Progress 07/01/05 to 06/30/10
Outputs
OUTPUTS: The last submitted progress report will serve as the final report. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The last submitted progress report will serve as the final report.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: During the year 2009, my laboratory has published 2 peer reviewed research articles and has started the preparation of a third manuscript. In addition, to publication in international journals, I presented our results at the International Congress for Parasitology in Holland and lab members presented work at the Annual Meeting of the American Society for Cell Biology. In total members of the laboratory including myself have given 2 research talks and 6 poster presentations. Finally, in 2009 two students completed and defended their graduate research projects. PARTICIPANTS: The funds provided helped in the training of graduate students and a post-doctoral fellow. Mark Lavine, Post doctoral fellow Ingrid Fruth, Graduate student (Completed PhD in May) Erin Garrison, Graduate student Maria Francia, Graduate student (COmpleted Ms in May) TARGET AUDIENCES: The results from our work are of special interest to scientists, medical doctors and public health professionals working with human parasite infections. Given that Toxoplasma gondii infect approximately a third of the human population regardless of geographical location the outcome of our research can potentially have a great impact on human health. Moreover, Toxoplasma is an important pathogen of animals, including sheep, thus our results are of interest to the agricultural field. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Obligate intracellular parasites such as Toxoplasma gondii are important human and animal pathogens. Since this type of parasites require to enter and exit cells in order to disseminate and survive in an infected individual, inhibiting either of these processes would lead to death of the parasite. Thus, a better understanding of the mechanisms by which the parasite enters and exits cell will lead to the identification of possible drug targets. In the last year we made particular progress in our understanding of how the parasite manipulates the host cell as to ensure its own survival. We made the significant discovery that the parasite manipulates the invaded cell to communicate with neighboring cells to make them more suitable for the parasite to invade once they need new host. This work was published and presented at meetings and has contributed to the general knowledge of how the parasites manipulates infected tissues so that it can more easily enter cells.
Publications
- Lavine MD and Arrizabalaga G. 2009. Induction of mitotic S-phase of host and neighboring cells by Toxoplasma gondii enhances parasite invasion. 164(1):95-99
- Garrison E and Arrizabalaga G. 2009. Disruption of a mitochondrial homolog of a MutS DNA Repair Enzyme confers drug resistance in the pathogenic parasite Toxoplasma gondii. Molecular Microbiology, 72(2):425-4
- Arrizabalaga, G. Disruption of a MutS DNA repair enzyme homologue confers drug resistance in Toxoplasma gondii International Congress on Toxoplasmosis, Amsterdam, Holland June 2009
- Garrison, E., Lavine, M.D. and Arrizabalaga G. Disruption of a Mitochondrial MutS Homolog Confers Drug Resistance in Toxoplasma gondii Western INBRE/COBRE meeting, Big Sky, Montana, September 2009
- Garrison, E., Lavine, M.D. and Arrizabalaga G. Disruption of a Mitochondrial MutS Homolog Confers Drug Resistance in Toxoplasma gondii American Society of Cell Biology Annual meeting, San Diego, December 2009
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: During the year 2008, my laboratory has published 2 peer reviewed research articles and contributed to another. An additional research manuscript is currently under review. In addition, to publication in international journals, the laboratory has presented our results at international meetings such as the Molecular Parasitology Meeting as well as in regional meetings such as Seattle Parasitology Meeting. In addition I have been invited to present my work at 3 different Universities during the past year. In total members of the laboratory including myself have given 5 research talks and over 10 poster presentations. PARTICIPANTS: The funds provided helped in the training of graduate students and a post-doctoral fellow. Mark Lavine, Post doctoral fellow Ingrid Fruth, Graduate student Erin Garrison, Graduate student Maria Francia, Graduate student TARGET AUDIENCES: The results from our work are of special interest to scientists, medical doctors and public health professionals working with human parasite infections. Given that Toxoplasma gondii infect approximately a third of the human population regardless of geographical location the outcome of our research can potentially have a great impact on human health. Moreover, Toxoplasma is an important pathogen of animals, including sheep, thus our results are of interest to the agricultural field. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Obligate intracellular parasites such as Toxoplasma gondii are important human and animal pathogens. Since this type of parasites require to enter and exit cells in order to disseminate and survive in an infected individual, inhibiting either of these processes would lead to death of the parasite. Thus, a better understanding of the mechanisms by which the parasite enters and exits cell will lead to the identification of possible drug targets. Accordingly, our work aims at elucidating the genes involved in egress and invasion using a combination of genetic and cell biology studies. In the past year we made significant progress on our investigations into the regulation of the parasite's motility as it exits and enters host cell. In specific we have isolated various mutants that misregulate motility. The study of these mutant strains will allow us to determine the proteins involved in this process. The preliminary analysis of these mutants was the basis for an American Cancer Society grant funded this year and lasting until 2012.
