On 2014 Mar 01, L David Sibley commented:

It is interesting to see that most of the comments are about why we choose to study AMA1 and whether or not AMA1 is essential. We feel that there may be some misunderstanding about the rationale for our study, so hopefully the following points will help to clarify a few things.

The first misunderstanding deals with the findings of our previous work on ALD mutants and binding to the MIC tail (Starnes et al 2009). When we in a previous comment that these studies “were not able to definitively separate the functions of energy metabolism from motility vs. invasion”, we meant that while the studies did identify a mutant (TgALD K41E-R42G) that separates the functions of energy production from invasion, they did not fully explain why such mutants had no effect on gliding. One plausible explanation was provided in that paper: decreased occupancy of ALD-MIC2 in the cell (a product of altered affinity and protein concentrations) might differentially affect gliding vs. invasion. However, an alternative explanation is that ALD might also bind to another adhesin that is important in invasion but not gliding. When it became apparent that mutants in the AMA1 tails (AMA1t) also affect ALD binding in vitro (i.e. FW/AA) (Sheiner and Soldati et al, 2010), this provided a logical candidate, since AMA1 was known to be involved in host cell invasion but not motility (Mital et al., 2005).

Perhaps a second point of confusion is that our study did not try to address the essentiality of AMA1, something that has been studied by others, who ascribe various roles to the protein including attachment, MJ formation, and cell penetration. We were not aware at the outset of our study of the data contained in Bargieri et al. 2013, as the paper was published online while our work was under review. However, this likely would not have changed our approach as were not concerned with whether AMA1 is essential, or what factors might compensate for its loss, but rather with how it functions when it is expressed. Because the FW/AA mutations are located in the tail of the protein, we reasoned they were more likely to be involved in functions in the cytosol, rather than influencing the roles of the extracellular domains. Hence, this mutant provided an excellent candidate to test whether decreased cell invasion was due to alteration in ALD binding. As it turned out, study of additional mutants did not provide support for this model. Moreover, by re-examining the role of ALD in energy production, our study newly revealed that the previously ascribed role in adhesin binding does not play an essential role in vivo. We believe this is the only aspect of the apicomplexan invasion model that is directly addressed by our studies. We also hope that our work will inspire further studies to figure out the real function of AMA1t, which in turn will help us to understand the invasion process better.

Bang Shen, David Sibley

On 2014 Feb 27, Robert Menard commented:

The goal of the paper was to see whether aldolase plays a mechanical role during motility and/or internalization, the motor-dependent processes of the zoite. AMA1, using AMA1 KOs, was shown by the Bargieri paper (not cited in the current paper) to have NO detectable role in these processes, which would imply that the putative aldolase-AMA1 interaction does not either. The sound rationale appears to be that of the Starnes paper, which asked the question using the MIC2-aldolase interaction, since MIC2 is a bona fide motor-binding protein and is clearly important for motile processes. The data of the Starnes paper clearly indicate that aldolase has no mechanical role. For example, mutants K41A and K41E:R42G bind to MIC2t with 18% and 5% efficiency, respectively, and to actin with 13% and 9% efficiency, respectively. Fig 5C shows that these mutants have no detectable defect in motility. It is surprising to see that the author now says the Starnes data ‘was not able to definitely separate the functions’. This is clearly contrary to the straightforward title and abstract, which reads: “we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity”.

Regarding the role of AMA1 in the TJ, the redundancy theory by AMA paralogs is presented incompletely. The author omits the crucial point that while AMA1 was not found to have any role in internalization at the TJ, it has an important role in adhesion to the host cell. AMA1 paralogs are expected to have a similar function, in promoting proper adhesion. Examination of Fig 1 shows overexpression of AMA1 paralogs in the AMA1 KO, but complete citation of the paper should also state increased paralog expression decreases the adhesion defect. In other words, AMA1 paralogs appear to be adhesins like AMA1, and are not expected to do at the TJ what AMA1 does not do itself. Lastly, we again point out that it is incorrect to state that inhibition experiments showed that the AMA1-RON interaction is important for TJ formation; they only indicate that its inhibition blocks invasion. This might occur by other scenarios than the interaction being the TJ, as further discussed in the Bargieri paper.

While we agree that it might be premature to draw a model of the TJ, it is equally dangerous to discard data that do not fit with the original model.

