Gary Ward

Comments 1 to 10 of 10

  • Gary Ward2014 Nov 09 10:54 a.m. 4 of 4 people found this helpful

    We reported in this paper that the enhancers of invasion and motility identified in the screen do not stimulate an increase in parasite intracellular calcium levels. This conclusion was based on semiquantitative microscopy-based measurements of the fluorescence intensity of the calcium reporter Fluo-4 in individual parasites, before and after treatment with enhancer. More recent quantitative experiments on populations of parasites using the ratiometric calcium reporter Fura-2 showed that treatment with three of the four enhancers tested does in fact stimulate an increase in parasite intracellular calcium levels (Tang Q, 2014); results with the fourth were inconclusive due to spectral overlap between the compound and indicator.

    The discovery that some, and perhaps all, of the enhancers identified in the screen increase parasite intracellular calcium levels provides a rationale for how this group of structurally diverse compounds can cause the same set of phenotypes, i.e, enhanced invasion, microneme secretion and motility.

  • Gary Ward2014 Nov 04 9:05 p.m. 3 of 3 people found this helpful

    This is a beautiful and clear demonstration of how Toxoplasma gondii can serve as both a useful model organism for the study of other apicomplexan parasites, and powerful surrogate system for small molecule screening. By complementing TgCDPK3 with Plasmodium falciparum CDPK1 (PfCDPK1), the group was able to confirm the functional localization dependence of PfCDPK1 and identify compounds that inhibit both PfCDPK1 and TgCDPK3, as well as those that inhibit PfCDPK1 alone. This work and the work of Sharling et al. PLOS Negl Trop Dis [2010] 4: e794 and others provide good examples of how studying T. gondii may be useful to understanding other apicomplexan parasites from a drug development standpoint.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Sam Ashley, Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal & Gary Ward

  • Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms.

    Andenmatten N.Nat Methods. 2013.4 commentsMarkus Meissner, Lilach Sheiner and 1 other also commented

    Gary Ward2013 Dec 10 10:14 p.m. 3 of 3 people found this helpful

    This paper presents a clever new way to generate gene knockouts in T. gondii, using a conditional dimerizable Cre recombinase (DiCre) system. Because the knockouts are experimentally induced with rapamycin, even essential genes can be disrupted for phenotypic characterization. In a first application of the technique, the authors show that two genes encoding proteins previously believed to be essential for parasite invasion, myosin A and the secreted adhesin MIC2, are in fact dispensable. In the case of the myoA knockout parasites, it would be interesting to see whether or not another parasite myosin is upregulated or now associates with the myosin motor complex (e.g., as assessed by a GAP45 IP) in the absence of myosin A.

    In contrast, parasites could not tolerate disruption of act1 (actin). Evidence is presented to suggest that the actin knockouts remain capable of invasion and the authors suggest that the most important defect is instead in apicoplast segregation. Have the authors attempted to isolate and maintain an act1 knockout clone in the presence of isopentenyl pyrophosphate (IPP)? Blood-stage Plasmodium falciparum can survive independently of the apicoplast as long as this isoprenoid precursor is provided (Yeh et al. PLOS Biol [2011] 9(8): e1001138). It would be interesting to look at the T. gondii act1 knockout’s ability (or perhaps inability) to glide and invade under similar conditions.

    The picture that emerges from these ground-breaking studies is that myosin A, MIC2 and actin are involved in host cell invasion, as the current model would posit, but that there is another previously unrecognized way for the parasites to invade independent of these proteins.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

  • Gary Ward2013 Dec 10 10:01 p.m. 2 of 2 people found this helpful

    This paper is a classic in the field of parasite cell biology. The stunning electron micrographs provide the first high-resolution look at the substructure of the conoid, a unique cytoskeletal structure found in a variety of apicomplexan parasites. The paper shows that the fibers of the conoid are composed of tubulin, but the protofilament arrangement within the individual fibers is unlike that of any other known tubulin-based structure: rather than a closed tube, the 9 protofilaments are arranged into a “comma” shape. This unique structure may enable the high degree of curvature required of these filaments within the thimble-shaped conoid (diameter 380 nm). FRAP experiments revealed the tubulin subunits are incorporated into the conoid during the early stages of daughter formation, but not in mature parasites. Remarkably, the authors also show that the pitch of the filaments changes as the conoid extends and retracts during parasite motility and host cell invasion.

