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Ionophore-Resistant Mutants of Toxoplasma gondii Reveal Host Cell Permeabilization as an Early Event in Egress

Black, Michael W.; Arrizabalaga, Gustavo; Boothroyd, John C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2000 EN
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Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca2+]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie− mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie− and Iid− phenotypes is supported by the observation that two-thirds of mutants selected as Iid− are also Iie−. Characterization of three distinct classes of IIE and IID mutants revealed that the Iie− phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie− parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie− mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie− mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.

icmT Is Essential for Pore Formation-Mediated Egress of Legionella pneumophila from Mammalian and Protozoan Cells

Molmeret, Maelle; Alli, O. A. Terry; Zink, Steven; Flieger, Antje; Cianciotto, Nicholas P.; Kwaik, Yousef Abu
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2002 EN
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The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA...

Toxoplasma gondii: Induction of egress by the potassium ionophore nigericin

Fruth, Ingrid A.; Arrizabalaga, Gustavo
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. Some of the devastating consequences of toxoplasmosis are in part due to the lysis of the host cell during parasite egress. The process of egress is poorly understood and since it is asynchronous in tissue culture its study has been limited to those conditions that induce it, such as artificial permeabilization of the host cell and induction of calcium fluxes with ionophores. Given that permeabilization leads to egress by the activation of motility upon a drop in host cell potassium concentration, we investigated whether the ionophore nigericin, which selectively causes efflux of potassium from the cell without the need for permeabilization, would cause egress. Nigericin effectively causes intracellular parasites to exit their host cell within 30 min of treatment with the drug. Our results show that nigericin-induced egress depends on an efflux of potassium from the cell and requires phospholipase C function and parasite motility. This novel method of inducing and synchronizing egress mimics the effect of artificial permeabilization in all respects. Nevertheless, since the membrane remains intact during the treatment, in our nigericin-induced egress we are able to detect parasite-dependent permeabilization of the host cell...

Glycoproteins Required for Entry Are Not Necessary for Egress of Pseudorabies Virus▿

Klupp, Barbara; Altenschmidt, Jan; Granzow, Harald; Fuchs, Walter; Mettenleiter, Thomas C.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition...

T-bet–dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow

Jenne, Craig N.; Enders, Anselm; Rivera, Richard; Watson, Susan R.; Bankovich, Alexander J.; Pereira, Joao P.; Xu, Ying; Roots, Carla M.; Beilke, Joshua N.; Banerjee, Arnob; Reiner, Steven L.; Miller, Sara A.; Weinmann, Amy S.; Goodnow, Chris C.; Lanier,
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 26/10/2009 EN
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27.26%
During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P5) transcript levels, and S1P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P1 also plays a role in NK cell egress from LNs. S1P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P5 as a T-bet–induced gene that is required for NK cell egress from LNs and BM.

Externally Triggered Egress Is the Major Fate of Toxoplasma gondii During Acute Infection1

Tomita, Tadakimi; Yamada, Tatsuya; Weiss, Louis M.; Orlofsky, Amos
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The apicomplexan parasite Toxoplasma gondii expands during acute infection via a cycle of invasion, intracellular replication and lytic egress. Physiological regulation has not yet been demonstrated for either invasion or egress. We now report that, in contrast to cell culture systems, in which egress occurs only after five or more parasite divisions (two to three days), intracellular residence is strikingly abbreviated in inflammatory cells in vivo, and early egress (after zero to two divisions) is the dominant parasite fate in acutely infected mice. Adoptive transfer experiments demonstrate rapid, reciprocal, kinetically uniform parasite transfer between donor and recipient compartments, with a half-life of approximately three hours. Inflammatory macrophages are major participants in this cycle of lytic egress and reinfection, which drives rapid macrophage turnover. Inflammatory triggering cells, principally macrophages, elicit egress in infected target macrophages, a process we term externally triggered egress (ETE). The mechanism of ETE does not require reactive oxygen or nitrogen species, the mitochondrial permeability transition pore, or a variety of signal transduction mediators, but is dependent on intracellular calcium and is highly sensitive to SB203580...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Coleman, Bradley I.; Gubbels, Marc-Jan
Fonte: MyJove Corporation Publicador: MyJove Corporation
Tipo: Artigo de Revista Científica
Publicado em 08/02/2012 EN
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The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11...

