A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

<p>Abstract</p> <p>Background</p> <p>Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators.</p> <p>Results</p> <p>Taking advantage of available genome sequences and annotation for <it>Toxoplasma gondii </it>(a pathogenic parasite responsible for illness in immunocompromised individuals) and <it>Plasmodium falciparum </it>(a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for <it>Toxoplasma</it>, and single-nucleotide polymorphism (SNP)-based genotyping of both <it>T. gondii </it>and <it>P. falciparum</it>. Expression profiling of the three clonotypic lineages dominating <it>T. gondii </it>populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional <it>T. gondii </it>isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform future array designs.</p> <p>Conclusions</p> <p>In addition to providing an initial global view of the <it>T. gondii </it>transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the <it>T. gondii </it>research community, and several aspects of the design strategy are likely to be useful for other pathogens.</p

Subcellular Antigen Location Influences T-Cell Activation during Acute Infection with Toxoplasma gondii

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8+ T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8+ T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8+ T-cell activation was assayed using ‘B3Z’ reporter cells in vitro, or adoptively-transferred OVA-specific ‘OT-I’ CD8+ T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8+ T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8+ T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis

Deux enzymes glycolytiques du protozoaire parasite Toxoplasma gondii (clonage, expression différentielle des gènes, caractérisation enzymatique et phylogénie)

L'interconversion du tachyzoite virulent et du bradyzoite quiescent est un processus important dans la pathogenese du protozoaire parasite toxoplasma gondii. Les bases moleculaires impliquees dans cette differenciation parasitaire ne sont pas definies. Une technique d'induction in vitro de la differenciation, conjuguee a la construction d'une banque soustractive, a permis de caracteriser des transcrits regules selon les stades parasitaires. Deux fragments d'adnc presentant de fortes homologies avec les enzymes glycolytiques glucose-6-phosphate isomerase (g6pi) et enolase (eno1) ont ete isoles. Nous avons alors clone les adnc complets codant ces deux enzymes. Les deux adnc complementent des mutants bacteriens deficients en g6pi et enolase, et generent des enzymes recombinantes actives produites chez e. Coli. Le gene eno1 est exclusivement transcrit chez le bradyzoite, alors que le transcrit du gene g6pi est retrouve dans les deux stades parasitaires avec un niveau d'expression plus important chez le bradyzoite. En revanche, les proteines g6pi et eno1 sont exclusivement exprimees par le bradyzoite. Les analyses structurales et phylogeniques montrent que les g6pi et enolases de t. Gondii et p. Falciparum sont tres proches de leurs homologues chez les plantes.Une etude structure-fonction de deux boucles peptidiques, typiques des enolases de t. Gondii et p. Falciparum et de celles de plantes superieures, demontre leur influence sur l'activite et la stabilite de l'enzyme. Enfin, nous avons egalement isole l'adnc codant une seconde enolase. Le gene eno2 est preferentiellement transcrit chez le tachyzoite virulent. L'isoforme eno2 est specifique de cette forme. Les caracteristiques enzymatiques des deux isoformes d'enolases de t. Gondii ont ete comparees, et leurs differences d'activites pourraient temoigner du maintien de la glycolyse en accord avec les besoins specifiques des deux stades asexues de t. Gondii dans le metabolisme des carbohydrates.LILLE1-BU (590092102) / SudocSudocFranceF

TgMORN1 is a key organizer for the basal complex of Toxoplasma gondii.

Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (DeltaTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in DeltaTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, DeltaTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis

Development of a System To Study CD4(+)-T-Cell Responses to Transgenic Ovalbumin-Expressing Toxoplasma gondii during Toxoplasmosis

The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4(+) T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4(+) T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-γ). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-γ(−/−) mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4(+)-T-cell responses during toxoplasmosis

Cutting Edge: Dendritic Cells Copulsed with Microbial and Helminth Antigens Undergo Modified Maturation, Segregate the Antigens to Distinct Intracellular Compartments, and Concurrently Induce Microbe-Specific Th1 and Helminth-Specific Th2 Responses

To examine the ability of dendritic cells (DC) to discriminate between helminth and microbial Ag and induce appropriately polarized Th responses, mouse DC were copulsed with the helminth Ag, schistosome egg Ag (SEA), along with the bacterium Proprionebacterium acnes, Pa, and transferred into wild-type mice. Strikingly, SEA/Pa-copulsed DC induced concurrent Pa-specific Th1 (but not Th2) responses and SEA-specific Th2 (but not Th1) responses. Although DC exposed to both Ag undergo many of the maturation-associated changes that accompany exposure to Pa alone, Pa-induced IL-12 production was inhibited by SEA. Examination of Ag uptake revealed that SEA and Pa are acquired via discrete pathways and enter non-overlapping intracellular compartments. Data suggest that segregation of SEA and Pa into distinct compartments, coupled with SEA-induced modifications of the DC maturation pathway, are significant components of the ability of DC to interpret signals inherent to SEA and Pa and induce appropriately polarized Th responses

Benefits and barriers to engagement of mental health caregivers in advisory roles: Results from a cross‐sectional survey

Abstract Background and Aims Mental health institutions and community organizations have had difficulty recruiting patients and caregivers onto their Patient, Family, and Community Advisory Committees (PFACs). Previous research has focused on barriers and enablers of engaging patients and caregivers who have advisory experience. This study acknowledges the experiential difference between patients and caregivers by focusing only on caregivers; further, we compare the barriers and enablers between advising versus non‐advising caregivers of loved ones with mental illness. Methods Data from a cross‐sectional survey codesigned by researchers, staff, clients, and caregiver affiliated with a tertiary mental health center were completed by n = 84 caregivers (n = 40 past/current PFAC advising caregivers; n = 44 non‐advising caregivers). Results Caregivers were disproportionately female and late middle‐aged. Advising and non‐advising caregivers differed on employment status. There were no differences of the demographics of their care‐recipients. More non‐advising caregivers reported being hindered from PFAC engagement by family‐related duties and interpersonal demands. Finally, more advising caregivers considered being publicly acknowledged as important. Conclusions Advising and non‐advising caregivers of loved ones with mental illness were similar in demographics and in reporting the enablers and hindrances that impact PFAC engagement. Nevertheless, our data highlights specific considerations that institutions/organizations should consider when recruiting and retaining caregivers on PFACs. Patient or Public Contribution This project was led by a caregiver advisor to address a need she saw in the community. The surveys were codesigned by a team of two caregivers, one patient, and one researcher. The surveys were reviewed by a group of five caregivers external to the project. The results of the surveys were discussed with two caregivers involved directly with the project