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Rab11A regulates the constitutive secretory pathway during Toxoplasma gondii invasion of host cells and parasite replication

Venugopal Kannan, Chehade Sylia, Werkmeister Elisabeth, Barois Nicolas, Periz Javier, Lafont Frank, Tardieux Isabelle, Khalife Jamal, Gordon Langsley, Meissner Markus, Marion Sabrina

Preprint posted on September 25, 2019 https://www.biorxiv.org/content/biorxiv/early/2019/09/25/782391

Rab11A: a central player in the T. gondii constitutive secretory pathway, and in vital steps of the parasites’ journey into the target host cell.

Selected by Mariana De Niz

Categories: cell biology, microbiology

Background

Toxoplasma gondii is an obligate intracellular parasite of the genus Apicomplexa that is thought to affect 25-30% of the world’s human population (1). Beyond its clinical importance, T. gondii has an extraordinary capacity to infect any warm-blooded vertebrate host (2). Although T. gondii infections generally develop into a harmless latent form in most infected humans, in immunocompromised patients reactivation of a latent infection can be lethal. In addition to its relevance for public health, T. gondii has several features inherent to its biology, that have made it an important model organism to investigate intracellular host-pathogen interactions.

A research topic of great interest in the T. gondii field of work, is the investigation of secretory organelles, and the role they play in different stages of the parasite’s life cycle. Secretory organelles where virulence factors are stored include micronemes, rhoptries, and dense granules (Figure 1). Effective invasion of and egress from host cells relies on the temporally and spatially coordinated exocytosis of these secretory organelles. While the molecular mechanisms triggering rhoptry and microneme release have been well studied, (Reviewed in 3-5) dense granule secretion remains a poorly explored aspect of T. gondii vesicular trafficking. However, dense granules are known to play a crucial role in T. gondii development and survival.

In mammalian cells, the constitutive exocytic route supports sorting of newly synthesized proteins from the endoplasmic reticulum, through the Golgi, to the plasma membrane. In T. gondii, dense granules are considered to be the default constitutive secretory pathway. In their work, Venugopal et al (6) investigated the role of the small GTPase Rab11A in dense granule exocytosis, and its impact in key events in T. gondii entry to its target host cell, including adhesion, motility, and invasion (Figure 1).

 

Key findings and developments

General findings

  • Rab11A regulates the constitutive secretory and recycling pathways, thus controlling secretion at the plasma membrane. Venugopal et al revealed an essential role of gondii Rab11A in:
    • Promoting the cytoskeleton driven transport of dense granules, and the release of their content into the vacuolar space.
    • Regulation of transmembrane protein trafficking and localization during parasite replication.
    • Extracellular parasite motility and adhesion to host cells.

 

Novel tools generated in this study

  • This study utilizes various tools to study Rab11A temporal and spatial distribution during the gondii life cycle, namely:
    • A polyclonal antibody in mice against Rab11A (newly developed).
    • A reporter Rab11A-mCherry parasite line which allows regulation of recombinant Rab11A levels using Shield-1.
    • Two reporter lines co-expressing SAG1DGPI-GFP (the major gondii surface antigen truncated of its GPI anchor, which accumulates in dense granules), and mCherry-Rab11A (WT and DN) to explore dense granule dynamics in relation to Rab11A.
    • A mathematical model of directed or diffusive motion to analyse vesicle dynamics.
    • Transiently transfected Rab11A-WT and Rab11A-DN parasites with plasmids encoding the transmembrane HA-tagged glucose transporter 1 (GT1) or the Ty-tagged rhomboid protease 4 (ROM4).

