Tuesday, March 29, 2011

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Traffic. 2011 Mar 25. doi: 10.1111/j.1600-0854.2011.01192.x. [Epub ahead of print]

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Gaji RY, Flammer HP, Carruthers VB.

Department of Microbiology and Immunology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 USA.

Abstract
Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto-myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable propeptide. Although MIC propeptides have been implicated in forward targeting to micronemes, the specific amino acids involved have not been identified. It was also not known if the propeptide has a general function in MICs trafficking in T. gondii and other apicomplexans. Here we show that propeptide domains are extensively interchangeable between T. gondii MICs and also with that of Eimeria tenella MIC5 (EtMIC5), suggesting a common mechanism of function. We also performed N-terminal deletion and mutational analysis of M2AP and MIC5 propeptides to show that a valine at position +3 (relative to signal peptidase cleavage) of proM2AP and a leucine at position +1 of proMIC5 are crucial for targeting to micronemes. Valine and leucine are closely related amino acids with similar side chains, implying a similar mode of function, a notion that was confirmed by correct trafficking of TgM2AP-V/L and TgMIC5-L/V substitution mutants. Propeptides of AMA1, MIC3, and EtMIC5 have valine or leucine at or near the N-termini and mutagenesis of these conserved residues validated their role in microneme trafficking. Collectively, our findings suggest that discrete, aliphatic residues at the extreme N-termini of proMICs facilitate trafficking to the micronemes.

© 2011 John Wiley & Sons A/S.
PMID: 21438967 [PubMed - as supplied by publisher]

Saturday, March 26, 2011

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence

oc Natl Acad Sci U S A. 2011 Mar 21. [Epub ahead of print]

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence

Reese ML, Zeiner GM, Saeij JP, Boothroyd JC, Boyle JP.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305.

Abstract
Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.

PMID: 21436047 [PubMed - as supplied by publisher]

T-cell production of matrix metalloproteinases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain

ASN Neuro. 2011 Jan 21;3(1). pii: e00049. doi: 10.1042/AN20100027.

T-cell production of matrix metalloproteinases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain

Clark RT, Philip Nance J, Noor S, Wilson EH.

Division of Biomedical Sciences, University of California, Riverside, CA 92521, U.S.A.

Abstract
Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodelling in the brain and a continuous requirement for peripheral leucocyte migration within the CNS (central nervous system). In the present study, we investigate the role of MMPs (matrix metalloproteinases) and their inhibitors in T-cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases-1), was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T-cells. In addition, infiltrating T-cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1-deficient mice had a decrease in perivascular accumulation of lymphocyte populations, yet an increase in the proportion of CD4+ T-cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Taken together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.

PMID: 21434872 [PubMed - in process]

Thursday, March 24, 2011

The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii

PLoS Pathog. 2011 Mar;7(3):e1002007. Epub 2011 Mar 10.

The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii

Straub KW, Peng ED, Hajagos BE, Tyler JS, Bradley PJ.

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

Abstract
The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.

PMID: 21423671 [PubMed - in process]

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids targeting farnesy

Bioorg Med Chem. 2011 Mar 16. [Epub ahead of print]

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase

Rosso VS, Szajnman SH, Malayil L, Galizzi M, Moreno SN, Docampo R, Rodriguez JB.

Departamento de Química Orgánica and UMYMFOR (CONICET-FCEyN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina; Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA.

Abstract
The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26 proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC(50) values at the nanomolar range. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting an IC(50) value of 0.67μM, while this compound showed an IC(50) value of 0.81μM against the target enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphosphate synthase TcSPPS showing an IC(50) value of 3.2μM. Interestingly, compound 26 was also very effective against T. gondii (tachyzoites) exhibiting IC(50) values of 6.23μM. This cellular activity was also related to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC(50)=0.093μM) As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control different tropical diseases.

Copyright © 2011 Elsevier Ltd. All rights reserved.
PMID: 21419634 [PubMed - as supplied by publisher]

Friday, March 11, 2011

Toxoplasma gondii Dense Granule Antigen 1 stimulates apoptosis of monocytes through autocrine TGF-β signaling

Apoptosis. 2011 Mar 9. [Epub ahead of print]

Toxoplasma gondii Dense Granule Antigen 1 stimulates apoptosis of monocytes through autocrine TGF-β signaling

D'Angelillo A, De Luna E, Romano S, Bisogni R, Buffolano W, Gargano N, Del Porto P, Del Vecchio L, Petersen E, Romano MF.

Department of Biochemistry and Medical Biotechnology, University of Naples "Federico II", Via S. Pansini 5, 80131, Naples, Italy.

