On 2014 Jun 24, Markus Meissner commented:

Comment by Markus Meissner, Isabelle Tardieux and Robert Menard:

In this study Lamarque et al., convincingly demonstrate a functional redundancy of AMA-like molecules that can partially complement for AMA1. Indeed they observe upregulation of these molecules in a mutant harbouring a gene disruption in ama1 and therefore validate our previous data (Bargieri et al., 2013). However, when it comes to the function of AMA1 (and AMA-like proteins) we disagree with the final conclusion of the authors for several reasons:

1. The authors conclude from an abortive invasion assay that in absence of AMA1 (but supposedly in presence of AMA2) parasites cannot remain attached to the host cell after secretion of the rhoptries because of the lack of a functional junction. These conclusions do not contradict the finding of a strong attachment phenotype and it remains to be seen what and when rhoptry secretion is triggered during attachment. One important control would have been to demonstrate that other (unrelated) attachment mutants (i.e. MIC2KO, Act1KO, etc.) do not show the same behaviour as AMA1KO in this assay. It is important to note that our own extensive analysis of host cell invasion events by AMA1KO parasites using real time imaging did not catch a scenario that could fit with this abortive invasion and would characterize the AMA1 deficient population. Therefore it is unknown at this point what causes the increased secretion of Rhop proteins into the host cell in absence of AMA1 or Ron2.

2. Using a tail mutation (FW>AA), the authors demonstrate that parasites remain attached, but do not invade, indicating an important role of the tail for invasion. It is important to note that this mutation has been described previously as disrupting the interaction to aldolase (Sheiner et al., 2010). However, a recent study (Shen et al., 2014) convincingly demonstrated that aldolase is not the crucial linker between micronemes and actin. Therefore in absence of knowledge of tail function the significance of this finding justifies several interpretations, such as signalling (indeed previous data demonstrate that the tail is phosphorylated during invasion) or simple structural consequences causing a dominant negative effect.

3. The demonstration that AMA1 is likely to be at the junction during invasion does not suggest a function of AMA1 during this process, since it could simply be a “waste” product after transmembrane processing. Indeed, processing mutants of AMA1 invade the host cell with slower kinetics (Parussini et al., 2012). Intriguingly, the authors did not make any attempts to characterise the kinetics of host cell entry of AMA1KO or AMA1KO/AMA2KO, which should be significantly reduced given the strength of the observed phenotype for AMA1KO/AMA2KO. In case of ama1KO we convincingly reported that the major effect is in host cell attachment and not downstream steps, such as host cell entry. How do the authors explain that there would be a full redundancy for downstream steps, but only a minor partial redundancy upstream?

We think that the data presented in this manuscript support a model, where AMA1/Ron2 interaction is important to stabilise parasite attachment to the host cell. AMA1 might then be involved in a signalling cascade that activates active entry into the host cell. Therefore, we suggest that AMA1 acts upstream of the actual entry event, as published previously (see Bargieri et al., 2013).

In summary, while AMA1/RON2 interaction is clearly important, its exact role remains unknown. A role in transducing force in the junction is not supported by available data. In agreement with a primary role of AMA1 in adhesion, AMA1 might further contribute to optimal positioning prior to entry by binding to RON2 in a formed junction. Alternatively, the interactions might ensure AMA1 processing, and possibly disengagement of its receptor, a step necessary for moving inside the cell. In all such cases, an important contribution of the AMA1 tail might be not as a mechanical link but through signalling.

On 2014 Jun 24, Markus Meissner commented:

Comment by Markus Meissner, Isabelle Tardieux and Robert Menard:

In this study Lamarque et al., convincingly demonstrate a functional redundancy of AMA-like molecules that can partially complement for AMA1. Indeed they observe upregulation of these molecules in a mutant harbouring a gene disruption in ama1 and therefore validate our previous data (Bargieri et al., 2013). However, when it comes to the function of AMA1 (and AMA-like proteins) we disagree with the final conclusion of the authors for several reasons:

1. The authors conclude from an abortive invasion assay that in absence of AMA1 (but supposedly in presence of AMA2) parasites cannot remain attached to the host cell after secretion of the rhoptries because of the lack of a functional junction. These conclusions do not contradict the finding of a strong attachment phenotype and it remains to be seen what and when rhoptry secretion is triggered during attachment. One important control would have been to demonstrate that other (unrelated) attachment mutants (i.e. MIC2KO, Act1KO, etc.) do not show the same behaviour as AMA1KO in this assay. It is important to note that our own extensive analysis of host cell invasion events by AMA1KO parasites using real time imaging did not catch a scenario that could fit with this abortive invasion and would characterize the AMA1 deficient population. Therefore it is unknown at this point what causes the increased secretion of Rhop proteins into the host cell in absence of AMA1 or Ron2.

2. Using a tail mutation (FW>AA), the authors demonstrate that parasites remain attached, but do not invade, indicating an important role of the tail for invasion. It is important to note that this mutation has been described previously as disrupting the interaction to aldolase (Sheiner et al., 2010). However, a recent study (Shen et al., 2014) convincingly demonstrated that aldolase is not the crucial linker between micronemes and actin. Therefore in absence of knowledge of tail function the significance of this finding justifies several interpretations, such as signalling (indeed previous data demonstrate that the tail is phosphorylated during invasion) or simple structural consequences causing a dominant negative effect.

3. The demonstration that AMA1 is likely to be at the junction during invasion does not suggest a function of AMA1 during this process, since it could simply be a “waste” product after transmembrane processing. Indeed, processing mutants of AMA1 invade the host cell with slower kinetics (Parussini et al., 2012). Intriguingly, the authors did not make any attempts to characterise the kinetics of host cell entry of AMA1KO or AMA1KO/AMA2KO, which should be significantly reduced given the strength of the observed phenotype for AMA1KO/AMA2KO. In case of ama1KO we convincingly reported that the major effect is in host cell attachment and not downstream steps, such as host cell entry. How do the authors explain that there would be a full redundancy for downstream steps, but only a minor partial redundancy upstream?

We think that the data presented in this manuscript support a model, where AMA1/Ron2 interaction is important to stabilise parasite attachment to the host cell. AMA1 might then be involved in a signalling cascade that activates active entry into the host cell. Therefore, we suggest that AMA1 acts upstream of the actual entry event, as published previously (see Bargieri et al., 2013).

In summary, while AMA1/RON2 interaction is clearly important, its exact role remains unknown. A role in transducing force in the junction is not supported by available data. In agreement with a primary role of AMA1 in adhesion, AMA1 might further contribute to optimal positioning prior to entry by binding to RON2 in a formed junction. Alternatively, the interactions might ensure AMA1 processing, and possibly disengagement of its receptor, a step necessary for moving inside the cell. In all such cases, an important contribution of the AMA1 tail might be not as a mechanical link but through signalling.