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Louis M. Weiss - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Characterization of Protein Effector Export in the Bradyzoite Stage of Toxoplasma gondii.
mBio, 2020Co-Authors: Joshua Mayoral, Peter Shamamian, Louis M. WeissAbstract:ABSTRACT The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii Bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with Bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host. IMPORTANCEToxoplasma Bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with Bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.
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In vitro characterization of protein effector export in the bradyzoite stage of Toxoplasma gondii
2020Co-Authors: Joshua Mayoral, Peter Shamamian, Louis M. WeissAbstract:ABSTRACT The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain a chronic infection of its host for prolonged periods. Despite this, little is known regarding if and how T. gondii Bradyzoites, a quasi-dormant life-stage residing within intracellular cysts, manipulate the host cell so as to maintain a persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. As MYR1 is known to be involved in the translocation of parasite derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show by immunofluorescence that most effectors accumulate in the host nucleus at early but not late timepoints post-infection during the tachyzoite to bradyzoite transition and when parasites farther along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon-gamma (IFN-γ) signaling, previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with Bradyzoites, and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications as to how this highly successful parasite maintains a persistent infection of its host. IMPORTANCE Toxoplasma Bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life-stage successfully establishes and maintains a persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different timepoints post-bradyzoite differentiation and found that they accumulate largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediates its function after prolonged infection with Bradyzoites and provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.
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Bradyzoite and sexual stage development
Toxoplasma gondii, 2020Co-Authors: Anthony P. Sinai, Laura J. Knoll, Louis M. WeissAbstract:Abstract Advances in our understanding of bradyzoite biology, the regulation of the tachyzoite to bradyzoite transition, and the programs driving differentiation have been facilitated by the identification of markers and the application of genetic approaches and imaging-based tools to this asexual life cycle stage involved in chronic infection. While originally thought to be static, evidence has accumulated that cysts are dynamic structures and that Bradyzoites within cysts are heterogeneous. The formation of the cyst wall is an early event in differentiation, and its composition is now being elucidated. Significant advances in our understanding of the sexual cycle have also occurred, resulting in the development of both cell culture and murine systems that recapitulate the sexual cycle. This advance addresses a major bottleneck for investigations and has opened the gates to a broader dissection of the sexual life cycle stages.
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Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Joshua Mayoral, Manlio Di Cristina, Vern B. Carruthers, Louis M. WeissAbstract:Toxoplasma gondii, a member of the Apicomplexa, is known for its ability to infect an impressive range of host species. It is a common human infection that causes significant morbidity in congenitally infected children and immunocompromised patients. This parasite can be transmitted by Bradyzoites, a slowly replicating life stage found within intracellular tissue cysts, and oocysts, the sexual life cycle stage that develops in domestic cats and other Felidae. T. gondii Bradyzoites retain the capacity to revert back to the quickly replicating tachyzoite life stage, and when the host is immune compromised unrestricted replication can lead to significant tissue destruction. Bradyzoites are refractory to currently available Toxoplasma treatments. Improving our understanding of bradyzoite biology is critical for the development of therapeutic strategies to eliminate latent infection. This chapter describes a commonly used protocol for the differentiation of T. gondii tachyzoites into Bradyzoites using human foreskin fibroblast cultures and a CO2-limited alkaline cell media, which results in a high proportion of differentiated Bradyzoites for further study. Also described are methods for purifying tissue cysts from chronically infected mouse brain using isopycnic centrifugation and a recently developed approach for measuring bradyzoite viability.
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identification of compounds that suppress toxoplasma gondii tachyzoites and Bradyzoites
PLOS ONE, 2017Co-Authors: Yuho Murata, Louis M. Weiss, Tatsuki Sugi, Kentaro KatoAbstract:Drug treatment for toxoplasmosis is problematic, because current drugs cannot eradicate latent infection with Toxoplasma gondii and can cause bone marrow toxicity. Because latent infection remains after treatment, relapse of infection is a problem in both infections in immunocompromised patients and in congenitally infected patients. To identify lead compounds for novel drugs against Toxoplasma gondii, we screened a chemical compound library for anti-Toxoplasma activity, host cell cytotoxicity, and effect on Bradyzoites. Of 878 compounds screened, 83 demonstrated >90% parasite growth inhibition. After excluding compounds that affected host cell viability, we further characterized two compounds, tanshinone IIA and hydroxyzine, which had IC50 values for parasite growth of 2.5 μM and 1.0 μM, respectively, and had no effect on host cell viability at 25 μM. Both tanshinone IIA and hydroxyzine inhibited parasite replication after invasion and both reduced the number of in vitro-induced Bradyzoites, whereas, pyrimethamine, the current therapy, had no effect on Bradyzoites. Both tanshinone IIA and hydroxyzine are potent lead compounds for further medicinal chemistry. The method presented for evaluating compounds for bradyzoite efficacy represents a new approach to the development of anti-Toxoplasma drugs to eliminate latency and treat acute infection.