On 2014 Feb 26, L David Sibley commented:

As was discussed in our paper, and the previous Starnes paper, prior mutants of aldolase or MIC2t were not able to definitively separate the functions of energy metabolism from motility vs. invasion. Indeed, several mutants made in other systems have shown these later two functions can be independently controlled. Hence showing that one behavior is affected while the other one is not, cannot be used to argue that the process is not essential at some point. Combined with the hypothesis that AMAt binding to aldolase might be important for invasion (while playing no role in motility), this dichotomy provided more than adequate rationale for our present study. We also do not agree that prior work on AMA1 rules out a role in invasion, it merely shows that individual genes may be dispensable under some circumstances. Examination of Figure 1D in Bargierri et al., reveals evidence for 15-fold upregulation of a paralog of AMA1 (incorrectly called a homologue) in the TgAMA1 KO, suggesting that this gene may mask the phenotype by compensating for AMA1. Whether Plasmodium has recognizable paralogs of AMA1, or uses another adapter, one could still argue that the mechanism is conserved since the MJ is preserved. Independent of these genetic studies, there is a strong body of work that AMA1 is critical to the MJ formation. At this point it is premature to discard the existing model based on an incomplete assessment of potential compensatory mechanisms for loss of individual genes.

On 2014 Feb 25, Robert Menard commented:

The demonstration that aldolase plays no bridging role between actin and the MIC2/TRAP tails during zoite motility was provided by Starnes et al, who introduced mutations in aldolase that specifically abolished its capacity to bind MIC2 but not its energy producing capacity; these mutations had no effect on tachyzoite motility, which demonstrated the point. Regarding AMA1, previous work in Toxoplasma and Plasmodium has conclusively demonstrated not just that AMA1 is not essential for internalization, but that it has no detectable role in the process: AMA1 KO tachyzoites form a normal TJ and enter cells at a normal speed. The argument of redundancy by AMA1 paralogs has no basis, since (i) we showed that AMA1 - and paralogs - play a role in adhesion to the host cell, not directly in invasion at the TJ, and (ii) Plasmodium expresses no AMA1 paralog. These genetic data (Giovannini et al, CHM, 2011; Bargieri et al, Nat Comm, 2013) strongly argue against any motor-dependent function of AMA1 in any zoite and thus question the rationale of the present study. R. Menard, M. Meissner and D. Bargieri

On 2014 Feb 22, L David Sibley commented:

We welcome the opportunity to clarify the reasoning behind our study. We chose the AMA1-aldolase interaction to study because it demonstrates the requirement of two residues in the tail (FW) of AMA1 for cell invasion in a conditional knockdown, as reported previously (Sheiner et al., 2010 (PMID:2054586)). This AMA1t mutant also fails to bind aldolase in vitro, leading to the testable hypothesis that these two phenotypes are linked. We chose AMA1 not to address whether it is essential or not (indeed the jury is still out on this important question), but rather to test the model that binding of adhesin tails to aldolase is critical for linking the motor complex (as suggested previously by Jewett et al., 2003 (PMID12718875), and Starnes et al., 2009 (PMID19380114)). We are aware that others have questioned the requirement for AMA1 during invasion, but these studies also failed to account for possible redundant roles of paralogs, for which there is clear evidence of up-regulation in at least the one study that looked (Bargieri et al., Nature Comm 2013 (PMID24108241)). The use of knockout strains also runs the risk that suppressor mutations may have arisen in the background, given the length of time required to obtain such knockouts, at least by conventional means. In contrast, the conditional system we employed is less prone to such issues of compensation.

By studying a broader collection of AMAt mutants than previously, we show that there is no correlation between AMA1t-aldolase binding in vitro and invasion. This led us to further investigate the role of aldolase using more efficient techniques for gene disruption (Andenmatten et al., Nat Meth 2013 (PMID23263690)). The results show conclusively that aldolase is required for energy metabolism, but not binding to adhesin tails during invasion. This insight could not have been provided by any of the previous studies, as outlined in the discussion to our paper. Although we have not tested MIC2t, or other adhesins shown to bind aldolase, this no longer seems worthwhile given the clear lack of a phenotype for aldolase negative cells when grown in the absence of glucose. As we further point out in the discussion, there are clearly important roles for conserved residues in the tails of adhesins and the search is on for what those functions might be.

