On 2016 Jan 21, Sebastian Lourido commented:

The conditional dimerizable Cre recombinase (DiCre) has been a powerful technique for conditional genome engineering in Toxoplasma, as first established in this article, and elaborated later (see Pieperhoff, et al. 2015. PLoS One). It has worked well in our hands for a variety of applications. Recently, we discovered that the reporter construct used in this study was cloned down stream and in frame of a Ty-tag (EVHTNQDPLD), such that the KillerRed expressed prior to recombination contains and N-terminal Ty-tag. This observation does not affect any of the experiments presented in the article. However, it might be important to note for future investigators planning further manipulations of the existing DiCre strains or reporter constructs.

On 2014 Jan 22, Lilach Sheiner commented:

Credit for IPP experiment: Boris Striepen and Carrie Brooks.

On 2014 Jan 22, Markus Meissner commented:

Hello Vermont JC. Thank you for the comment and the intriguing questions raised. We fully agree that it will be very interesting to see the composition of the remaining motor complex in absence of MyoA and we currently perform these experiments. You might be interested in our current study, submitted as prepub (Egarter et al., 2014 bioRxiv 01/2014; DOI:10.1101/001800) that adresses some of your questions. With regards to maintaining the act1 KO in presence of IPP we planned this experiment until we were assured from experts working on the apicoplast in Toxoplasma gondii that IPP cannot complement apicoplast loss in this parasite (Sheiner et al. personal communication). I also agree with your conclusion that there must be an actin-myosin-MIC2 independent invasion mechanism and it will be very interesting to see if it plays a major role in wild type parasites as well. Future will tell.

On 2013 Dec 11, Gary Ward commented:

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

On 2013 Dec 11, Gary Ward commented:

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

On 2016 Jan 21, Sebastian Lourido commented:

The conditional dimerizable Cre recombinase (DiCre) has been a powerful technique for conditional genome engineering in Toxoplasma, as first established in this article, and elaborated later (see Pieperhoff, et al. 2015. PLoS One). It has worked well in our hands for a variety of applications. Recently, we discovered that the reporter construct used in this study was cloned down stream and in frame of a Ty-tag (EVHTNQDPLD), such that the KillerRed expressed prior to recombination contains and N-terminal Ty-tag. This observation does not affect any of the experiments presented in the article. However, it might be important to note for future investigators planning further manipulations of the existing DiCre strains or reporter constructs.