Analysis of Bacterial Pilus Assembly by Shearing and Immunofluorescence Microscopy
Bacterial surface appendages of the type 4 pilus superfamily play diverse roles in adherence, aggregation, motility, signaling, and macromolecular transport. Here we describe two analytical approaches to study assembly of type 4 pili and of pseudopili produced by type 2 protein secretion systems: the shearing assay and immunofluorescence microscopy. These complementary antibody-based methods allow for semiquantitative analysis of fiber assembly. The shearing assay can be scaled up to yield crude extracts of pili that can be further analyzed by electron and atomic force microscopy or by mass spectrometry.
Key wordsType 4 pili Type 2 secretion pseudopili Pilus assembly Shearing assay Immuno-detection Tricine SDS-PAGE Immunofluorescence microscopy
The work in our group is funded by the Institut Pasteur, CNRS and ANR grant 14-CE09-0004. A.L.R. was funded by the Pasteur-Paris University PhD program. J.L.T. was funded by the ANR grant 14-CE09-0004 and by the NSERC postdoctoral fellowship. We thank Nadia Izadi-Pruneyre, Daniel Ladant and members of the NMR of Biomolecules and Biochemistry of Macromolecular Interactions Units for interest and support. We thank Servier Medical Art (http://www.servier.com/ Powerpoint-image-bank) as a source of drawings used in Fig. 1.
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