In terms of the model for invasion, our studies indicate that some other protein(s) must be responsible for linking the adhesin tails to the motor complex, and that if aldolase participates, it is redundant. This finding is consistent with the view that redundancy in biology is likely the norm for essential pathways. The work by Andenmatten et al., Nat Meth 2013 (PMID23263690) offers an exciting new tool to explore such redundancy with greater precision than previously possible, and indeed this was the basis for the final test in our study. Andenmatten et al., show that under conditions where genes can be rapidly excised by inducible Cre, it is possible to delete some of the core components of the glideosome. However, this is not without consequence as such mutants are severely impaired in gliding and invasion. Hence, we would interpret the available data as providing strong support for the current glideosome model for motility and invasion, rather than cause to abandon it. What remains unanswered by current studies is what is the backup or alternative mechanism(s), by which such deletion parasites still invade? There are a number of possibilities including: 1) residual levels of proteins remaining in knockout cells; 2) redundancy (i.e. paralogs that are upregulated), as are clearly evident in the genome, or 3) alternative mechanisms that serve as backup, albeit clearly less efficient. The remaining challenge for the field is to fill in these details, at which point it may be possible to provide a revised model for gliding and invasion by apicomplexans.

On 2014 Feb 20, Markus Meissner commented:

This nice study re-adresses the role of the glycolytic enzyme aldolase as a critical component of the gliding and invasion machinery of apicomplexan parasites as suggested previously by the same group. Aldolase has been described as a critical linker molecule between the tail domain of microcemal transmembrane proteins, such as AMA1 or MIC2 and the actin-myosin motor, thereby playing a crucial role for force transmission during motility and invasion. Here the authors tested if the interaction between AMA1 and ALD is important for host cell invasion. However, previous analysis of AMA1 knockdown (the same mutant as used in this study) and knockout mutants ruled out an important role of AMA1 as force transmitter during gliding motility (Mital et al., 2005) and host cell invasion (Giovannini et al., CHM 2011; Bargieri et al., Nature Comm 2013). Why did the authors expect a critical role of aldolase binding to AMA1, when AMA1 itself is not required for force transmission? Did the authors also investigate the role of MIC2 in binding to aldolase?

The authors continued to analyse the phenotype of an ALD knockout and demonstrate that these parasites can invade the host cell normally, ruling out an important function of aldolase during this process.

It would have been very informative for the reader if the authors would have discussed their finding in a more holistic view that also incorporates recent findings on the gliding and invasion machinery. The authors mention that the current model needs to be revised, which is certainly true, since several of the core components for invasion appear to be not essential for invasion, including actin itself (Andenmatten et al., Nat Meth 2013). It would have been very interesting to hear the opinion of the senior author how he currently perceives the molecular mechanisms involved in gliding and invasion. As it stands now it seems that we do not have a model that is sufficiently backed up by experimental data.

On 2014 Feb 20, Markus Meissner commented:

This nice study re-adresses the role of the glycolytic enzyme aldolase as a critical component of the gliding and invasion machinery of apicomplexan parasites as suggested previously by the same group. Aldolase has been described as a critical linker molecule between the tail domain of microcemal transmembrane proteins, such as AMA1 or MIC2 and the actin-myosin motor, thereby playing a crucial role for force transmission during motility and invasion. Here the authors tested if the interaction between AMA1 and ALD is important for host cell invasion. However, previous analysis of AMA1 knockdown (the same mutant as used in this study) and knockout mutants ruled out an important role of AMA1 as force transmitter during gliding motility (Mital et al., 2005) and host cell invasion (Giovannini et al., CHM 2011; Bargieri et al., Nature Comm 2013). Why did the authors expect a critical role of aldolase binding to AMA1, when AMA1 itself is not required for force transmission? Did the authors also investigate the role of MIC2 in binding to aldolase?

The authors continued to analyse the phenotype of an ALD knockout and demonstrate that these parasites can invade the host cell normally, ruling out an important function of aldolase during this process.

It would have been very informative for the reader if the authors would have discussed their finding in a more holistic view that also incorporates recent findings on the gliding and invasion machinery. The authors mention that the current model needs to be revised, which is certainly true, since several of the core components for invasion appear to be not essential for invasion, including actin itself (Andenmatten et al., Nat Meth 2013). It would have been very interesting to hear the opinion of the senior author how he currently perceives the molecular mechanisms involved in gliding and invasion. As it stands now it seems that we do not have a model that is sufficiently backed up by experimental data.

On 2014 Feb 22, L David Sibley commented:

We welcome the opportunity to clarify the reasoning behind our study. We chose the AMA1-aldolase interaction to study because it demonstrates the requirement of two residues in the tail (FW) of AMA1 for cell invasion in a conditional knockdown, as reported previously (Sheiner et al., 2010 (PMID:2054586)). This AMA1t mutant also fails to bind aldolase in vitro, leading to the testable hypothesis that these two phenotypes are linked. We chose AMA1 not to address whether it is essential or not (indeed the jury is still out on this important question), but rather to test the model that binding of adhesin tails to aldolase is critical for linking the motor complex (as suggested previously by Jewett et al., 2003 (PMID12718875), and Starnes et al., 2009 (PMID19380114)). We are aware that others have questioned the requirement for AMA1 during invasion, but these studies also failed to account for possible redundant roles of paralogs, for which there is clear evidence of up-regulation in at least the one study that looked (Bargieri et al., Nature Comm 2013 (PMID24108241)). The use of knockout strains also runs the risk that suppressor mutations may have arisen in the background, given the length of time required to obtain such knockouts, at least by conventional means. In contrast, the conditional system we employed is less prone to such issues of compensation.