 

Specific findings: Rab11A plays a role in dense granule exocytosis and transmembrane protein localization at the parasite plasma membrane

  • Live microscopy in combination with various parasite markers for the actin cytoskeleton or the inner membrane complex led to several important conclusions (summarised in Figure 1) including:
    • Rab11A participates in cargo transport between the apical and basal poles of the parasites and vesicular budding from the Golgi/ELC compartments.
    • Rab11A-dependent vesicular transport depends on the actin cytoskeleton
    • Rab11A may regulate actin-dependent material exchanges between parasites.
  • Live imaging also showed that Rab11A-positive vesicles dynamically co-distribute with dense granules in replicating tachyzoites.
  • Analysis of dense granule trajectories in Rab11A-WT and Rab11A-DN parasites suggested a role for Rab11A in regulating dense granule directed transport along the parasite cortical cytoskeleton. Rapid restoration of Rab11A functions by washing out Shield-1 and therefore the expression of Rab11A-DN suggested that Rab11A is also required for the steps of dense granule docking/tethering at the parasite plasma membrane.
  • Accordingly, Rab11A-DN parasites display a drastic block in the release of several dense granule (GRA) proteins into the vacuolar space.
  • The authors propose that Rab11A plays a broad role in exocytosis, and is involved in separate regulatory pathways: on one hand for the trafficking of proteins to the parasite plasma membrane; on the other hand, for the release of dense granule proteins into the vacuolar space during parasite replication.
Figure 1. Rab11a involvement in the constitutive secretory pathway during T. gondii replication.

Specific findings: Rab11A plays a role in host cell adhesion, motility and invasion

  • A role for Rab11A in parasite entry into the host cell has been previously demonstrated. In the present study the authors investigated which steps of parasite entry (i.e. adhesion, motility, and invasion) were altered in Rab11A-DN parasites. They demonstrated that Rab11A-DN tachyzoites showed impaired attachment to human fibroblasts, as well as impaired motility correlated with altered morphology compared to their Rab11A-WT counterparts. However, despite the defects in adhesion and motility, Rab11A-DN parasites showed only mild defects in host cell invasion, and were able to successfully form a moving junction.
  • Rab11A-DN parasites show impaired MIC2 secretion upon induction of microneme exocytosis by ethanol. The authors demonstrated that this was not the result of a defect in MIC2 protein synthesis or MIC2-positive microneme apical localisation.
  • The authors went on to analyse Rab11A dynamic localization in extracellular motile parasites. They showed that Rab11A regulates the apically polarized accumulation of dense granules during the early steps of parasite adhesion and entry into host cells.

 

What I like about this paper

I found this an exciting piece, because it investigates a topic that seems relatively understudied in T. gondii, and the authors do this in a very elegant and step-wise manner. Equally, this study incorporates many modern tools to effectively answer relevant cell biological questions. I found the findings on Rab11A exciting and a promising topic for future work in this field. Furthermore, the research presented in this pre-print complements, and is coherent with, various recent and novel studies in T. gondii, successfully fitting a missing piece of a complex puzzle.

 

Open questions

*Note: questions with answers by authors are found at the bottom section of this highlight.

  1. You found in your work novel roles for Rab11A in T. gondii dense granule secretion, as well as parasite adhesion to the host cell, and motility. A very general question I have is that the previous study focusing specifically on Rab11A in T. gondii was published over a decade ago (7), with already promising findings and follow-up questions. In your work you are now addressing multiple roles of Rab11A. I am curious as to why the role of Rab11A in T. gondii seems to not have been a focus of research for a long time?
  2. Also a general question: you and others state that while the mechanisms of secretion for rhoptries and micronemes is well studied, constitutive secretion in T. gondii is a relatively understudied topic. Rab11A is only one member of this secretory pathway, and you have already shown exciting biology with relevance to pathology. Are you envisaging to address other members of the secretory pathway (eg. other Rabs or SNAREs), hypothesizing they might play equally important roles?
  3. You showed in your work that Rab11A dynamics suggest polarized transport of de novo synthesized material during daughter cell emergence and extracellular motility. Do you have a hypothesis on what the material being transported might be in each case, and whether it might be a different cargo specific to each of the processes?
  4. From the previous question, are you interested in investigating potential interactions between host and parasite organelles? Do you think this could shed further insights into the T. gondii constitutive secretory pathway and its effect and interactions with the target host cell?
  5. Given your findings, you suggest that Rab11A may contribute to the regulation of the actin network function and dynamics. Actin has been the focus of various labs, and has been shown to be key for multiple aspects of T. gondii biology. If Rab11A plays an important role in actin regulation, do you hypothesize this might expand the relevance of this Rab protein beyond the one you have studied/revealed in your work?
  6. You briefly mention a mode of cellular transport called hitchhiking, as a mechanism controlling organelle movement. Can you expand a bit further on this mechanism, specific to your findings and relevance in parasitology?
  7. Out of curiosity, do you also envisage studying membrane contact sites between T. gondii organelles and between T. gondii and the target host cell to further explore the role of Rab11A and the constitutive secretory pathway?