    In the years since this paper was published, additional isoforms of α- and β-tubulin have been discovered for a total of three each (Hu et al. PLOS Pathog [2006] 2(2): e13). It would be interesting to see whether any of these isoforms localize specifically to the conoid or to the other tubulin-based structures within the parasite.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

  • Gary Ward2013 Dec 06 11:22 p.m. 1 of 1 people found this helpful

    This is a descriptive study of the ultrastructure of the residual body (RB) formed during T. gondii replication, highlighting the continuity between the parasite cytosol and RB as well as the intravacuolar connections between parasites. The authors argue that the dense granule protein GRA2 plays a role in maintaining these connections.

    The EM images confirm the presence of previously observed structures and organelles within the RB, including portions of the nucleus. It is unclear what is meant by the author’s statement that “Antibodies against SAG1, the parasite major surface protein, labelled the plasma membrane of proliferating parasites but not the RB membrane; probably the availability of the RB membrane was limited by the binding of the tachyzoites”. We were left wondering why the RB doesn't stain for SAG1 even though there is clearly membrane present.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

  • Gary Ward2013 Nov 20 9:32 p.m. 1 of 1 people found this helpful

    This report describes a simple yet elegant series of experiments demonstrating that the T. gondii parasitophorous vacuole membrane (PVM) acts as a sieve, allowing small molecules (up to ~1300 Da) to pass from the cytosol into the vacuolar space. The researchers microinjected a panel of fluorescent probes either into the cytosol of infected cells or into the parasitophorous vacuole and observed bi-directional, passive transport across the PVM. These experiments provided an important new insight into the interface between the intracellular parasite and the host cell. The likely presence of nonselective membrane channels in the PVM is analogous to the outer membrane of Gram-negative bacteria, in which membrane porins allow passive transport of nutrients and metabolites into the bacterial periplasmic space.

    These results from these experiments are consistent with characterization of the vacuole membrane in host cells infected with the related apicomplexan parasites, Plasmodium falciparum [Desai et al, Nature 362 (1993); Desai and Rosenberg, PNAS 94 (1997)] and Eimeria nieschulzi [Werner-Meier and Entzeroth, Parasitol Res 83 (1997)]. The obligate intracellular microsporidian Encephalitozoon cuniculi also resides within an intracellular vacuole capable of passive transport of fluorescent peptides up to 1100 Da while 10 kDa fluorescently-labeled dextrans are excluded [Rönnebäumer et al, Eukaryot Cell 7 (2008)]. The presence of non-selective membrane channels and the non-fusogenic nature of the parasitic vacuoles established by these phylogenetically distant organisms point to a similar adaptation to an intracellular parasitic lifestyle.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

  • Gary Ward2013 Nov 17 10:32 p.m. 1 of 1 people found this helpful

    This paper describes a clever high-throughput assay to identify small molecules that disrupt the interaction of Plasmodium falciparum AMA1 and RON2, and can thereby block merozoite invasion of erythrocytes. The data provide promising proof-of-concept for the development of novel antimalarials that disrupt protein-protein interactions critical for invasion. This particular interaction is thought to happen extracellularly, within the blood, which could facilitate access of such drugs to their targets.

    The three inhibitors described have IC50 values in the 20-30 uM range. The authors state that "small-molecule inhibitors will result in reduced or no emission signal depending on the strength of the inhibition". Our group was interested to know what the dynamic range of the AlphaScreen assay is and whether it can capture binding at both ends of the affinity spectrum (subnanomolar to millimolar).

