Abstract
Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time–lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif–tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.
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Acknowledgements
We thank C. Parsot for reagents and helpful discussions, and we acknowledge all other members of P. Sansonetti's laboratory for their support. Furthermore, we thank P. Roux, M. Marchand and S. Shorte at the “Plate-forme d'Imagerie Dynamique” (PFID) at the Institut Pasteur for invaluable experimental help, discussion and advice on data processing. Finally, we are grateful to M. Mavris and J. Rohde for critically reading the manuscript, and to K. Schauer for help with the figure layout. P.S. is a Howard Hughes International Scholar, and J.E. received successive fellowships from the Fondation pour la Recherche Médicale (FRM), the European Molecular Biology Organization (EMBO) and the Human Frontiers Science Program (HFSP).
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Supplementary information
Supplementary Fig. 1
Fusion between IpaC and EGFP prevents the secretion of the resulting protein chimera and of other type III effectors. (PDF 519 kb)
Supplementary Fig. 2
Fluorescent labelling of bacterial proteins with biarsenic components. (PDF 578 kb)
Supplementary Fig. 3
Fluorescent labelling of intrabacterial IpaB–4Cys and IpaC–4Cys is dependent on the FlAsH concentration. (PDF 789 kb)
Supplementary Fig. 4
IpaB–4Cys or IpaC–4Cys are secreted with similar efficiencies independently of FlAsH-incorporation. (PDF 961 kb)
Supplementary Fig. 5
Ethanedithiol (EDT) negatively affects T3S mediated cellular invasion by Shigella flexneri. (PDF 414 kb)
Supplementary Fig. 6
The appearance of actin foci formation occurs with similar kinetics for M90T, ipaB/IpaB–4Cys, and ipaC/IpaC–4Cys. (PDF 221 kb)
Supplementary Video 1
Time lapse of IpaB secretion. (MOV 616 kb)
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Enninga, J., Mounier, J., Sansonetti, P. et al. Secretion of type III effectors into host cells in real time. Nat Methods 2, 959–965 (2005). https://doi.org/10.1038/nmeth804
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DOI: https://doi.org/10.1038/nmeth804
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