Wolfgang Bohne - One of the best experts on this subject based on the ideXlab platform.
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Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures
International journal for parasitology, 2002Co-Authors: Nathalie Vonlaufen, Arunasalam Naguleswaran, Wolfgang Bohne, Norbert Muller, Camilla Bjorkman, Nadine Keller, Milton M. Mcallister, Eliane J. Müller, Reto Caldelari, Andrew HemphillAbstract:Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and Bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum Bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 mM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of Bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
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targeted disruption of the bradyzoite specific gene bag1 does not prevent tissue cyst formation in toxoplasma gondii
Molecular and Biochemical Parasitology, 1998Co-Authors: Wolfgang Bohne, David J P Ferguson, Uwe Gross, Michael W. White, Christopher A. Hunter, David S RoosAbstract:Abstract Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite ‘tissue cyst’ form of Toxoplasma gondii, first appearing ∼2–3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii Bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of Bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.
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Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii.
Molecular and biochemical parasitology, 1998Co-Authors: Wolfgang Bohne, David J P Ferguson, Uwe Gross, Michael W. White, Christopher A. Hunter, David S RoosAbstract:Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite 'tissue cyst' form of Toxoplasma gondii, first appearing approximately 2-3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii Bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of Bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.
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stage specific expression of a selectable marker in toxoplasma gondii permits selective inhibition of either tachyzoites or Bradyzoites
Molecular and Biochemical Parasitology, 1997Co-Authors: Wolfgang Bohne, David S RoosAbstract:The establishment of culture conditions suitable for inducing differentiation of Toxoplasma gondii tachyzoites into parasites resembling the latent bradyzoite form has opened this important developmental transition to experimental analysis. In order to develop a genetic marker suitable for positive and negative selection during parasite differentiation. the T. gondii HXGPRT gene was placed under control of 5' flanking sequences derived from two bradyzoite-specific genes: BAG1 and LDH2. Random transgene integration at undefined genomic loci resulted in modest regulation (approximately 5-6-fold induction) above relatively high background levels (approximately 4% of wild-type controls). Integration of transgenes at a defined genomic position was achieved by targeting the uracil phosphoribosyl transferase (UPRT) locus using flanking homologous sequences and fluorouracil selection. This strategy was found to provide the added advantage of enhancing bradyzoite induction frequencies under conditions of pyrimidine starvation (low CO2). Constructs integrated in the direction of normal UPRT transcription exhibited moderate levels of inducibility, but transgenes integrated in the opposite direction were dramatically induced under differentiation conditions: 50-100-fold above the very low levels observed in tachyzoites (< 1% control). Positive selection (using mycophenolic acid) was shown to inhibit tachyzoites but not Bradyzoites, while negative selection (using 8-azahypoxanthine) inhibited Bradyzoites only. Stage-specific regulation of the HXGPRT selectable marker should permit genetic selections for the identification of mutants in the bradyzoite differentiation process.
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Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements
Molecular and Biochemical Parasitology, 1997Co-Authors: Wolfgang Bohne, Anne Wirsing, Udo GroßAbstract:Abstract BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to Bradyzoites. In order to define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5′ flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1 / cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/ cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5′ flanking sequences of BAG1 . Stage-specific regulation of BAG1/CAT was independent of the 3′-flanking region, since constructs containing 3′-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3′-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5′-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences.
David J P Ferguson - One of the best experts on this subject based on the ideXlab platform.
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Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii Bradyzoites.