Abstract
Monocyte/macrophages represent the first line of defense against protozoan parasites. Different mechanisms of monocyte suppression by Toxoplasma gondii that sustain parasite invasion and persistence have been described, including apoptosis. In the present study, we investigated the effect of microbial excretory-secretory polypeptides, namely the microneme protein MIC3 and the dense granule antigen GRA1, on apoptosis of monocytes from patients with congenital toxoplasmosis and healthy individuals. We found that GRA1 but not MIC3 could induce apoptosis of monocytes, observing the effect in samples from both Toxoplasma-infected and uninfected individuals, thus ruling out involvement of mechanisms of apoptosis linked to adaptive immunity or a cellular context related to infection. Selective inhibition of TGF-β type I receptors reversed GRA1-induced apoptosis, indicating that this apoptosis involved canonical TGF-β signaling. By using TGF-β-neutralizing antibodies, we showed that monocyte apoptosis required endogenous TGF-β and that GRA1 stimulation activated TGF-β transcription and expression in monocytes but not lymphocytes, suggesting involvement of an autocrine TGF-β-mediated mechanism in GRA1-induced apoptosis.

PMID: 21390541 [PubMed - as supplied by publisher]

Thursday, March 10, 2011

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation

J Biol Chem. 2011 Mar 7. [Epub ahead of print]

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation

Jaffe EK, Shanmugan D, Gardberg A, Dieterich S, Sankaran B, Stewart LJ, Myler PJ, Roos DS.

Fox Chase Cancer Center, United States;

Abstract
Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.

PMID: 21383008 [PubMed - as supplied by publisher]

Modulation of early {beta}-defensin-2 production as a mechanism developed by Type I Toxoplasma gondii to evade human intestinal immunity

Infect Immun. 2011 Mar 7. [Epub ahead of print]

Modulation of early {beta}-defensin-2 production as a mechanism developed by Type I Toxoplasma gondii to evade human intestinal immunity

Morampudi V, Braun MY, D'Souza S.

Toxoplasmosis Laboratory, Operational Direction Communicable and Infectious Diseases, Scientific Institute of Public Health, 1180 Brussels, Belgium; Institute for Medical Immunology, Faculty of Medicine, Universite Libre de Bruxelles (ULB), 6041 Gosselies, Belgium.

Abstract
We investigated the early innate immune responses induced in human intestinal epithelial cells (IEC) by the three defined Toxoplasma gondii genotype strains. Transcriptome analysis revealed that among differentially expressed genes, β-defensins distinguished the most IEC infected by fast or slow replicating T. gondii genotypes. Although β-defensin 1 and 3 genes were not expressed in host cells early post-infection, the slow replicating Type II and III parasites induced high levels of β-defensin 2 gene expression. Notably, no β-defensin 2 gene expression occurred early after infection with the fast replicating Type I parasite. However, activation of this gene in IEC by Poly I:C treatment prior to infection substantially decreased parasite viability, and pretreatment of parasites with synthetic β-defensin 2 significantly reduced their infectivity of IEC. These findings strongly support the modulation of early β-defensin 2 expression as a mechanism used by Type I T. gondii parasites to mediate immune evasion.

PMID: 21383053 [PubMed - as supplied by publisher]

Toxoplasma gondii protects against H(2) O(2) -induced apoptosis in ARPE-19 cells

Acta Ophthalmol. 2011 Feb 25. doi: 10.1111/j.1755-3768.2011.02113.x. [Epub ahead of print]

Toxoplasma gondii protects against H(2) O(2) -induced apoptosis in ARPE-19 cells through the transcriptional regulation of apoptotic elements and downregulation of the p38 MAPK pathway

Choi SH, Park SJ, Cha GH, Quan JH, Chang NS, Ahn MH, Shin DW, Lee YH.

Department of Ophthalmology, Chungnam National University School of Medicine, Daejeon, Korea Department of Infection Biology and Research Institute for Medical Sciences, Chungnam National University School of Medicine, Daejeon, Korea Department of Environmental Biology and Medical Parasitology, College of Medicine, Hanyang University, Seoul, Korea.

Abstract
Purpose:  Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, can lead to severe visual impairment. T. gondii inhibits or delays programmed cell death caused by various apoptotic triggers; however, the mechanisms involved in the T. gondii-induced suppression of apoptosis in retinal cells have not been analysed in detail. Methods:  We investigated the role of T. gondii infection in H(2) O(2) -induced apoptosis in human retinal pigment epithelial cells (ARPE-19) by monitoring the activities of apoptosis-regulating molecules and mitogen-activated protein kinases (MAPKs), including p38 MAPK. We also examined the gene downstream from p38 MAPK. Results:  T. gondii infection significantly inhibited the cellular toxicity of H(2) O(2) (500 μm) and increased cell viability in a multiplicity of infection (MOI)-dependent manner by reducing DNA fragmentation and reactive oxygen species (ROS) generation in ARPE-19 cells. Western blot analysis also showed that T. gondii infection prevented the host cell expression of pro-apoptotic factors, such as Bad and Bax, and the activation of caspase-3. Infection with T. gondii increased the expression of the anti-apoptotic factor Bcl-2 in ARPE-19 cells under oxidative stress. In accordance with these findings, Toxoplasma infection was protective enough to suppress the phosphorylation of p38 MAPK following H(2) O(2) treatment. Exposure to H(2) O(2) increased the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells, and its expression was significantly inhibited in H(2) O(2) -treated infected cells. Conclusion:  The protective function of T. gondii infection against ROS-induced apoptosis results from changes in the expression of apoptotic molecules and the downregulation of stress-induced intracellular signalling.

© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.
PMID: 21385331 [PubMed - as supplied by publisher]

Tuesday, March 08, 2011

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis

PLoS Pathog. 2011 Feb;7(2):e1001286. Epub 2011 Feb 17.

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis

Tawk L, Dubremetz JF, Montcourrier P, Chicanne G, Merezegue F, Richard V, Payrastre B, Meissner M, Vial HJ, Roy C, Wengelnik K, Lebrun M.

UMR 5235 CNRS, Université Montpellier 1 & 2, Montpellier, France.

Abstract
Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

PMID: 21379336 [PubMed - in process]

Thursday, March 03, 2011

Rapid discovery of inhibitors of Toxoplasma gondii using hybrid structure-based computational approach

J Comput Aided Mol Des. 2011 Feb 26. [Epub ahead of print]

Rapid discovery of inhibitors of Toxoplasma gondii using hybrid structure-based computational approach

Kortagere S, Mui E, McLeod R, Welsh WJ.

University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ, 08854, USA.

Abstract
Toxoplasma (T.) gondii, the causative agent of toxoplasmosis, is a ubiquitous opportunistic pathogen that infects individuals worldwide, and is a leading cause of severe congenital neurologic and ocular disease in humans. No vaccine to protect humans is available, and hypersensitivity and toxicity limit the use of the few available medicines. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. Using the Hybrid Structure Based (HSB) method, we have previously identified small molecule inhibitors of P. falciparum that seem to target a novel protein-protein interaction between the Myosin tail interacting protein and myosin light chain. This pathway has been hypothesized to be involved in invasion of host erythrocytes by the parasite and is broadly conserved among the apicomplexans. Guided by similar computational drug design approaches, we investigated this series of small molecules as potential inhibitors of T. gondii. Compound C3-21, identified as the most active inhibitor in this series, exhibited an IC(50) value ~500 nM against T. gondii. Among the 16 structural analogs of C3-21 tested thus far, nine additional compounds were identified with IC(50) values <10.0 μM. In vitro assays have revealed that C3-21 markedly limits intracellular growth of T. gondii tachyzoites, but has no effect on host cell human foreskin fibroblasts (HFF) at concentrations more than a log greater than the concentration that inhibits the parasites.

PMID: 21359560 [PubMed - as supplied by publisher]

Tuesday, March 01, 2011

Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii

DNA Repair (Amst). 2011 Feb 23. [Epub ahead of print]

Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii

Onyango DO, Naguleswaran A, Delaplane S, Reed A, Kelley MR, Georgiadis MM, Sullivan WJ Jr.

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, United States.

Abstract
DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis.

Copyright © 2011 Elsevier B.V. All rights reserved.
PMID: 21353648 [PubMed - as supplied by publisher]

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo

Exp Parasitol. 2011 Feb 24. [Epub ahead of print]

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo

Christodoulou V, Messaritakis I, Svirinaki E, Tsatsanis C, Antoniou M.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece.

Abstract
Although macrophages have a microbicidal role in the immune system they themselves can be infected by pathogens. Often a simultaneous infection by more than one microbe may occur in a single cell. This is the first report of coinfection of macrophages with Toxoplasma gondii and Leishmania infantum, in vitro and in vivo. L. infantum does not cause severe disease in mice but T. gondii, RH strain, is lethal. Cell culture studies using THP-1 macrophages dually infected in vitro revealed that 4.3% harbored both parasites 24hrs after infection. When mice were infected with both parasites on the same day 7.3% of the infected cells carried both parasites 7 days later. Yet, if mice were first infected with L. infantum and then with Toxoplasma (5 days post infection) 18.7% of the macrophages hosted either parasite but concomitant infection could not be found and mice, already harboring L. infantum, survived Toxoplasma's lethal effect.

Copyright © 2011. Published by Elsevier Inc.
PMID: 21354140 [PubMed - as supplied by publisher]

Improved Techniques for Endogenous Epitope Tagging and Gene Deletion in Toxoplasma gondii

J Microbiol Methods. 2011 Feb 22. [Epub ahead of print]

Improved Techniques for Endogenous Epitope Tagging and Gene Deletion in Toxoplasma gondii

Upadhya R, Kim K, Hogue-Angeletti R, Weiss LM.

Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park, Avenue, Bronx, NY 10461.

Abstract
Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.

Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21352857 [PubMed - as supplied by publisher]