References

  1. Robert-Gangneux F., and Darde ML, Epidemiology of and diagnostic strategies for toxoplasmosis, Clin Microb Rev,2012, doi: 10.1128/CMR.05013-11.
  2. Dupont CD, Christian DA, and Hunter CA, Immune response and immunopathology during toxoplasmosis, Semin Immunopathol., 2012, doi: 10.1007/s00281-012-0339-3.
  3. Bullen HE, Bisio H, Soldati-Favre D, The triumvirate of signalling molecules controlling Toxoplasma microneme exocytosis: cyclic GMP, calcium, and phosphatidic acid, PloS Pathogens, 2019, doi: 10.1371/journal.ppat.1007670.
  4. Frenal K, Dubremetz JF, Lebrun M, Soldati-Favre D, Gliding motility powers invasion and egress in Apicomplexa, Nat Rev Microbiol, 2017, doi: 10.1038/nmicro.2017.86
  5. Dubremetz JF, Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction, Cell Microb, 2007, doi: 10.1111/j.1462-5822.2007.00909.x
  6. Venugopal K., et al., Rab11a regulates the constitutive secretory pathway during Toxoplasma gondii invasion of host cells and parasite replication, bioRxiv, 2019, doi:10.1101/782391.
  7. Agop-Nersesian C, et al., Rab11a-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis, Plos Pathogens, 2009, e1000270. doi:10.1371/journal.ppat.1000270

Acknowledgements

I thank Dr. Sabrina Marion and Dr. Kannan Venugopal for their engagement, time and input on this highlight, and for providing helpful insight and scientific discussions about their preprint. I thank also Mate Palfy for helpful comments on this highlight.

Tags: #toxoplasma, rab11a

Posted on: 18th December 2019

doi: https://doi.org/10.1242/prelights.15727

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  • Author's response

    Sabrina Marion and Kannan Venugopal shared

    1.You found in your work novel roles for Rab11A in T. gondii dense granule secretion, as well as parasite adhesion to the host cell, and motility. A very general question I have is that the previous study focusing specifically on Rab11A in T. gondii was published over a decade ago (7), with already promising findings and follow-up questions. In your work you are now addressing multiple roles of Rab11A. I am curious as to why the role of Rab11A in T. gondii seems to not have been a focus of research for a long time?

    In my opinion the toxoplasma research community has a limited number of research groups that work on hardcore protein trafficking. The ever expanding list of unknown / “hypothetical” protein trafficking pathway members that result from protein-protein interaction studies such as IPs and pull downs have traditionally drawn the interest of these research groups in dissecting the functional roles of “novel” protein molecules or complexes than digging deep into the functions of already existing ones. In addition, owing to the essential role of moving junction formation in triggering parasite invasion into host cells, past studies have focused on dissecting the mechanisms of rhoptry and microneme secretion. However, it is important to mention that important discoveries have been made on the mechanisms by which dense granules proteins are translocated through the vacuole limiting membrane towards the host cell cytosol. Also, recent advances in imaging tools such as super resolution microscopy available to dissect fine details of protein localisation and dynamics within any given cell type, and the respective molecular markers and transgenic parasite lines enabling co-localisation, has aided filling the gaps and addressing previously unanswered questions.

