Analysis of Bacterial Pilus Assembly by Shearing and Immunofluorescence Microscopy

Areli Luna-Rico (1) , Jenny-Lee Thomassin (1) , Olivera Francetic (1)

1 Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions

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.

Keywords: Immuno-detection; Immunofluorescence microscopy; Pilus assembly; Shearing assay; Tricine SDS-PAGE; Type 2 secretion pseudopili

Domains: Life Sciences [q-bio]; Microbiology and Parasitology; Bacteriology

Areli Luna-Rico, Jenny-Lee Thomassin, Olivera Francetic. Analysis of Bacterial Pilus Assembly by Shearing and Immunofluorescence Microscopy. Springer. Methods in Molecular Biology, 1764, Humana Press, New York, NY, pp.291-305, 2018, Protein Complex Assembly, 978-1-4939-7758-1. ⟨10.1007/978-1-4939-7759-8_18⟩. ⟨pasteur-02559622⟩