CNS-derived CCL21 is both sufficient to drive homeostatic CD4+ T cell proliferation and necessary for efficient CD4+ T cell migration

Brain Behav Immun. 2010 Sep 21. [Epub ahead of print]

CNS-derived CCL21 is both sufficient to drive homeostatic CD4+ T cell proliferation and necessary for efficient CD4+ T cell migration into the CNS parenchyma following Toxoplasma gondii infection

Ploix CC, Noor S, Crane J, Masek K, Carter W, Lo DD, Wilson EH, Carson MJ.

Roche Ltd., Basel, Switzerland.

Abstract
Injury, infection and autoimmune triggers increase CNS expression of the chemokine CCL21. Outside the CNS, CCL21 contributes to chronic inflammatory disease and autoimmunity by three mechanisms: recruitment of lymphocytes into injured or infected tissues, organization of inflammatory infiltrates into lymphoid-like structures and promotion of homeostatic CD4+ T-cell proliferation. To test if CCL21 plays the same role in CNS inflammation, we generated transgenic mice with astrocyte-driven expression of CCL21 (GFAP-CCL21 mice). Astrocyte-produced CCL21 was bioavailable and sufficient to support homeostatic CD4+ T-cell proliferation in cervical lymph nodes even in the absence of endogenous CCL19/CCL21. However, lymphocytes and glial-activation were not detected in the brains of uninfected GFAP-CCL21 mice, although CCL21 levels in GFAP-CCL21 brains were higher than levels expressed in inflamed Toxoplasma-infected nontransgenic brains. Following Toxoplasma infection, T-cell extravasation into submeningeal, perivascular and ventricular sites of infected CNS was not CCL21-dependent, occurring even in CCL19/CCL21-deficient mice. However, migration of extravasated CD4+, but not CD8+ T cells from extra-parenchymal CNS sites into the CNS parenchyma was CCL21-dependent. CD4+ T cells preferentially accumulated at perivascular, submeningeal and ventricular spaces in infected CCL21/CCL19-deficient mice. By contrast, greater numbers of CD4+ T cells infiltrated the parenchyma of infected GFAP-CCL21 mice than in wild-type or CCL19/CCL21-deficient mice. Together these data indicate that CCL21 expression within the CNS has the potential to contribute to T cell-mediated CNS pathology via: (a) homeostatic priming of CD4+ T-lymphocytes outside the CNS and (b) by facilitating CD4+ T-cell migration into parenchymal sites following pathogenic insults to the CNS.

PMID: 20868739 [PubMed - as supplied by publisher]

Topological journey of parasite-derived antigens for presentation by MHC class I molecules

Trends Immunol. 2010 Sep 22. [Epub ahead of print]

Topological journey of parasite-derived antigens for presentation by MHC class I molecules

Blanchard N, Shastri N.

Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA; Current address: Physiopathology Research Centre of Toulouse-Purpan/INSERM U563, Department of Immunology and Infectious diseases, CHU Purpan, BP3028, 31024 Toulouse Cedex, France.

Abstract
Within cells of their host, many bacteria and parasites inhabit specialized compartments, such as a modified phagosome for Mycobacterium tuberculosis or a parasitophorous vacuole for Toxoplasma gondii. These locations could exclude microbial material from entry into the MHC class I surveillance pathway. Remarkably, however, under these circumstances, cells can still signal the presence of invading pathogens to circulating CD8(+) T cells, which typically play a key role in protection against such intracellular organisms. Here, we review MHC I presentation pathways in various contexts, ranging from model antigens in non-infectious settings to pathogen-infected cells. We suggest that presentation of intracellular pathogens can be described as not just one, but several distinct pathways; perhaps because diverse pathogens have evolved different strategies to interact with host cells.

PMID: 20869317 [PubMed - as supplied by publisher]

{alpha}-tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties to Confer Dinitroaniline Resistance

Eukaryot Cell. 2010 Sep 24. [Epub ahead of print]

{alpha}-tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties to Confer Dinitroaniline Resistance

Lyons-Abbott S, Sackett DL, Wloga D, Gaertig J, Morgan RE, Werbovetz KA, Morrissette NS.

Department of Molecular Biology and Biochemistry, University of CA, Irvine 92617; Laboratory of Integrative and Medical Biophysics, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA 20892; Department of Cellular Biology, University of Georgia, 30602; Division of Medicinal Chemistry & Pharmacognosy, Ohio State University, 43210.

Abstract
Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provides additional support for the proposed dinitroaniline binding site on α-tubulin and validates the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.

PMID: 20870876

Coordinated Progression through Two Subtranscriptomes Underlies the Tachyzoite Cycle of Toxoplasma gondii

PLoS One. 2010 Aug 26;5(8). pii: e12354.

Coordinated Progression through Two Subtranscriptomes Underlies the Tachyzoite Cycle of Toxoplasma gondii

Behnke MS, Wootton JC, Lehmann MM, Radke JB, Lucas O, Nawas J, Sibley LD, White MW.

Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America.

Abstract
BACKGROUND: Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission.

METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities.

CONCLUSION/SIGNIFICANCE: Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.

PMID: 20865045 [PubMed - in process]

Congenital infections with Trypanosoma cruzi or Toxoplasma gondii are associated with decreased serum interferon-γ and interleukin-18

Ann Trop Med Parasitol. 2010 Sep;104(6):485-92.

Congenital infections with Trypanosoma cruzi or Toxoplasma gondii are associated with decreased serum concentrations of interferon-γ and interleukin-18 but increased concentrations of interleukin-10

Mayer JP, Biancardi M, Altcheh J, Freilij H, Weinke T, Liesenfeld O.

Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin, Campus Benjamin Franklin, Hindenburgdamm 27, 12203 Berlin; Germany, Klinik für Kinderchirurgie, Helios Klinik Berlin-Buch, Schwanebecker Chaussee 50, 13125 Berlin, Germany.

Abstract
Little is known about the immune responses of newborns with congenital Chagas disease (CCD) or congenital toxoplasmosis (CT) but they probably differ to those seen in adults with Chagas disease or toxoplasmosis, leading to differences in pathology. The concentrations of interleukin-18 (IL-18), interferon-γ (IFN-γ) and interleukin 10 (IL-10) in the sera of infants with CCD or CT were determined and compared with those in the sera of uninfected controls (born to mothers who were seropositive or seronegative for Trypanosoma cruzi). The infants with CCD or CT were found to have lower IL-18 and IFN-γ concentrations but higher IL-10 concentrations than the uninfected controls. The IL-18 and IFN-γ concentrations were also significantly lower in the infants with CCD than in those with CT. Although the infants with symptomatic CT had significantly higher serum concentrations of IL-18 than those with asymptomatic infection with Toxoplasma, the infants with symptomatic CCD had similar serum concentrations of IL-18 to the infants with asymptomatic Tr. cruzi infection. Taken together, these results indicate that IL-10 contributes to the suppression of pro-inflammatory immune responses and therefore, perhaps, to clinically overt CCD and CT.

PMID: 20863437 [PubMed - in process]

UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

PLoS Pathog. 2010 Aug 26;6(8). pii: e1001071.

UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

Melo MB, Kasperkovitz P, Cerny A, Könen-Waisman S, Kurt-Jones EA, Lien E, Beutler B, Howard JC, Golenbock DT, Gazzinelli RT.

University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Abstract
UNC93B1 associates with Toll-Like Receptor (TLR) 3, TLR7 and TLR9, mediating their translocation from the endoplasmic reticulum to the endolysosome, hence allowing proper activation by nucleic acid ligands. We found that the triple deficient '3d' mice, which lack functional UNC93B1, are hyper-susceptible to infection with Toxoplasma gondii. We established that while mounting a normal systemic pro-inflammatory response, i.e. producing abundant MCP-1, IL-6, TNFα and IFNγ, the 3d mice were unable to control parasite replication. Nevertheless, infection of reciprocal bone marrow chimeras between wild-type and 3d mice with T. gondii demonstrated a primary role of hemopoietic cell lineages in the enhanced susceptibility of UNC93B1 mutant mice. The protective role mediated by UNC93B1 to T. gondii infection was associated with impaired IL-12 responses and delayed IFNγ by spleen cells. Notably, in macrophages infected with T. gondii, UNC93B1 accumulates on the parasitophorous vacuole. Furthermore, upon in vitro infection the rate of tachyzoite replication was enhanced in non-activated macrophages carrying mutant UNC93B1 as compared to wild type gene. Strikingly, the role of UNC93B1 on intracellular parasite growth appears to be independent of TLR function. Altogether, our results reveal a critical role for UNC93B1 on induction of IL-12/IFNγ production as well as autonomous control of Toxoplasma replication by macrophages.

PMID: 20865117 [PubMed - in process]

Advances in imaging the innate and adaptive immune response to Toxoplasma gondii

Future Microbiol. 2010 Sep;5:1321-8.

Advances in imaging the innate and adaptive immune response to Toxoplasma gondii

John B, Weninger W, Hunter CA.

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract
Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts and can have devastating effects in the developing fetus as well as the immunocompromised host. An appreciation of how this organism interacts with the host immune system is crucial to understanding the pathogenesis of this disease. The last decade has been marked by the application of various imaging techniques, such as bioluminescent imaging as well as confocal and multiphoton microscopy to study toxoplasmosis. The ability to manipulate parasites to express fluorescent/bioluminescent markers or model antigens/enzymes combined with the development of reporter mice that allow the detection of distinct immune populations have been crucial to the success of many of these studies. These approaches have permitted the visualization of parasites and immune cells in real-time and provided new insights into the nature of host-pathogen interactions. This article highlights some of the advances in imaging techniques, their strengths and weaknesses, and how these techniques have impacted our understanding of the interaction between parasites and various immune populations during toxoplasmosis.

PMID: 20860479 [PubMed - in process]

Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

Proc Natl Acad Sci U S A. 2010 Sep 20. [Epub ahead of print]

Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

Joyce BR, Queener SF, Wek RC, Sullivan WJ Jr.

Departments of Pharmacology and Toxicology and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202.

Abstract
While seeking a new host cell, obligate intracellular parasites, such as the protozoan Toxoplasma gondii, must be able to endure the stress of an extracellular environment. The mechanisms Toxoplasma use to remain viable while deprived of a host cell are not understood. We have previously shown that phosphorylation of Toxoplasma eukaryotic initiation factor-2α (TgIF2α) is a conserved response to stress. Here we report the characterization of Toxoplasma harboring a point mutation (S71A) in TgIF2α that prevents phosphorylation. Results show that TgIF2α phosphorylation is critical for parasite viability because the TgIF2α-S71A mutants are ill-equipped to cope with life outside the host cell. The TgIF2α-S71A mutants also showed a significant delay in producing acute toxoplasmosis in vivo. We conclude that the phosphorylation of TgIF2α plays a crucial role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell.

PMID: 20855600 [PubMed - as supplied by publisher]

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

PLoS One. 2010 Sep 14;5(9). pii: e12736.

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

Bakszt R, Wernimont A, Allali-Hassani A, Mok MW, Hills T, Hui R, Pizarro JC.

The Structural Genomics Consortium (SGC), University of Toronto, Toronto, Ontario, Canada.

Abstract
BACKGROUND: Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population.

METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers.

CONCLUSIONS: We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

PMID: 20856875 [PubMed - as supplied by publisher]

Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif is Required for Recognition by Toll-like Receptor 11

J Mol Biol. 2010 Sep 16. [Epub ahead of print]

Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif is Required for Recognition by Toll-like Receptor 11

Kucera K, Koblansky AA, Saunders LP, Frederick KB, De La Cruz EM, Ghosh S, Modis Y.

Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.

Abstract
Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin, and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa like Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor 11 (TLR11) generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long β-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the IL-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the β-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and β-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite C. parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from P. falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin, and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the β-hairpin, and diverges widely from the actin-binding surfaces of vertebrate profilins.

PMID: 20851125 [PubMed - as supplied by publisher]

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

PLoS Pathog. 2010 Sep 9;6(9). pii: e1001094.

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Beck JR, Rodriguez-Fernandez IA, Cruz de Leon J, Huynh MH, Carruthers VB, Morrissette NS, Bradley PJ.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America.

Abstract
Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.

PMID: 20844581 [PubMed - in process]

Toxoplasma gondii infection in first-episode and inpatient individuals with schizophrenia

Int J Infect Dis. 2010 Sep 13. [Epub ahead of print]

Toxoplasma gondii infection in first-episode and inpatient individuals with schizophrenia

Hamidinejat H, Ghorbanpoor M, Hosseini H, Alavi SM, Nabavi L, Jalali MH, Borojeni MP, Jafari H, Mohammadaligol S.

Department of Pathobiology, Shahid Chamran University, Ahvaz, Iran.

Abstract
BACKGROUND: A high seroprevalence of Toxoplasma gondii infection has been detected in psychiatric patients, particularly in schizophrenia cases.

METHODS: In the present study 98 patients suffering from schizophrenia (58 inpatients and 40 first-episode patients) and 96 control patients (50 healthy volunteers and 46 with a depressive disorder) were examined for the presence of both IgG and IgM antibodies against T. gondii by enzyme-linked immunosorbent assay (ELISA). We applied the Bradford Hill criteria to identify the weight of causal inference.

RESULTS: The positivity rate of anti-T. gondii IgG antibodies among individuals with schizophrenia (57.1%) was significantly higher than in healthy controls (29.2%). There were no associations between immune status ratio (ISR) values and the risk of schizophrenia. The weight of evidence approach using the Bradford Hill criteria revealed a 92% probability of a causal association.

CONCLUSION: Our results show that exposure to T. gondii may lead to schizophrenia.

PMID: 20843718 [PubMed - as supplied by publisher]

Apicomplexan parasite adhesins: novel strategies for targeting host cell carbohydrates

Curr Opin Struct Biol. 2010 Sep 13. [Epub ahead of print]

Apicomplexan parasite adhesins: novel strategies for targeting host cell carbohydrates

Boulanger MJ, Tonkin ML, Crawford J.

Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, STN CSC, Victoria, BC, Canada V8W 3P6.

Abstract
Apicomplexan parasites such as Plasmodium spp. (malaria) and Toxoplasma gondii (toxoplasmosis) are significant global pathogens of humans and animals. Unlike many intracellular bacterial and viral pathogens that rely on host cell uptake machinery to gain entry, apicomplexan parasites promote recognition, attachment and ultimately invasion of host cells through an orchestrated delivery of adhesins. While several of these adhesins are now known to target host cell glycans, only recently have atomic level insights been forthcoming. Here we review recent developments in defining detailed molecular blueprints used by these widespread pathogens to drive host cell adhesion and promote infectivity.

PMID: 20843678 [PubMed - as supplied by publisher]

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

PLoS One. 2010 Aug 19;5(8). pii: e12302.

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

Lorestani A, Sheiner L, Yang K, Robertson SD, Sahoo N, Brooks CF, Ferguson DJ, Striepen B, Gubbels MJ.

Department of Biology, Boston College, Chestnut Hill, Massachusetts, United States of America.

Abstract
The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.

PMID: 20808817 [PubMed - in process]

Identification of attractive drug targets in neglected-disease pathogens using an in silico approach

PLoS Negl Trop Dis. 2010 Aug 24;4(8). pii: e804.

Identification of attractive drug targets in neglected-disease pathogens using an in silico approach

Crowther GJ, Shanmugam D, Carmona SJ, Doyle MA, Hertz-Fowler C, Berriman M, Nwaka S, Ralph SA, Roos DS, Van Voorhis WC, Agüero F.

Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America.

Abstract
BACKGROUND: The increased sequencing of pathogen genomes and the subsequent availability of genome-scale functional datasets are expected to guide the experimental work necessary for target-based drug discovery. However, a major bottleneck in this has been the difficulty of capturing and integrating relevant information in an easily accessible format for identifying and prioritizing potential targets. The open-access resource TDRtargets.org facilitates drug target prioritization for major tropical disease pathogens such as the mycobacteria Mycobacterium leprae and Mycobacterium tuberculosis; the kinetoplastid protozoans Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi; the apicomplexan protozoans Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni.

METHODOLOGY/PRINCIPAL FINDINGS: Here we present strategies to prioritize pathogen proteins based on whether their properties meet criteria considered desirable in a drug target. These criteria are based upon both sequence-derived information (e.g., molecular mass) and functional data on expression, essentiality, phenotypes, metabolic pathways, assayability, and druggability. This approach also highlights the fact that data for many relevant criteria are lacking in less-studied pathogens (e.g., helminths), and we demonstrate how this can be partially overcome by mapping data from homologous genes in well-studied organisms. We also show how individual users can easily upload external datasets and integrate them with existing data in TDRtargets.org to generate highly customized ranked lists of potential targets.

CONCLUSIONS/SIGNIFICANCE: Using the datasets and the tools available in TDRtargets.org, we have generated illustrative lists of potential drug targets in seven tropical disease pathogens. While these lists are broadly consistent with the research community's current interest in certain specific proteins, and suggest novel target candidates that may merit further study, the lists can easily be modified in a user-specific manner, either by adjusting the weights for chosen criteria or by changing the criteria that are included.

PMID: 20808766 [PubMed - in process]

CXCL10 is required to maintain T cell populations and control parasite replication during chronic ocular toxoplasmosis

Invest Ophthalmol Vis Sci. 2010 Sep 1. [Epub ahead of print]

CXCL10 is required to maintain T cell populations and control parasite replication during chronic ocular toxoplasmosis

Norose K, Kikumura A, Luster AD, Hunter CA, Harris TH.

Infection and Host Defense, Chiba University, Chiba, Japan.

Abstract
PURPOSE. Toxoplasma gondii is a major cause of ocular disease, which can lead to permanent vision loss in humans. T cells are critically involved in parasite control, but little is known about the molecules that promote T cell trafficking and migration in the retina. Thus, the aim of this study was to image and dissect the T cell response during chronic toxoplasmic retinochoroiditis. METHODS. C57BL/6 mice were infected with the Me49 strain of T. gondii and T cells that infiltrated the eye were analyzed by flow cytometry and imaged using multi-photon microscopy. IFN-gamma, CXCL9, CXCL10, and CXCR3 mRNA levels were measured by real-time PCR. To investigate the role of CXCL10, mice were treated with anti-CXCL10 antibodies and histopathology and immunohistochemistry were performed to monitor changes in pathology, cellular infiltration, and parasite burden in the eye. RESULTS. Infection with T. gondii leads to the infiltration of highly-activated, motile T cells into the eye. These cells express CXCR3, are capable of producing IFN-gamma and TNF-alpha, and CD8+ T cells express granzyme B. The expression of CXCL9 and CXCL10 in the retina was significantly upregulated during chronic infection. Treatment of chronically infected mice with anti-CXCL10 antibodies led to decreases in the number of CD3+, CD4+, and CD8+ T cells and IFN-gamma mRNA expression in the retina and an increase in replicating parasites and ocular pathology. CONCLUSION. The maintenance of the T cell response and control of T. gondii in the eye during chronic infection is dependent on CXCL10.

PMID: 20811054 [PubMed - as supplied by publisher]