Publications
- Lavine, M.D. and Arrizabalaga, G., 2008. Induction of mitotic S-phase of host and neighboring cells by Toxoplasma gondii enhances parasite invasion. Molecular and Biochemical Parasitology, In press. Saeij, J., Arrizabalaga, G. and Boothroyd J.C., 2008. A cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence. Immunity and infection, 76(6):2402-10. Lavine, M and Arrizabalaga, G., 2008 Exit from host cells by the pathogenic parasite Toxoplasma gondii does not require motility. Eukaryotic Cell, 7(1): 131-40.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: During the year 2007, my laboratory has published 2 peer reviewed research articles and a review article. An additional research manuscript is currently in press. In addition, to publication in international journals, the laboratory has presented our results at international meetings such as the Molecular Parasitology Meeting and the International Conference on Toxoplasmosis as well as in regional meetings such as Seattle Parasitology Meeting. In total members of the laboratory have given 3 research talks and over 10 poster presentations.
PARTICIPANTS: The funds provided helped in the training of graduate students and a post-doctoral fellow. Mark Lavine, Post doctoral fellow Ingrid Fruth, Graduate student Erin Garrison, Graduate Student.
TARGET AUDIENCES: The results from our work are of special interest to scientists, medical doctors and public health professionals working with human parasite infections. Given that Toxoplasma gondii infect approximately a third of the human population regardless of geographical location the outcome of our research can potentially have a great impact on human health.
PROJECT MODIFICATIONS: No major changes were made.
Impacts
Obligate intracellular parasites such as Toxoplasma gondii are important human and animal pathogens. Since this type of parasites require to enter and exit cells in order to disseminate and survive in an infected individual, inhibiting either of these processes would lead to death of the parasite. Thus, a better understanding of the mechanisms by which the parasite enters and exits cell will lead to the identification of possible drug targets. Accordingly, our work aims at elucidating the genes involved in egress and invasion using a combination of genetic and cell biology studies. In the past year we made significant progress on our investigations into egress. Our two main discoveries are that egress can be induced with the antibiotic nigericin and that non-induced egress proceeds by mechanical tension exerted by the parasite on the host cell membrane. These discoveries shed light on the mechanisms behind egress, a poorly understood process central to Toxoplasma
pathogenesis.
Publications
- Lavine, M and Arrizabalaga, G., 2008 Exit from host cells by the pathogenic parasite Toxoplasma gondii does not require motility. Eukaryotic Cell, In press, to be published January 2008
- Lavine, M.D., Knoll, L.J., Rooney, P.J. and Arrizabalaga, G. 2007. A Toxoplasma gondii mutant defective in responding to calcium fluxes shows reduced in vivo pathogenicity. Molecular and Biochemical Parasitology,155(2): 113-122
- Fruth, I.A. and Arrizabalaga G., 2007. Toxoplasma gondii: Induction of egress by the potassium ionophore nigericin. International Journal of Parasitology, International Journal of Parasitology, 37(14): 1559-67
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Progress 01/01/06 to 12/31/06
Outputs
The overall goal of this project is to elucidate the mechanisms and genes involved in the invasion and egress of human cells by the pathogenic parasite Toxoplasma gondii. This past year we made progress on our second objective and third objectives, which entailed isolating and characterizing mutants defective in responses to changes in potassium concentration. In a high [K+] buffer (K+ buffer) wild type parasites are blocked from invading but they recover and invade if [K+] is returned to normal. Recently, we concluded a genetic selection aimed at isolating mutants that invade host cells despite a high [K+]. After six rounds of selection for parasites that invade in the potassium buffer from two independently created mutant populations we established 24 clones from each population. In a preliminary screen of all clones established, ten clones from population 1 and 6 clones from population 2 exhibited an increase ability to invade cells in the presence of high [K+]
when compared to the parental strain. We have analyzed one of these mutants, IKC4, in detail. Mutant IKC4 has a 35% efficiency of invasion under a [K+] that allows only 1% of parasites from the parental strain to invade. This mutant's enhanced ability to invade cells is specific to high [K+] conditions, as this effect is not seen when the studies are performed in standard buffers. On the contrary, careful analysis of invasion under normal conditions revealed that mutant IKC4 exhibits an approximately 50% decrease in its ability to invade cells as compared to the parental strain. Together these results indicate that this novel mutant can invade cells in a K+ concentration that normally blocks motility and invasion but has a compromised ability to complete a successful invasion under normal conditions. Given IKC4's ability to invade at high potassium concentration, we tested this mutant's sensitivity to nigericin, which we have shown can induce egress by inducing K+ efflux from the host
cell (see Aim 2). IKC4 is more sensitive to nigericin than the parental strain. We propose that this enhanced sensitivity to nigericin is due to the mutant's ability to move in high [K+], thus less K+ needs to be extruded from the cell for it to undergo egress. Using standard plasmid rescue methods we have identified the genomic region where pHANA is inserted in mutant IKC4. Four different gene finder systems predict a gene within this region and searches through the Toxoplasma database indicate the existence of expressed sequence tags (ESTs) corresponding to this predicted gene. The putative protein encoded in this region is predicted to have several trans-membrane domains and a signal sequence. Homology searches with the predicted proteins do not shed light to the function of this protein but its possible location within a membrane could indicate a role in either signaling or ion homeostasis. Further molecular and phenotypic characterization of the mutant parasite will aid the
understanding of the role of this uncharacterized gene in the detection and response to K+ fluxes.