Replication of Herpes Simplex Virus: Egress of Progeny Virus at Specialized Cell Membrane Sites

Mingo, Rebecca M.; Han, Jun; Newcomb, William W.; Brown, Jay C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2012 EN
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In the final stages of the herpes simplex virus 1 (HSV-1) life cycle, a viral nucleocapsid buds into a vesicle of trans-Golgi network (TGN)/endosome origin, acquiring an envelope and an outer vesicular membrane. The virus-containing vesicle then traffics to the plasma membrane where it fuses, exposing a mature virion. Although the process of directed egress has been studied in polarized epithelial cell lines, less work has been done in nonpolarized cell types. In this report, we describe a study of HSV-1 egress as it occurs in nonpolarized cells. The examination of infected Vero cells by electron, confocal, and total internal reflection fluorescence (TIRF) microscopy revealed that HSV-1 was released at specific pocket-like areas of the plasma membrane that were found along the substrate-adherent surface and cell-cell-adherent contacts. Both the membrane composition and cytoskeletal structure of egress sites were found to be modified by infection. The plasma membrane at virion release sites was heavily enriched in viral glycoproteins. Small glycoprotein patches formed early in infection, and virus became associated with these areas as they expanded. Glycoprotein-rich areas formed independently from virion trafficking as confirmed by the use of a UL25 mutant with a defect in capsid nuclear egress. The depolymerization of the cytoskeleton indicated that microtubules were important for the trafficking of virions and glycoproteins to release sites. In addition...

A Forward Genetic Screen Reveals that Calcium-dependent Protein Kinase 3 Regulates Egress in Toxoplasma

Garrison, Erin; Treeck, Moritz; Ehret, Emma; Butz, Heidi; Garbuz, Tamila; Oswald, Benji P.; Settles, Matt; Boothroyd, John; Arrizabalaga, Gustavo
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.26%
Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.

New stages in the program of malaria parasite egress imaged in normal and sickle erythrocytes

Glushakova, Svetlana; Humphrey, Glen; Leikina, Evgenia; Balaban, Amanda; Miller, Jeffrey; Zimmerberg, Joshua
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
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The apicomplexan parasite Plasmodium falciparum causes malignant malaria. The mechanism of parasite egress from infected erythrocytes that disseminate parasites in the host at the end of each asexual cycle is unknown [1]. Two new stages of the egress program are revealed: 1) swelling of the parasitophorus vacuole accompanied by shrinkage of the erythrocyte compartment and 2) poration of the host cell membrane seconds before erythrocyte rupture due to egress. Egress was inhibited in dehydrated cells from patients with sickle cell disease, in accord with experimental dehydration of normal cells [2], suggesting that vacuole swelling involves intake of water from the erythrocyte compartment. Erythrocyte membrane poration occurs in relaxed cells, thus excluding involvement of osmotic pressure in this process. Poration does not depend on cysteine protease activity because protease inhibition blocks egress [3–5] but not poration, and poration is required for the parasite cycle because the membrane sealant P1107 interferes with egress. We suggest the following egress program: parasites initiate water influx into the vacuole from the erythrocyte cytosol to expand the vacuole for parasite separation and vacuole rupture upon its critical swelling. Separated parasites leave the erythrocyte by breaching its membrane...

Distinct signalling pathways control Toxoplasma egress and host-cell invasion

Lourido, Sebastian; Tang, Keliang; Sibley, L David
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.36%
Calcium signalling coordinates motility, cell invasion, and egress by apicomplexan parasites, yet the key mediators that transduce these signals remain largely unknown. One underlying assumption is that invasion into and egress from the host cell depend on highly similar systems to initiate motility. Using a chemical-genetic approach to specifically inhibit select calcium-dependent kinases (CDPKs), we instead demonstrate that these pathways are controlled by different kinases: both TgCDPK1 and TgCDPK3 were required during ionophore-induced egress, but only TgCDPK1 was required during invasion. Similarly, microneme secretion, which is necessary for motility during both invasion and egress, universally depended on TgCDPK1, but only exhibited TgCDPK3 dependence when triggered by certain stimuli. We also demonstrate that egress likely comes under a further level of control by cyclic GMP-dependent protein kinase and that its activation can induce egress and partially compensate for the inhibition of TgCDPK3. These results demonstrate that separate signalling pathways are integrated to regulate motility in response to the different signals that promote invasion or egress during infection by Toxoplasma gondii.