By studying a broader collection of AMAt mutants than previously, we show that there is no correlation between AMA1t-aldolase binding in vitro and invasion. This led us to further investigate the role of aldolase using more efficient techniques for gene disruption (Andenmatten et al., Nat Meth 2013 (PMID23263690)). The results show conclusively that aldolase is required for energy metabolism, but not binding to adhesin tails during invasion. This insight could not have been provided by any of the previous studies, as outlined in the discussion to our paper. Although we have not tested MIC2t, or other adhesins shown to bind aldolase, this no longer seems worthwhile given the clear lack of a phenotype for aldolase negative cells when grown in the absence of glucose. As we further point out in the discussion, there are clearly important roles for conserved residues in the tails of adhesins and the search is on for what those functions might be.

In terms of the model for invasion, our studies indicate that some other protein(s) must be responsible for linking the adhesin tails to the motor complex, and that if aldolase participates, it is redundant. This finding is consistent with the view that redundancy in biology is likely the norm for essential pathways. The work by Andenmatten et al., Nat Meth 2013 (PMID23263690) offers an exciting new tool to explore such redundancy with greater precision than previously possible, and indeed this was the basis for the final test in our study. Andenmatten et al., show that under conditions where genes can be rapidly excised by inducible Cre, it is possible to delete some of the core components of the glideosome. However, this is not without consequence as such mutants are severely impaired in gliding and invasion. Hence, we would interpret the available data as providing strong support for the current glideosome model for motility and invasion, rather than cause to abandon it. What remains unanswered by current studies is what is the backup or alternative mechanism(s), by which such deletion parasites still invade? There are a number of possibilities including: 1) residual levels of proteins remaining in knockout cells; 2) redundancy (i.e. paralogs that are upregulated), as are clearly evident in the genome, or 3) alternative mechanisms that serve as backup, albeit clearly less efficient. The remaining challenge for the field is to fill in these details, at which point it may be possible to provide a revised model for gliding and invasion by apicomplexans.

On 2014 Feb 25, Robert Menard commented:

The demonstration that aldolase plays no bridging role between actin and the MIC2/TRAP tails during zoite motility was provided by Starnes et al, who introduced mutations in aldolase that specifically abolished its capacity to bind MIC2 but not its energy producing capacity; these mutations had no effect on tachyzoite motility, which demonstrated the point. Regarding AMA1, previous work in Toxoplasma and Plasmodium has conclusively demonstrated not just that AMA1 is not essential for internalization, but that it has no detectable role in the process: AMA1 KO tachyzoites form a normal TJ and enter cells at a normal speed. The argument of redundancy by AMA1 paralogs has no basis, since (i) we showed that AMA1 - and paralogs - play a role in adhesion to the host cell, not directly in invasion at the TJ, and (ii) Plasmodium expresses no AMA1 paralog. These genetic data (Giovannini et al, CHM, 2011; Bargieri et al, Nat Comm, 2013) strongly argue against any motor-dependent function of AMA1 in any zoite and thus question the rationale of the present study. R. Menard, M. Meissner and D. Bargieri

On 2014 Feb 26, L David Sibley commented:

As was discussed in our paper, and the previous Starnes paper, prior mutants of aldolase or MIC2t were not able to definitively separate the functions of energy metabolism from motility vs. invasion. Indeed, several mutants made in other systems have shown these later two functions can be independently controlled. Hence showing that one behavior is affected while the other one is not, cannot be used to argue that the process is not essential at some point. Combined with the hypothesis that AMAt binding to aldolase might be important for invasion (while playing no role in motility), this dichotomy provided more than adequate rationale for our present study. We also do not agree that prior work on AMA1 rules out a role in invasion, it merely shows that individual genes may be dispensable under some circumstances. Examination of Figure 1D in Bargierri et al., reveals evidence for 15-fold upregulation of a paralog of AMA1 (incorrectly called a homologue) in the TgAMA1 KO, suggesting that this gene may mask the phenotype by compensating for AMA1. Whether Plasmodium has recognizable paralogs of AMA1, or uses another adapter, one could still argue that the mechanism is conserved since the MJ is preserved. Independent of these genetic studies, there is a strong body of work that AMA1 is critical to the MJ formation. At this point it is premature to discard the existing model based on an incomplete assessment of potential compensatory mechanisms for loss of individual genes.