    A 1000-fold molar excess of inhibitor was required to disrupt RON2L-AMA1 interaction in the screen, perhaps because the compound is added after RON2L-AMA1 complex formation and must essentially displace the RON2L from AMA1. The assay may have been done this way purposely, to recapitulate what happens in the blood, i.e., secreted RON2 is probably not exposed on the surface of the red cell for long before it is bound by AMA1. It would nonetheless be interesting to know what happens to the IC50 if compound is added to AMA1 before the addition of RON2L.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelson, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

  • Gary Ward2013 Oct 20 11:01 p.m.

    This is a seminal paper in the field, for two reasons:

    1) It reported the development of a new, Tet-transactivator-based system for controlling gene expression in T. gondii. This system has since been widely adopted as a way to make conditional knockout parasites and is particularly useful for studying the function of essential genes in this haploid organism. For a powerful extension of this system based on promoter replacement, see Sheiner et al PLOS Pathogens 7 (2011) e1002392.

    2) In the first application of the new system, the authors demonstrated directly the importance of the parasite’s Class XIV myosin, TgMyoA, for gliding motility, host cell invasion and host cell egress. This observation led to a great deal of subsequent work on the myosin motor complex and its role in the biology of T. gondii and other apicomplexan parasites.

    Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelson, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

  • Gary Ward2013 Jun 24 4:23 p.m. 2 of 2 people found this helpful

    Thanks to the authors for this timely reanalysis of the current model of apicomplexan invasion. Recent work from the Meissner group has shown that several of the parasite proteins previously thought to be essential for invasion can be knocked out, casting some doubt on the model. I agree with much of what they write, but have three comments:

    1) Non-essential does not mean non-important. In several of these knockouts, invasion and motility are dramatically impaired (e.g., ~80-85% inhibition of invasion) and defects of this magnitude can translate into greatly reduced virulence in vivo (e.g., Meissner et al, Science [2002] 298, 837). While the new data show clearly that MyoA and AMA1 are not essential for invasion, they at the same time show them to be important for invasion. Whether they are important for the reasons predicted by the current model is a separate question.

    2) In the authors' view, the data either show that the "parasites use a single entry mechanism and hence the current invasion model is wrong and needs to be replaced by a new model, [or] the current model is overall valid but an additional, motor independent invasion mechanism is at work that facilitates host cell invasion in KO mutants of the glideosome". Redundant mechanisms are a common feature of parasite biology (e.g., erythrocyte-binding proteins of malaria parasites, Trends in Parasitology [2012] 28, 23), so I agree with these two possibilities. However, I would add a third: by knocking out a parasite protein that plays an important role in a process that is so critical to the parasite lytic cycle, enormous selective pressure is put on the parasites to come up with a way around the problem. Compensatory mutations or changes in the expression of other proteins that can "fill in" for the missing protein may occur. In fact, we have seen an improvement over time in the ability of some of these mutants to invade, which likely represents some advantageous mutation or change in gene expression that is being selected for in culture. Such changes will be hard to track down and even harder to rule out, but the phenotype observed in these knockouts will be due to some combination of the knockout itself and whatever the parasite does to overcome it.

    3) The collar seen around the invading tachyzoite in Fig. 2 is intriguing, and unlike what we typically see by EM during invasion of human foreskin fibroblasts. How rare are these events, and are they specific to this cell type? This profile could reflect an alternative invasion mechanism, as proposed. On the other hand, these cells might have an unusually impenetrable cortical cytoskeleton and the parasites push but can't get in. In this case the moving junction would in fact really be a moving junction and would be pulled along the body of the stationary parasite, much like occurs during Cryptosporidium invasion and consistent with the standard model of invasion.

  • Gary Ward2013 Jun 24 1:08 p.m.

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