Impacts
The poorly understood process of egress is essential for the survival and pathogenesis of Toxoplasma. Thus inhibition of this process would control the dissemination of the parasite and the resulting disease. Consequently, our work, which aims at elucidating the genes involved in egress, will identify novel targets for drug design. Moreover, the study of Toxoplasma egress, which involves cytoskeletal rearrangements, organellar secretion and ion fluxes, is likely to shed light not only on this critical pathogenic event but also on processes relevant to a wide variety of eukaryotic organisms. Finally, given that the library of mutants we have designed and established can be screened for virtually any phenotype, it will be useful to the entire Toxoplasma community.
Publications
- Lavine, M.D., and Arrizabalaga, G., 2006. Invasion and egress by the obligate intracellular parasite Toxoplasma gondii: potential targets for the development of new antiparasitic drugs. Current Pharmaceutical Design, In press
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Progress 01/01/05 to 12/31/05
Outputs
The overall goal of this project is to elucidate the mechanisms and genes involved in the invasion of human cells by the pathogenic parasite Toxoplasma gondii. The specific objectives are: Objective 1: Isolate Mutants with a Delay in Egress and Defects in Invasion Objective 2: Select Mutants Resistant to K+ Buffer Invasion Block. Objective 3: Identify Gene Products Disrupted in the Egress, Invasion and K+ Block Mutants. A.Generation of independent insertional and chemical mutants: We have been able to readily establish and maintain 3,000 insertional mutants of a Toxoplasma strain using the protocol described in the original proposal. Our ultimate goal is to generate a total of 7,000 insertional mutants of this parental strain. We are also generating independent chemical point mutants to diversify the mutant library. We utilized the methods described in the original proposal to establish 1,000 independent chemical mutants and will create another 2,000 to complete our
library. B.Screen for mutants with slow rate of propagation: Defects in any of the steps of the lytic cycle, including egress, will result in slower propagation through the infected tissue. Accordingly, egress and invasion mutants can be screened for by first identifying those clones with a reduced propagation phenotype. We have established a protocol for rapidly measuring the rate of propagation of the mutant clones in 96 well tissue culture plates using ? galactosidase (?gal) as a marker and have begun to screen though the chemical and insertional mutants. We currently have approximately 30 mutants with a defect in propagation and will investigate the specific defects in these mutants with a series of phenotypic tests. C.Select mutants resistant to K+ buffer invasion block: It has been shown that a loss of K+ within the host cell triggers the egress process, while high [K+] in the outside environment inhibits Toxoplasma motility and invasion The mechanisms by which the parasite
perceives ionic changes in its environment and then translates them to either egress or invasion are not known and therefore, their identification is one of the goals of our research. To elucidate this process we designed a selection scheme to isolate mutants that can invade cells in spite of a high [K+], a condition normally 98% efficient at blocking invasion. Utilizing the methods we described in the original proposal we have established 7 mutants clones that have an increased ability to invade host cells in the presence of high [K+] as compared to the parental strain. When allowed to invade cells for 30 minutes in the presence of high [K+], only 1.8% of parasites from the parental strain enter the cells. In contrast, under the same conditions, 8.0 % of parasites of mutant KB1.11 and 16 % of mutant KB2.9 parasites are able to invade. Since these two novel mutant strains are from independent populations they are likely to represent distinct disruptions. We are currently mapping the
site of insertion in both of these mutants to determine the genes disrupted.
Impacts
The poorly understood process of egress is essential for the survival and pathogenesis of Toxoplasma. Thus inhibition of this process would control the dissemination of the parasite and the resulting disease. Consequently, our work, which aims at elucidating the genes involved in egress, will identify novel targets for drug design. Moreover, the study of Toxoplasma egress, which involves cytoskeletal rearrangements, organellar secretion and ion fluxes, is likely to shed light not only on this critical pathogenic event but also on processes relevant to a wide variety of eukaryotic organisms. Finally, given that the library of mutants we have designed and established can be screened for virtually any phenotype, it will be useful to the entire Toxoplasma community.
Publications
- Karasov, A, Boothroyd J.C. and Arrizabalaga, G, 2005. Identification and disruption of a rhoptry localized Sodium Hydrogen Exchanger in Toxoplasma gondii. International Journal of Parasitology, 35 (3); 285-291
- Saeij, J. P. J., Boyle, J. P., Grigg, M.E., Arrizabalaga G., and Boothroyd J. C. 2005. Bioluminescence imaging of Toxoplasma infection in living mice reveals dramatic differences between strains. Infect Immun 73(2); 695-702
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