New Roles for Perforins and Proteases in Apicomplexan Egress

Roiko, Marijo S.; Carruthers, Vern B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.32%
Egress is a pivotal step in the lifecycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late stage schizonts activate and secrete a subtlilisin, PfSUB1, which processes enigmatic putative proteases called SERAs that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.

CXCR4 Is Dispensable for T Cell Egress from Chronically Inflamed Skin via the Afferent Lymph

Geherin, Skye A.; Wilson, R. Paul; Jennrich, Silke; Debes, Gudrun F.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 21/04/2014 EN
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27.32%
T cell recirculation through extralymphoid tissues is essential to immune surveillance, host defense and inflammation. In this process, T cells enter the tissue from the blood and subsequently leave via the afferent lymph. In the absence of inflammation, T cells require CCR7 expression to egress from the skin or lung, which is consistent with the constitutive expression of the CCR7 ligand CCL21 on lymphatic endothelium. However, during chronic inflammation alternative chemoattractants come into play, allowing Ccr7-deficient (Ccr7−/−) T cells to egress efficiently from affected skin. As T cell egress from inflamed sites is a potential control point of the inflammatory response, we aimed to determine alternative T cell exit receptors using a mouse and a sheep model. We show that CCR7+ and CCR7– T cells exiting from the chronically inflamed skin were highly responsive to the CXCR4 ligand CXCL12, which was induced in the lymphatics in the inflamed site. Based on these findings, we hypothesized that CXCR4 mediates T cell egress from inflamed skin. However, pharmacological inhibition of CXCR4 did not affect the tissue egress of wildtype or Ccr7−/− CD4 and CD8 T cells after adoptive transfer into chronically inflamed skin. Similarly...

Acidification Activates Toxoplasma gondii Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity

Roiko, Marijo S.; Svezhova, Nadezhda; Carruthers, Vern B.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 06/11/2014 EN
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27.32%
Pathogenic microbes rely on environmental cues to initiate key events during infection such as differentiation, motility, egress and invasion of cells or tissues. Earlier investigations showed that an acidic environment activates motility of the protozoan parasite T. gondii. Conversely, potassium ions, which are abundant in the intracellular milieu that bathes immotile replicating parasites, suppress motility. Since motility is required for efficient parasite cell invasion and egress we sought to better understand its regulation by environmental cues. We found that low pH stimulates motility by triggering Ca2+-dependent secretion of apical micronemes, and that this cue is sufficient to overcome suppression by potassium ions and drive parasite motility, cell invasion and egress. We also discovered that acidification promotes membrane binding and cytolytic activity of perforin-like protein 1 (PLP1), a pore-forming protein required for efficient egress. Agents that neutralize pH reduce the efficiency of PLP1-dependent perforation of host membranes and compromise egress. Finally, although low pH stimulation of microneme secretion promotes cell invasion, it also causes PLP1-dependent damage to host cells, suggesting a mechanism by which neutral extracellular pH subdues PLP1 activity to allow cell invasion without overt damage to the target cell. These findings implicate acidification as a signal to activate microneme secretion and confine cytolytic activity to egress without compromising the viability of the next cell infected.

Viral Mimicry of Cdc2/cyclin-dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

Hamirally, Sofia; Kamil, Jeremy P.; Ndassa-Colday, Yasmine M.; Jahng, Wan Jin; Baek, Moon-Chang; Noton, Sarah; Silva, Laurie A.; Simpson-Holley, Martha; Lin, Alison J.; Knipe, David Mahan; Golan, David Eric; Marto, Jarrod; Coen, Donald Mark
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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37.19%
The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser[super]22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser[super]22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly...