Eukaryotic cell, 2011Co-Authors: Kerry R. Buchholz, David J P Ferguson, Heather M. Fritz, Xiucui Chen, Blythe Durbin-johnson, David M. Rocke, Patricia A. Conrad, John C. BoothroydAbstract:The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro Bradyzoites 4 days postinfection, and in vivo Bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by Bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo Bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall. Asexual replication of the protozoan parasite Toxoplasma gondii occurs through two developmental forms, the rapidly growing tachyzoite and the bradyzoite, which is slow growing and forms tissue cysts. Tachyzoites replicate during acute infection, but after about a week, conversion to Bradyzoites oc
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A novel rhoptry protein in Toxoplasma gondii Bradyzoites and merozoites
Molecular and biochemical parasitology, 2005Co-Authors: Jodi A. Schwarz, David J P Ferguson, Ashley E. Fouts, Craig A. Cummings, John C. BoothroydAbstract:The secretory organelles of Toxoplasma gondii orchestrate invasion of the host cell and establish the parasitophorous vacuole. Although much has been learned about the roles played by these organelles in invasion by the tachyzoite stage, little is known about the contents or functions of these organelles during bradyzoite development or pathogenesis. We identified a novel protein that localizes to the rhoptries of the bradyzoite stage, but is absent from the tachyzoite stage. This protein, BRP1, first appears in the nascent rhoptries during the first division of bradyzoite stage development. We observed secretion of BRP1 and other rhoptry proteins into the parasitophorous vacuole during bradyzoite development in vitro, but there was no evidence that this occurs in vivo. Brp1 knockout parasites did not appear to have any developmental or growth defects in vitro, and were able to establish infections in mice both as tachyzoites (via intraperitoneal injection of in vitro-derived tachyzoites) or Bradyzoites (via oral gavage using cysts harvested from mouse brain). Mice infected using brain cysts from the brp1 knockout or the control strain developed similar numbers and sizes of brain cysts. Thus BRP1 does not appear to play an essential role in development of the bradyzoite stage, development of brain cysts, or oral infection of new hosts, at least in the mouse model used here. Since we also observed that BRP1 is expressed in the merozoite stages in the gut of infected cats, the coccidian phase of the life cycle may be where BRP1 plays its most important role.
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use of molecular and ultrastructural markers to evaluate stage conversion of toxoplasma gondii in both the intermediate and definitive host
International Journal for Parasitology, 2004Co-Authors: David J P FergusonAbstract:Toxoplasma gondii has a complex life cycle involving definite (cat) and intermediate (all warm blooded animals) hosts. This gives rise to four infectious forms each of which has a distinctive biological role. Two (tachyzoite and merozoite) are involved in propagation within a host and two (bradyzoite and sporozoite) are involved in transmission to new hosts. The various forms can be identified by their structure, host parasite relationship and distinctive developmental processes. In the present in vivo study, the various stages have been evaluated by electron microscopy and immunocytochemistry using a panel of molecular markers relating to surface and cytoplasmic molecules, metabolic iso-enzymes and secreted proteins that can differentiate between tachyzoite, bradyzoite and coccidian development. Tachyzoites were characterised as being positive for surface antigen 1, enolase isoenzyme 2, lactic dehydrogenase isoenzyme 1 and negative for bradyzoite antigen 1. In contrast, Bradyzoites were negative for SAG1 but positive for BAG1, ENO1 and LDH2. When stage conversion was followed in brain lesion at 10 and 15 days post-infection, tachyzoites were predominant but a number of single intermediate organisms displaying tachyzoite and certain bradyzoite markers were observed. At later time points, small groups of organisms displaying only bradyzoite markers were also present. A number (9) of dense granule proteins (GRA1-8, NTPase) have also been identified in both tachyzoites and Bradyzoites but there were differences in their location during parasite development. All the dense granule proteins extensively label the parasitophorous vacuole during tachyzoite development. In contrast the tissue cyst wall displays variable staining for the dense granule proteins, which also expresses an additional unique cyst wall protein. The molecular differences could be identified at the single cell stage consistent with conversion occurring at the time of entry into a new cell. These molecular differences were reflected in the structural differences in the parasitophorous vacuoles observed by electron microscopy. Stage conversion to enteric (coccidian) development was limited to the enterocytes of the cat small intestine. Although no specific markers were available, this form of development can be identified by the absence of specific tachyzoite (SAG1) and bradyzoite (BAG1) markers although the isoenzymes ENO2 and LHD1 were expressed. There was also a significant difference in the expression of the dense granule proteins. The coccidian stages and merozoites only expressed two (GRA7 and NTPase) of the nine dense granule proteins and this was reflected in significant differences in the structure of the parasitophorous vacuole. The coccidian stages also undergo conversion from asexual to sexual development. The mechanism controlling this process is unknown but does not involve any change in the host cell type or parasitophorous vacuole and may be pre-programmed, since the number of asexual cycles was self-limiting. In conclusion, it was possible using a combination of molecular markers to identify tachyzoite, bradyzoite and coccidian development in tissue sections.