On 2014 Feb 27, Robert Menard commented:

The goal of the paper was to see whether aldolase plays a mechanical role during motility and/or internalization, the motor-dependent processes of the zoite. AMA1, using AMA1 KOs, was shown by the Bargieri paper (not cited in the current paper) to have NO detectable role in these processes, which would imply that the putative aldolase-AMA1 interaction does not either. The sound rationale appears to be that of the Starnes paper, which asked the question using the MIC2-aldolase interaction, since MIC2 is a bona fide motor-binding protein and is clearly important for motile processes. The data of the Starnes paper clearly indicate that aldolase has no mechanical role. For example, mutants K41A and K41E:R42G bind to MIC2t with 18% and 5% efficiency, respectively, and to actin with 13% and 9% efficiency, respectively. Fig 5C shows that these mutants have no detectable defect in motility. It is surprising to see that the author now says the Starnes data ‘was not able to definitely separate the functions’. This is clearly contrary to the straightforward title and abstract, which reads: “we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity”.

Regarding the role of AMA1 in the TJ, the redundancy theory by AMA paralogs is presented incompletely. The author omits the crucial point that while AMA1 was not found to have any role in internalization at the TJ, it has an important role in adhesion to the host cell. AMA1 paralogs are expected to have a similar function, in promoting proper adhesion. Examination of Fig 1 shows overexpression of AMA1 paralogs in the AMA1 KO, but complete citation of the paper should also state increased paralog expression decreases the adhesion defect. In other words, AMA1 paralogs appear to be adhesins like AMA1, and are not expected to do at the TJ what AMA1 does not do itself. Lastly, we again point out that it is incorrect to state that inhibition experiments showed that the AMA1-RON interaction is important for TJ formation; they only indicate that its inhibition blocks invasion. This might occur by other scenarios than the interaction being the TJ, as further discussed in the Bargieri paper.

While we agree that it might be premature to draw a model of the TJ, it is equally dangerous to discard data that do not fit with the original model.

On 2014 Mar 01, L David Sibley commented:

It is interesting to see that most of the comments are about why we choose to study AMA1 and whether or not AMA1 is essential. We feel that there may be some misunderstanding about the rationale for our study, so hopefully the following points will help to clarify a few things.

The first misunderstanding deals with the findings of our previous work on ALD mutants and binding to the MIC tail (Starnes et al 2009). When we in a previous comment that these studies “were not able to definitively separate the functions of energy metabolism from motility vs. invasion”, we meant that while the studies did identify a mutant (TgALD K41E-R42G) that separates the functions of energy production from invasion, they did not fully explain why such mutants had no effect on gliding. One plausible explanation was provided in that paper: decreased occupancy of ALD-MIC2 in the cell (a product of altered affinity and protein concentrations) might differentially affect gliding vs. invasion. However, an alternative explanation is that ALD might also bind to another adhesin that is important in invasion but not gliding. When it became apparent that mutants in the AMA1 tails (AMA1t) also affect ALD binding in vitro (i.e. FW/AA) (Sheiner and Soldati et al, 2010), this provided a logical candidate, since AMA1 was known to be involved in host cell invasion but not motility (Mital et al., 2005).

Perhaps a second point of confusion is that our study did not try to address the essentiality of AMA1, something that has been studied by others, who ascribe various roles to the protein including attachment, MJ formation, and cell penetration. We were not aware at the outset of our study of the data contained in Bargieri et al. 2013, as the paper was published online while our work was under review. However, this likely would not have changed our approach as were not concerned with whether AMA1 is essential, or what factors might compensate for its loss, but rather with how it functions when it is expressed. Because the FW/AA mutations are located in the tail of the protein, we reasoned they were more likely to be involved in functions in the cytosol, rather than influencing the roles of the extracellular domains. Hence, this mutant provided an excellent candidate to test whether decreased cell invasion was due to alteration in ALD binding. As it turned out, study of additional mutants did not provide support for this model. Moreover, by re-examining the role of ALD in energy production, our study newly revealed that the previously ascribed role in adhesin binding does not play an essential role in vivo. We believe this is the only aspect of the apicomplexan invasion model that is directly addressed by our studies. We also hope that our work will inspire further studies to figure out the real function of AMA1t, which in turn will help us to understand the invasion process better.

Bang Shen, David Sibley