Sunday, January 31, 2010

Chromatin Modifications: Implications in the Regulation of Gene Expression in Toxoplasma gondii

Cell Microbiol. 2010 Jan 26. [Epub ahead of print]

Chromatin Modifications: Implications in the Regulation of Gene Expression in Toxoplasma gondii

Bougdour A, Braun L, Cannella D, Hakimi MA.

Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163 - ATIP+ group, Université Joseph Fourier, BP 170, F-38042 Grenoble cedex 9, France.

Summary The apicomplexan Toxoplasma gondii completes its life cycle by successive processes of parasite differentiation that rely on a tight control of gene expression to ensure appropriate protein profiles on time. During the last five years, several groups have pioneered this field of investigation suggesting that epigenetics could play an important role in the control of parasite gene expression. Histone modifications serve as an effective way to regulate gene transcription but they do not operate alone; rather, they act in concert with other putative epigenetic information carriers (histone variants, small RNAs) and DNA-sequence-specific transcription factors to modulate the higher order structure of the chromatin fiber and govern the on-time recruitment of the transcriptional machinery to specific genes. Regarding the 'histone code' hypothesis, the parasite is endowed with a rich repertoire of histone-modifying enzymes catalyzing site-selective modifications, which are subsequently interpreted by effector proteins that recognize specific covalent marks. Still, several peculiarities seem unique to T. gondii. This review is a synthesis of the current knowledge of how epigenetics contribute to the control of gene expression in T. gondii and, likely, other Apicomplexa.

PMID: 20109158 [PubMed - as supplied by publisher]

Coordinated Loading of IRG Resistance GTPases on to the Toxoplasma gondii Parasitophorous Vacuole

Cell Microbiol. 2010 Jan 26. [Epub ahead of print]

Coordinated Loading of IRG Resistance GTPases on to the Toxoplasma gondii Parasitophorous Vacuole

Khaminets A, Hunn JP, Könen-Waisman S, Zhao YO, Preukschat D, Coers J, Boyle JP, Ong YC, Boothroyd JC, Reichmann G, Howard JC.

Institute for Genetics University of Cologne Cologne 50674 Germany.

The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasmagondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about an hour. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

PMID: 20109161 [PubMed - as supplied by publisher]

The unexpected role for the aryl hydrocarbon receptor on susceptibility to experimental toxoplasmosis

J Biomed Biotechnol. 2010;2010:505694. Epub 2010 Jan 11.

The unexpected role for the aryl hydrocarbon receptor on susceptibility to experimental toxoplasmosis

Sanchez Y, de Dios Rosado J, Vega L, Elizondo G, Estrada-Muñiz E, Saavedra R, Juárez I, Rodríguez-Sosa M.

Unidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México (UNAM), CP 54090, o. Tlalnepantla, Edo. de México, Mexico.

The aryl hydrocarbon receptor (AhR) is part of a signaling system that is mainly triggered by xenobiotic agents. Increasing evidence suggests that AhR may regulate immunity to infections. To determine the role of AhR in the outcome of toxoplasmosis, we used AhR-/- and wild-type (WT) mice. Following an intraperitoneal infection with Toxoplasma gondii (T. gondii), AhR-/- mice succumbed significantly faster than WT mice and displayed greater liver damage as well as higher serum levels of tumor necrosis factor (TNF)-alpha, nitric oxide (NO), and IgE but lower IL-10 secretion. Interestingly, lower numbers of cysts were found in their brains. Increased mortality was associated with reduced expression of GATA-3, IL-10, and 5-LOX mRNA in spleen cells but higher expression of IFN-gamma mRNA. Additionally, peritoneal exudate cells from AhR-/- mice produced higher levels of IL-12 and IFN-gamma but lower TLR2 expression than WT mice. These findings suggest a role for AhR in limiting the inflammatory response during toxoplasmosis.

PMID: 20111744 [PubMed - in process]

High-intensity signals in the basal ganglia from gadolinium-enhanced T1-weighted MRI as an early change in toxoplasma encephalitis

J Infect Chemother. 2010 Jan 29. [Epub ahead of print]

High-intensity signals in the basal ganglia from gadolinium-enhanced T1-weighted MRI as an early change in toxoplasma encephalitis in an AIDS patient

Suzuki K, Masuya M, Matsumoto T, Ito N, Ohishi K, Maeda M, Katayama N.

Department of Hematology and Oncology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.