Calcium ionophore-induced egress of toxoplasma gondii shortly after host cell invasion

Caldas, Lucio Ayres; Souza, Wanderley de; Attias, Márcia
Fonte: Inmetro - Instituto Nacional de Metrologia, Qualidade e Tecnologia Publicador: Inmetro - Instituto Nacional de Metrologia, Qualidade e Tecnologia
Tipo: Artigo de Revista Científica
ENG
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37.26%
11 p. : il.; Calcium plays crucial roles in important events of Toxoplasma gondii life cycle, including motility, invasion and egress from the host cell. Calcium ionophore has been used to artificially trigger release of the parasites from infected cells. In this report we describe that calcium ionophore A21387 induced T. gondii egress from LLC-MK2 cells at times as early as 2 h after entry. Addition of kinase inhibitors as staurosporine, wortmanine and genistein to the incubation medium significantly reduced ionophore-induced egress. The same occurred when the actin inhibitor cytochalasin D was used. Parasites egressed 2 h post-infection from ionophoretreated cultures were unable of establishing infection in a new cell. S-VHS recording of egressing parasites showed that they assume an hourglass shape as they cross the plasma membrane, similar to the moving junction constriction observed during active invasion, and extrudes the conoid, similarly to what is also observed during invasion. Transmission and high resolution scanning electron microscopy revealed that the egressing tachyzoites are free from host cell derived membranes. These include plasma membrane and parasitophorous vacuole membranes as well as associated endoplasmic reticulum membranes. Taken together...

CHARACTERIZING THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 2 UL21 PROTEIN IN VIRAL NUCLEAR EGRESS

Nassiri, Arash
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
EN; EN
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37.32%
The herpes simplex virus type 2 (HSV-2) is an important human pathogen that is the main cause of genital herpes infections and has a significant and wide-ranging impact on human health. HSV-2 infections are one of the most common sexually transmitted diseases and are highly prevalent worldwide, thus making understanding their infection mechanisms all the more relevant. The UL21 gene, which is conserved amongst members of the Alphaherpesvirinae subfamily, encodes a tegument protein that is essential for HSV-2 propagation. More specifically, UL21 plays a critical role in the primary envelopment of capsids during nuclear egress, however the precise mechanism by which it does this is unclear. To investigate the role of UL21 in HSV-2 nuclear egress, we focused on events upstream of primary envelopment. First, we examined the role of UL21 in disrupting the localization of nuclear lamins during infection. Nuclear rim localization of lamins A, C, B1, and B2 was indistinguishable between the wild-type (WT), UL21 null (∆UL21), or repaired (∆UL21R) HSV-2 strains, suggesting UL21 is not implicated in the disassembly of nuclear lamins at the nuclear membrane. Additionally, UL21 did not influence the localization of the nuclear egress complex (NEC)...

In Vivo Egress of an Alphaherpesvirus from Axons

Tomishima, Mark J.; Enquist, Lynn W.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2002 EN
Relevância na Pesquisa
27.32%
Many alphaherpesviruses establish a latent infection in the peripheral nervous systems of their hosts. This life cycle requires the virus to move long distances in axons toward the neuron's cell body during infection and away from the cell body during reactivation. While the events underlying entry of the virion into neurons during infection are understood in principle, no such consensus exists regarding viral egress from neurons after reactivation. In this study, we challenged two different models of viral egress from neurons by using pseudorabies virus (PRV) infection of the rat retina: does PRV egress solely from axon terminals, or can the virus egress from axon shafts as well as axon terminals? We took advantage of PRV gD mutants that are not infectious as extracellular particles but are capable of spreading by cell-cell contact. We observed that both wild-type virus and a PRV gD null mutant are capable of spreading from axons to closely apposed nonneuronal cells within the rat optic nerve after intravitreal infection. However, infection does not spread from these infected nonneuronal cells. We suggest that viral egress can occur sporadically along the length of infected axons and is not confined solely to axon terminals. Moreover...

Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress

Gaji, Rajshekhar Y.; Johnson, Derrick E.; Treeck, Moritz; Wang, Mu; Hudmon, Andy; Arrizabalaga, Gustavo
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 06/11/2015 EN
Relevância na Pesquisa
27.32%
Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover...

The Cytoplasmic Terminus of Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein B Is Not Essential for Virion Egress and Infectivity▿

Subramanian, R.; D'Auvergne, O.; Kong, Haixia; Kousoulas, K. G.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.45%
Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress...