Oral Presentation 2014 International Biophysics Congress

Analysis of bacterial type III effector secretion mechanism based on the observation of rotation of type III secretion apparatus (#149)

Takashi Ohgita 1 , Naoki Hayashi 1 , Naomasa Gotoh 1 , Kentaro Kogure 1
  1. Kyoto Pharmaceutical University, Kyoto, Japan

Gram negative bacteria (e.g. Pseudomonas aeruginosa) inject effector proteins involved in infection and toxicity into host cells via needle-like type III secretion apparatus (T3SA). However, the mechanism of effector secretion through T3SA is unknown. T3SA shows high genetic and structural similarities to flagellum. The effector secretion via T3SA requires proton-motive force, like flagellar rotation. Based on such high similarities between T3SA and flagellum, we hypothesized that T3SA would generate driving-force for effector secretion by rotating its needle structure depending on proton-motive force.

To prove this hypothesis, we first constructed a novel observation system for T3SA rotation. In this system, streptavidin-coated fluorescent microbead was attached specifically to the tip of T3SA via strep tag II peptide. Therefore, T3SA rotation was expected to be observed as microbead rotation. By using this system, we succeeded in observing T3SA rotation in the effector secretion condition for the first time. The rotation was inhibited by a protonophore (Carbonyl cyanide-m-chlorophenyl hydrazone) which decreases proton-motive force1). Moreover, we found out that effector secretion was suppressed, when T3SA rotation was inhibited physicochemically by viscous polymers (e.g. polyethylene glycol 8000)2).

Consequently, in this study, we succeeded in observing T3SA rotation for the first time, and it was suggested that the proton-motive force dependent T3SA rotation plays an important role in effector secretion process. Based on this, we proposed “rotational-secretion model” as a novel hypothetical mechanism of effector secretion through the T3SA.

  1. Ohgita T. et al, FASEB J., 27, 2862-2872 (2013)
  2. Ohgita T., et al, Open Biol, 3, 130133 (2013)