A 30-year-old Brazilian man hospitalized with AIDS developed a high-grade fever. Neither culture studies nor radiological examinations revealed the cause; small yet highly intense signals in the basal ganglia were detected upon gadolinium (Gd)-enhanced T1-weighted magnetic resonance imaging (MRI) of the head. This finding was equivocal at that time but obviously abnormal for his age. A week later, he developed a movement disorder in his right arm, speech apraxia, and a worsening disturbance of consciousness. Repeated Gd-enhanced T1-weighted MRI demonstrated incredible changes in the brain; enhanced lesions in the basal ganglia deteriorated over time, multiple nodular and ring-enhanced lesions were observed in almost the entire brain. A diagnosis of toxoplasma encephalitis (TE) was confirmed by the detection of Toxoplasma gondii DNA in the cerebrospinal fluid. After initiation of intravenous trimethoprim-sulfamethoxazole (TMP-SMX; 10 mg/kg/day of TMP and 50 mg/kg/day of SMX) treatment, his symptoms and radiological findings improved dramatically. Our case suggests that high-intensity signals seen in the basal ganglia of a Gd-enhanced T1-weighted MRI, even at the preclinical stage, is indicative of TE. Because the use of MRI in general has become more widespread, it is predicted that preclinical lesions of TE will be found in various clinical settings more frequently.

PMID: 20111977 [PubMed - as supplied by publisher]

Apicomplexa, trypanosoma and parasitic nematode protein kinases as antiparasitic therapeutic targets

Curr Opin Investig Drugs. 2010 Feb;11(2):147-56.

Apicomplexa, trypanosoma and parasitic nematode protein kinases as antiparasitic therapeutic targets

Liotta F, Siekierka JJ.

Montclair State University, Sokol Institute of Pharmaceutical Life Sciences and Department of Chemistry and Biochemistry, 1 Normal Avenue, Montclair, NJ 07043, USA.

Parasitic infections caused by Plasmodium, Trypanosoma, Leishmania, Toxoplasma and parasitic nematodes affect hundreds of millions of individuals worldwide and are the cause of significant mortality and morbidity, particularly in developing countries. These diseases also have an impact on individuals from developed countries; for example, some US troops in Iraq and Afghanistan have been infected with Leishmania. The annual mortality associated with parasitic infections is estimated to be 1.5 million deaths. The socioeconomic impact of the morbidity associated with parasitic infections is significant, and the development of new drugs, aimed at novel targets, is urgently needed to develop effective treatments for these diseases. The small-molecule inhibitors discussed in this review constitute useful tools with which to explore the relevance of kinase inhibition in inducing antiparasitic activity. The aim of recent target-based approaches used in the development of parasite kinase inhibitors is to identify novel antiparasitic agents with therapeutic potential.

PMID: 20112164 [PubMed - in process

Friday, January 29, 2010

Is there any role of Toxoplasma gondii in the etiology of obsessive-compulsive disorder?

Psychiatry Res. 2010 Jan 26. [Epub ahead of print]

Is there any role of Toxoplasma gondii in the etiology of obsessive-compulsive disorder?

Miman O, Mutlu EA, Ozcan O, Atambay M, Karlidag R, Unal S.

Department of Microbiology, Afyon Kocatepe University Medical Faculty, Afyonkarahisar, Turkey.

Obsessive-compulsive disorder (OCD) is a common psychiatric illness. Although the aetiology of OCD is still unknown, the family-genetic data show that familial forms of OCD may be associated with a specific genetic susceptibility. Recent investigations have associated development of OCD with infectious illness. Toxoplasmic encephalitis (TE) is a common presentation of Toxoplasma gondii infection of the central nervous system (CNS). The most commonly affected CNS region in TE is the cerebral hemisphere, followed by the basal ganglia, cerebellum and brain stem. The basal ganglia has been implicated in the development of OCD. Therefore, in this study, it was aimed to investigate a possible association between Toxoplasma infection and OCD. We selected 42 patients with OCD and 100 healthy volunteers, and investigated the sero-positivity rate for anti-Toxoplasma IgG antibodies by Enzyme Linked Immunosorbent Assay (ELISA). The sero-positivity rate for anti-T. gondii IgG antibodies among OCD patients (47.62%) was found to be significantly higher than the rate in healthy volunteers (19%). This is the first report to examine a potential association between Toxoplasma infection and OCD. The main finding of the present study is an increased level of IgG antibodies to T. gondii in OCD patients when compared with the level in healthy controls. There might be a causal relationship between chronic toxoplasmosis and the aetiology of OCD. Copyright © 2010 Elsevier Ltd. All rights reserved.

PMID: 20106536 [PubMed - as supplied by publisher]

Outbreak of acquired ocular toxoplasmosis involving 248 patients

Arch Ophthalmol. 2010 Jan;128(1):28-32.

Outbreak of acquired ocular toxoplasmosis involving 248 patients

Balasundaram MB, Andavar R, Palaniswamy M, Venkatapathy N.

Uvea Clinic, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, 641014, India.

Comment in:

Arch Ophthalmol. 2010 Jan;128(1):126-8.

OBJECTIVE: To describe the demographic profile and clinical and laboratory findings of 248 patients with acquired retinitis caused by systemic infection with toxoplasmosis in a presumed outbreak of the disease. DESIGN: Retrospective observational case series. RESULTS: Most patients (209) were residents of one city in Southern India. A total of 35 patients had a prodrome of fever, and 242 patients had unilateral retinitis without associated old retinochoroidal scars. All had laboratory evidence of acquired systemic infection with Toxoplasma gondii, and all favorably responded to antitoxoplasma therapy. Toxoplasma IgM and IgG antibodies were detected, suggesting recently acquired systemic disease. Complications seen were macular scars in 50 eyes (25.1%); epiretinal membranes, 23 eyes (11.5%); cataract, 5 eyes (2.5%); posterior vitreous detachment, 12 eyes (6%); and retinal detachment, 12 eyes (6%). One recurrence has been seen. The suspected source of infection is municipal drinking water. CONCLUSION: Large numbers of residents of any age in a population are at risk of acquiring ocular disease during an outbreak of toxoplasmosis, which can go unnoticed, and can cause significant ocular morbidity.

PMID: 20065213 [PubMed - indexed for MEDLINE]

Friday, January 22, 2010

Toxoplasma gondii infection specifically increases the levels of key host microRNAs

PLoS One. 2010 Jan 15;5(1):e8742.

Toxoplasma gondii infection specifically increases the levels of key host microRNAs

Zeiner GM, Norman KL, Thomson JM, Hammond SM, Boothroyd JC

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

BACKGROUND: The apicomplexan parasite Toxoplasma gondii can infect and replicate in virtually any nucleated cell in many species of warm-blooded animals; thus, it has evolved the ability to exploit well-conserved biological processes common to its diverse hosts. Here we have investigated whether Toxoplasma modulates the levels of host microRNAs (miRNAs) during infection. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray profiling and a combination of conventional molecular approaches we report that Toxoplasma specifically modulates the expression of important host microRNAs during infection. We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase. The abundance of mature miR-17 family members, which are derived from these two miRNA clusters, remains unchanged in host cells infected with the closely related apicomplexan Neospora caninum; thus, the Toxoplasma-induced increase in their abundance is a highly directed process rather than a general host response to infection. CONCLUSIONS/SIGNIFICANCE: Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown. Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.

PMID: 20090903 [PubMed - in process]

Wednesday, January 20, 2010

Apicoplast: keep it or leave it

Microbes Infect. 2010 Jan 15. [Epub ahead of print]

Apicoplast: keep it or leave it

Fleige T, Limenitakis J, Soldati-Favre D.

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1 Rue Michel Servet, 1211 Geneva, Switzerland.

Most Apicomplexans possess a relic plastid named apicoplast, originating from secondary endosymbiosis of a red algae. This non-photosynthetic organelle fulfils important metabolic functions and confers sensitivity to antibiotics. The tasks of this organelle is compared across the phylum of Apicomplexa, highlighting its role in metabolic adaptation to different intracellular niches. Copyright © 2010. Published by Elsevier SAS.

PMID: 20083219

IL-6 Promotes NK Cell Production of IL-17 during Toxoplasmosis

J Immunol. 2010 Jan 18. [Epub ahead of print]

IL-6 Promotes NK Cell Production of IL-17 during Toxoplasmosis

Passos ST, Silver JS, O'Hara AC, Sehy D, Stumhofer JS, Hunter CA.

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

Previous studies have implicated T cell production of IL-17 in resistance to Toxoplasma gondii as well as the development of immune-mediated pathology during this infection. Analysis of C57BL/6 and C57BL/6 RAG(-/-) mice challenged with T. gondii-identified NK cells as a major innate source of IL-17. The ability of soluble Toxoplasma Ag to stimulate NK cells to produce IL-17 was dependent on the presence of accessory cells and the production of IL-6, IL-23, and TGF-beta. In contrast, these events were inhibited by IL-2, IL-15, and IL-27. Given that IL-6 was one of the most potent enhancers of NK cell production of IL-17, further studies revealed that only a subset of NK cells expressed both chains of the IL-6R, IL-6 upregulated expression of the Th17-associated transcription factor RORgammat, and that IL-6(-/-) mice challenged with T. gondii had a major defect in NK cell production of IL-17. Together, these data indicate that many of the same cytokines that regulate Th17 cells are part of a conserved pathway that also control innate production of IL-17 and identify a major role for IL-6 in the regulation of NK cell responses.

PMID: 20083665 [PubMed - as supplied by publisher]

Toxoplasma gondii infection positively modulates the macrophages migratory molecular complex by increasing matrix metalloproteinases

Vet Parasitol. 2010 Jan 7. [Epub ahead of print]

Toxoplasma gondii infection positively modulates the macrophages migratory molecular complex by increasing matrix metalloproteinases, CD44 and alphavbeta3 integrin

Seipel D, de Lima Oliveira BC, Resende TL, Schuindt SH, de Oliveira Pimentel PM, Kanashiro MM, Arnholdt AC.

Laboratório de Biologia do Reconhecer, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, LBR 210/P4, Horto, Campos dos Goytacazes, Rio de Janeiro, CEP 28015-620, Brazil.

Toxoplasmosis is a world wide spread zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells for dispersal throughout the body. However, the molecular principals or outcomes of the subversion of the host cell are largely unknown. We evaluated the involvement of host invasive machinery in the migration of T. gondii infected murine cells from a monocytic/macrophage lineage. Migration in Matrigel of infected macrophages was augmented after 48h of infection, and inhibition of metalloproteinases abolished migration. We also demonstrated that T. gondii infection induces a decreasing of CD44 at cell surface independent of the ERK signaling pathway, and that secretion of active MMP9 is augmented upon infection. Infected macrophages showed increased expression of MT1-MMP and ADAM10 membrane matrix metalloproteinases. Furthermore, processing of pro-alphav and pro-beta3 in T. gondii infected cells seems to depend on metalloproteinases to generate functional mature integrin alphavbeta3 molecules, with no evidence of the involvement of proprotein convertase pathway. Copyright © 2010 Elsevier B.V. All rights reserved.

PMID: 20080350 [PubMed - as supplied by publisher]

A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity

PLoS Pathog. 2010 Jan 15;6(1):e1000720.

A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity

Heaslip AT, Leung JM, Carey KL, Catti F, Warshaw DM, Westwood NJ, Ballif BA, Ward GE.

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.

Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.

PMID: 20084115 [PubMed - in process]

Wednesday, January 06, 2010

Toxoplasma gondii: Fluconazole and itraconazole activity against toxoplasmosis in a murine model

Exp Parasitol. 2009 Dec 31. [Epub ahead of print]

Toxoplasma gondii: Fluconazole and itraconazole activity against toxoplasmosis in a murine model

Martins-Duarte ES, Lemgruber L, Souza WD, Vommaro RC.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, CCS Universidade Federal do Rio de Janeiro, 21941-902 - Rio de Janeiro, Brazil.

Toxoplasma gondii is an important opportunistic pathogen affecting immunocompromised patients with AIDS. Toxoplasmic encephalitis is responsible for high morbidity and mortality. In this study, we investigated the activity of the antifungals fluconazole (FLZ) and itraconazole (ITZ) against T. gondii in mice infected with the Me49 strain. As previously reported for ITZ, FLZ also demonstrated a selective effect against T. gondiiin vitro; the IC(50) values obtained for FLZ were 8.9 muM and 3.1 muM after 24 h and 48 h of treatment, respectively. A 10-day treatment of mice with orally or intraperitoneally administered 20 mg/kg/day FLZ showed a significant survival difference compared to untreated mice. The administration of 20 mg/kg/day ITZ significantly reduced the brain cyst burden compared to untreated mice but did not exert significant protection against death. The results obtained in this work are rather promising as ITZ and FLZ are safe and low-cost drugs available on the market. Copyright © 2009. Published by Elsevier Inc.

PMID: 20045696

Azithromycin reduces ocular infection during congenital transmission of toxoplasmosis in the Calomys callosus model

J Parasitol. 2009 Aug;95(4):1005-10.

Azithromycin reduces ocular infection during congenital transmission of toxoplasmosis in the Calomys callosus model

Lopes CD, Silva NM, Ferro EA, Sousa RA, Firminot ML, Bernardes ES, Roque-Barreira MC, Pena JD.

Ocular Immunology Laboratory, Institute of Biomedical Sciences, Universidade Federal de Uberlândia, Av. Pará 1720, Uberlândia, MG, Brazil

Toxoplasma gondii is a widely distributed obligatory intracellular parasite that causes severe disease to the fetus when transmitted during pregnancy. Drugs used to avoid congenital transmission have shown side effects, and their efficacy is controversial. The most widely used treatment for acute toxoplasmosis during pregnancy is pyrimethamine plus sulfadiazine, which has several side effects. In this work, we tested the efficacy of azithromycin in reducing congenital transmission of T. gondii in the large vesper mouse, Calomys callosus, a rodent. Females of C callosus were inoculated perorally with 20 cysts of ME49 strain of T. gondii on the day of fertilization, and fetuses were collected from the 15th to the 19th day of gestation. Azithromycin (300 mg/kg), in association with pyrimethamine (100 or 50 mg/kg) plus sulfadiazine (100 or 75 mg/kg) and folinic acid (15 mg/kg) (SPAf), or vehicle, were administered orally on different days after infection. Brain and ocular tissues were removed and processed for immunohistochemistry using a polyclonal antibody against T. gondii, or were processed for parasite DNA quantification. Toxoplasma gondii was detected in the brains of all females and in fetuses' eyes of C. callosus treated with SPAf. On the other hand, in females treated with azithromycin, there was a reduction of T. gondii in the brains of mothers, and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy.

PMID: 20050006

Long-term survival of Toxoplasma gondii sporulated oocysts in seawater

J Parasitol. 2009 Aug;95(4):1019-20.

Long-term survival of Toxoplasma gondii sporulated oocysts in seawater

Lindsay DS, Dubey JP.

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, Virginia 24061, USA.

Toxoplasma gondii is now recognized as an important pathogen in costal marine mammals. Oocysts from cat feces are believed to be washed into seawater and serve as a source of infection via transport hosts. Experimentally, it has been demonstrated that T. gondii oocysts can sporulate in seawater and remain infectious for mice for up to 6 mo. The present study examined the long-term survival of T. gondii in seawater (15 ppt NaCl) kept at 4 C or at room temperature. Oocysts kept at 4 C for 24 mo were orally infectious for mice, while those kept at room temperature for 24 mo were not.

PMID: 20050010

Friday, January 01, 2010

Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin

J Biol Chem. 2009 Dec 30. [Epub ahead of print]

Toxoplasma gondii actin depolymerizing factor acts primarily to sequester G-actin

Mehta S, Sibley LD.

Washington Univ. Sch. Med., United States;

Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Parasites in this phylum utilize a unique process of motility termed gliding, which is dependent on parasite actin filaments. Surprisingly, 98% of parasite actin is maintained as G-actin, suggesting that filaments are rapidly assembled and turned over. Little is known about the regulated disassembly of filaments in the Apicomplexa. In higher eukaryotes, the Actin Depolymerizing Factor/Cofilin (AC) proteins are essential regulators of actin filament turnover. ADF is one of the few actin binding proteins conserved in apicomplexan parasites. In this study we examined the mechanism by which T. gondii ADF (TgADF) regulates actin filament turnover. Unlike other members of the AC family, apicomplexan ADFs lack key F-actin binding sites. Surprisingly, this promotes their enhanced disassembly of actin filaments. Restoration of the C-terminal F-actin binding site to TgADF stabilized its interaction with filaments, but reduced its net filament disassembly activity. Analysis of severing activity revealed that TgADF is a weak severing protein, requiring much higher concentrations than typical AC proteins. Investigation of TgADF interaction with T. gondii actin (TgACT) revealed that TgADF disassembled short TgACT oligomers. Kinetic and steady-state polymerization assays demonstrated that TgADF has strong monomer sequestering activity, inhibiting TgACT polymerization at very low concentrations. Collectively these data indicate that TgADF promoted the efficient turnover of actin filaments via weak severing of filaments and strong sequestering of monomers. This suggests a dual role for TgADF in maintaining high G-actin concentrations and effecting rapid filament turnover.

PMID: 20042603