Myc by Toxoplasma tachyzoites, suggesting the existence of one or more exported “effector” proteins. The identity of such an effector, however, has not previously been known. Here, we show that a previously known secreted protein, GRA16, plays a crucial role in c-Myc upregulation. This finding will enable further dissection of the precise mechanism and role of c-Myc upregulation in Toxoplasma-infected cells.

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.

scholarly journals Microsporidia Interact with Host Cell Mitochondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Bing Han ◽  
Yanfen Ma ◽  
Vincent Tu ◽  
Tadakimi Tomita ◽  
Joshua Mayoral ◽  
...  
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Surface Protein ◽  
Host Cells ◽  
Anion Channels ◽  
Polar Tube ◽  
Content Type ◽  
Proteomic Approach ◽  
Voltage Dependent ◽  
Microsporidian Infection

ABSTRACT Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV. IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

scholarly journals CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e01001-20
Author(s):  
Paul-Christian Burda ◽  
Hugo Bisio ◽  
Jean-Baptiste Marq ◽  
Dominique Soldati-Favre ◽  
Volker T. Heussler
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Infected Host ◽  
Protein Subunit ◽  
Host Cell Membrane ◽  
Guanine Nucleotide Binding Protein ◽  
Liver Stage ◽  
Content Type ◽  
Apicomplexan Parasites

ABSTRACTToxoplasma gondii and members of the genus Plasmodium are obligate intracellular parasites that leave their infected host cell upon a tightly controlled process of egress. Intracellular replication of the parasites occurs within a parasitophorous vacuole, and its membrane as well as the host plasma membrane need to be disrupted during egress, leading to host cell lysis. While several parasite-derived factors governing egress have been identified, much less is known about host cell factors involved in this process. Previously, RNA interference (RNAi)-based knockdown and antibody-mediated depletion identified a host signaling cascade dependent on guanine nucleotide-binding protein subunit alpha q (GNAQ) to be required for the egress of Toxoplasma tachyzoites and Plasmodium blood stage merozoites. Here, we used CRISPR/Cas9 technology to generate HeLa cells deficient in GNAQ and tested their capacity to support the egress of T. gondii tachyzoites and Plasmodium berghei liver stage parasites. While we were able to confirm the importance of GNAQ for the egress of T. gondii, we found that the egress of P. berghei liver stages was unaffected in the absence of GNAQ. These results may reflect differences between the lytic egress process in apicomplexans and the formation of host cell-derived vesicles termed merosomes by P. berghei liver stages.IMPORTANCE The coordinated release of apicomplexan parasites from infected host cells prior to reinvasion is a critical process for parasite survival and the spread of infection. While Toxoplasma tachyzoites and Plasmodium blood stages induce a fast disruption of their surrounding membranes during their egress from host cells, Plasmodium liver stages keep the host cell membrane intact and leave their host cell in host cell-derived vesicles called merosomes. The knockout of GNAQ, a protein involved in G-protein-coupled receptor signaling, demonstrates the importance of this host factor for the lytic egress of T. gondii tachyzoites. Contrastingly, the egress of P. berghei is independent of GNAQ at the liver stage, indicating the existence of a mechanistically distinct strategy to exit the host cell.

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