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Assembly of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli

Tomoaki Ogino ,^1,3,, Ryuta Ohno,^1, Kachiko Sekiya,² Asaomi Kuwae,^1,3 Takeshi Matsuzawa,^1,3 Takashi Nonaka,^4, Hiroyuki Fukuda,⁴ Shinobu Imajoh-Ohmi,⁴ and Akio Abe^1,3*

Laboratory of Bacterial Infection, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan,¹ Laboratory of Electron Microscopy, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan,² The Kitasato Institute, Tokyo, Japan,³ Laboratory Center for Proteomics Research, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo, Japan⁴

Received 18 June 2005/ Accepted 3 February 2006

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

Present address: Department of Molecular Genetics/Section of Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.

These authors contributed equally to this work.

Present address: Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8, Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan.

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