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targeted disruption of the bradyzoite specific gene bag1 does not prevent tissue cyst formation in toxoplasma gondii
Molecular and Biochemical Parasitology, 1998Co-Authors: Wolfgang Bohne, David J P Ferguson, Uwe Gross, Michael W. White, Christopher A. Hunter, David S RoosAbstract:Abstract Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite ‘tissue cyst’ form of Toxoplasma gondii, first appearing ∼2–3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii Bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of Bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.
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Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii.
Molecular and biochemical parasitology, 1998Co-Authors: Wolfgang Bohne, David J P Ferguson, Uwe Gross, Michael W. White, Christopher A. Hunter, David S RoosAbstract:Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite 'tissue cyst' form of Toxoplasma gondii, first appearing approximately 2-3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii Bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of Bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.
John C. Boothroyd - One of the best experts on this subject based on the ideXlab platform.
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Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii Bradyzoites.
Eukaryotic cell, 2011Co-Authors: Kerry R. Buchholz, David J P Ferguson, Heather M. Fritz, Xiucui Chen, Blythe Durbin-johnson, David M. Rocke, Patricia A. Conrad, John C. BoothroydAbstract:The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro Bradyzoites 4 days postinfection, and in vivo Bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by Bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo Bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall. Asexual replication of the protozoan parasite Toxoplasma gondii occurs through two developmental forms, the rapidly growing tachyzoite and the bradyzoite, which is slow growing and forms tissue cysts. Tachyzoites replicate during acute infection, but after about a week, conversion to Bradyzoites oc
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A cluster of four surface antigen genes specifically expressed in Bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence.
Infection and immunity, 2008Co-Authors: Jeroen P. J. Saeij, Gustavo Arrizabalaga, John C. BoothroydAbstract:Toxoplasma gondii is one of the most successful protozoan parasites of warm-blooded animals. Stage-specific expression of its surface molecules is thought to be key to its ability to establish chronic infection in immunocompetent animals. The rapidly dividing tachyzoite stage displays a different subset of family of surface antigen 1 (SAG1)-related sequences (SRSs) from that displayed by the encysted bradyzoite stage. It is possible that this switch is necessary to protect the Bradyzoites against an immune response raised against the tachyzoite stage. Alternatively, it might be that bradyzoite SRSs evolved to facilitate invasion of different cell types, such as those found in the brain, where cysts develop, or the small intestine, where Bradyzoites must enter after oral infection. Here we studied the function of a cluster of four tandem genes, encoding bradyzoite SRSs called SAG2C, -D, -X, and -Y. Using bioluminescence imaging of mice infected with parasites expressing firefly luciferase (FLUC) driven by the SAG2D promoter, we show stage conversion for the first time in living animals. A truncated version of the SAG2D promoter (SAG2Dmin) gave efficient expression of FLUC in both tachyzoites and Bradyzoites, indicating that the bradyzoite specificity of the complete SAG2D promoter is likely due to an element(s) that normally suppresses expression in tachyzoites. Comparing mice infected with the wild type or a mutant where the SAG2CDXY cluster of genes has been deleted (ΔSAG2CDXY), we demonstrate that whereas ΔSAG2CDXY parasites are less capable of maintaining a chronic infection in the brain, they do not show a defect in oral infectivity.
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The BSR4 protein is up-regulated in Toxoplasma gondii Bradyzoites, however the dominant surface antigen recognised by the P36 monoclonal antibody is SRS9.
International journal for parasitology, 2007Co-Authors: Tam T Van, John C. Boothroyd, Seon-kyeong Kim, Manel Camps, Laura J. KnollAbstract:The protozoan parasite, Toxoplasma gondii, interconverts between fast-growing tachyzoites and slow-growing Bradyzoites within intermediate hosts. The surface of T. gondii is covered by the SAG1-related sequence (SRS) superfamily of glycosyl phosphatidyl inositol-anchored proteins, many of which are stage-specific. Previous transient transfection of BSR4, a member of the SRS superfamily, showed reactivity with the bradyzoite-specific P36 mAb by immunofluorescene assay. BSR4 mRNA levels were equally abundant in tachyzoites and Bradyzoites, suggesting post-transcriptional regulation of the protein. In this study, we show that BSR4 protein is present in both tachyzoites and Bradyzoites, but up-regulated in Bradyzoites. However, stable expression of BSR4 in two BSR4-negative T. gondii strains shows minimal reactivity to the P36 mAb by Western immunoblotting, even though the BSR4 protein is abundant. We discovered that the SRS9 protein, a bradyzoite-specific member of the SRS superfamily and encoded immediately downstream of BSR4, was also ablated in the BSR4-negative strains, suggesting that SRS9 is the surface antigen recognised by the P36 mAb. Stable expression of SRS9 in the BSR4 mutant strains shows robust reactivity to the P36 mAb. Immunoprecipitation experiments confirm that the P36 mAb interacts with the SRS9 protein. These data indicate that while the BSR4 protein is up-regulated in Bradyzoites, the dominant antigen that the P36 mAb recognises is SRS9.