    2. Also a general question: you and others state that while the mechanisms of secretion for rhoptries and micronemes is well studied, constitutive secretion in T. gondii is a relatively understudied topic. Rab11A is only one member of this secretory pathway, and you have already shown exciting biology with relevance to pathology. Are you envisaging to address other members of the secretory pathway (eg. other Rabs or SNAREs), hypothesizing they might play equally important roles?

    Yes indeed we do plan to follow up by studying the functional roles of other components of this secretory pathway that we identified to interact with Rab11A (in the GDP as well as the activated GTP bound states) by means of IP and GST pull down experiments. We did fish out SNAREs and potential regulatory factors of exocytosis, which we intend to work upon in the future.

    3. You showed in your work that Rab11A dynamics suggest polarized transport of de novo synthesized material during daughter cell emergence and extracellular motility. Do you have a hypothesis on what the material being transported might be in each case, and whether it might be a different cargo specific to each of the processes?

    Indeed, while the material being transported during daugther cell emergence may be related to building the new daughter cell scaffold made out of components of the IMC and the glideosome, the cargos apically transported in extracellular parasites may control parasite motility or prepare the parasite for its journey into the host cell by anticipating the burst of dense granule protein secretion that occurs upon entry. However, we believe that the mechanisms regulating Rab11A-positive vesicle anchoring at the apical pole may be similar during daughter cell division and in extracellular parasites and may involve components of the conoid.

    4. From the previous question, are you interested in investigating potential interactions between host and parasite organelles? Do you think this could shed further insights into the T. gondii constitutive secretory pathway and its effect and interactions with the target host cell?

    Yes, we have started to exploit the major defect of the Rab11A DN parasite line in the release of important parasite effectors to better understand host cell manipulation by T. gondii and discover new modulated pathways.

    5. Given your findings, you suggest that Rab11A may contribute to the regulation of the actin network function and dynamics. Actin has been the focus of various labs, and has been shown to be key for multiple aspects of T. gondii biology. If Rab11A plays an important role in actin regulation, do you hypothesize this might expand the relevance of this Rab protein beyond the one you have studied/revealed in your work?

    Rab11A seems to be tightly connected with the actin cytoskeleton activity. While we demonstrated that Rab11A vesicle transport requires the actin cytoskeleton, we still need to investigate whether Rab11A regulates actin-dependent mechanisms, such as material exchanges between parasites and some key acto-myosin dependent steps of cytokinesis.

    6. You briefly mention a mode of cellular transport called hitchhiking, as a mechanism controlling organelle movement. Can you expand a bit further on this mechanism, specific to your findings and relevance in parasitology?

    This mode of transport has recently emerged as a novel mechanism to control organelle movement. During this process, the “hitchhiker” benefits from distinct molecular motors present at the surface of the “vehicle” and thereby is shuttled to particular cell locations that can be also defined by “trafficking signals” present at the surface of the vehicle. Co-movement of cargo may facilitate interactions at membrane contact sites important for organelle maturation or fusion. Further studies are now required to decipher whether this process is specific to dense granules or is employed for other vesicular transport events in the parasite. T. gondii expresses a limited number of Rabs compared to mammalian cells, thus, it is likely that each Rab may interact with numerous partners under specific regulatory signals and be involved in distinct trafficking pathways.

     

    7. Out of curiosity, do you also envisage studying membrane contact sites between T. gondii organelles and between T. gondii and the target host cell to further explore the role of Rab11a and the constitutive secretory pathway?                         

    In mammalian cells, Rab11A has been shown to interact with the exocyst complex and SNARE proteins promoting vesicle docking and fusion. Likewise, we would like to develop imaging tools allowing the direct visualization of cargo release (vesicle fusion) after the dense granule has docked to the plasma membrane of the parasite and thereby assess the putative role of Rab11A in this process. Some tools have been developed in mammalian cells to vizualise exocytic events but quite some work has to be invested now to adapt them to replicating parasites or moving extracellular parasites.  Indeed, one can imagine that such tools could be also used to further study rhoptry contact with the host cell plasma membrane and the subsequent step of fusion and rhoptry protein release.

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