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Infection with Toxoplasma gondii Bradyzoites Has a Diminished Impact on Host Transcript Levels Relative to Tachyzoite Infection
Infection and immunity, 2006Co-Authors: Ashley E. Fouts, John C. BoothroydAbstract:Toxoplasma gondii, an intracellular pathogen, has the potential to infect nearly every warm-blooded animal but rarely causes morbidity. The ability for the parasite to convert to the bradyzoite stage and live inside slow-growing cysts that can go unnoticed by the host immune system allows for parasite persistence for the life of the infected host. This intracellular survival likely necessitates host cell modulation, and tachyzoites are known to modify a number of signaling cascades within the host to promote parasite survival. Little is known, however, about how Bradyzoites manipulate their host cell. Microarrays were used to profile the host transcriptional changes caused by bradyzoite infection and compared to those of tachyzoite-infected and uninfected hosts cells 2 days postinfection in vitro. Infection resulted in chemokine, cytokine, extracellular matrix, and growth factor transcript level changes. A small group of genes were specifically induced by tachyzoite infection, including granulocyte-macrophage colony-stimulating factor, BCL2-related protein A1, and interleukin-24. Bradyzoite infection yielded only about half the changes seen with tachyzoite infection, and those changes that did occur were almost all of lower magnitude than those induced by tachyzoites. These results suggest that Bradyzoites lead a more stealthy existence within the infected host cell.
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A novel rhoptry protein in Toxoplasma gondii Bradyzoites and merozoites
Molecular and biochemical parasitology, 2005Co-Authors: Jodi A. Schwarz, David J P Ferguson, Ashley E. Fouts, Craig A. Cummings, John C. BoothroydAbstract:The secretory organelles of Toxoplasma gondii orchestrate invasion of the host cell and establish the parasitophorous vacuole. Although much has been learned about the roles played by these organelles in invasion by the tachyzoite stage, little is known about the contents or functions of these organelles during bradyzoite development or pathogenesis. We identified a novel protein that localizes to the rhoptries of the bradyzoite stage, but is absent from the tachyzoite stage. This protein, BRP1, first appears in the nascent rhoptries during the first division of bradyzoite stage development. We observed secretion of BRP1 and other rhoptry proteins into the parasitophorous vacuole during bradyzoite development in vitro, but there was no evidence that this occurs in vivo. Brp1 knockout parasites did not appear to have any developmental or growth defects in vitro, and were able to establish infections in mice both as tachyzoites (via intraperitoneal injection of in vitro-derived tachyzoites) or Bradyzoites (via oral gavage using cysts harvested from mouse brain). Mice infected using brain cysts from the brp1 knockout or the control strain developed similar numbers and sizes of brain cysts. Thus BRP1 does not appear to play an essential role in development of the bradyzoite stage, development of brain cysts, or oral infection of new hosts, at least in the mouse model used here. Since we also observed that BRP1 is expressed in the merozoite stages in the gut of infected cats, the coccidian phase of the life cycle may be where BRP1 plays its most important role.
J Heesemann - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of a bradyzoite specifically expressed gene hsp30 bag1 of toxoplasma gondii related to genes encoding small heat shock proteins of plants
Molecular Microbiology, 1995Co-Authors: Wolfgang Bohne, David J P Ferguson, U. Gross, J HeesemannAbstract:Summary Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite-specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C-terminal region of small heat-shock proteins from plants. Stage-specific expression of the naturally occurring 30kDa antigen in Bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen, Immuno-electron microscopy indicated a cytosolic location of this antigen in Bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite-specific primers amplified transcripts in Bradyzoites only, not in tachyzoites.
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Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants.
Molecular Microbiology, 1995Co-Authors: Wolfgang Bohne, David J P Ferguson, U. Gross, J HeesemannAbstract:Summary Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite-specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C-terminal region of small heat-shock proteins from plants. Stage-specific expression of the naturally occurring 30kDa antigen in Bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen, Immuno-electron microscopy indicated a cytosolic location of this antigen in Bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite-specific primers amplified transcripts in Bradyzoites only, not in tachyzoites.