Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli

Abstract

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.

FIG. 1.

Purification of the EPEC NCs. (A) Purification scheme for the EPEC NCs. Whole-cell lysate was prepared from an EPEC ΔespA/A strain grown in DMEM, and the lysate was clarified by centrifugation. The resulting supernatant fraction (sup fr.) was ultracentrifuged, and then a pellet fraction (ppt fr.) (NC fr. I) was recovered. NC fr. I was layered on a sucrose cushion and then ultracentrifuged. The resulting pellet fraction (NC fr. II) was subjected to CsCl density gradient centrifugation, and then 20 fractions (CsCl frs. 1 to 20) were collected from the top of the gradient. For more details, see Materials and Methods. (B) Electron micrographs of negatively stained EPEC NCs. NC frs. I and II and CsCl fractions 3, 7, 11, and 14 were negatively stained and observed by TEM. White arrows indicate the basal bodies of the EPEC NCs, and black arrows indicate ring structures that were presumed to be the BfpB-BfpG complexes (29). Bar, 250 nm.

FIG. 2.

Identification of EscC and EspA as components of the EPEC NC. (A) NC fr. I (4.5 μg), NC fr. II (0.3 μg), and CsCl fractions 1 to 20 (6 μl each), shown in Fig. 1, were analyzed by 12% SDS-PAGE followed by silver staining. The positions of marker proteins are shown on the left. The positions of p56/EscC, p24/EspA, p84/AceF, and p55/BfpB are shown on the right. (B) Western blot analysis of NC fr. I, NC fr. II, and the CsCl fractions. Samples corresponding to those shown in panel A were immunoblotted with anti-EscC (upper panel) or -EspA (lower panel) antibodies. (C) Distribution of EspA and EscC in the CsCl fractions. Relative amounts of EspA and EscC in the CsCl fractions were estimated from the band densities observed on the Western blots (panel B) by using the NIH Image program. Densities (g/cm³) of the fractions were determined by refractometry.

FIG. 3.

Supermolecular structures of the EPEC NCs. (A) Alignment of the EPEC and Shigella NCs detected in NC frs. I and CsCl fractions 3, 7, 11, and 14. Bar, 100 nm. (B) Presumed model of the EPEC NC core structure. The size of each structure is given in nanometers. (C) Distribution of needle length in the NCs, as detected in CsCl fractions 3, 7, 11, and 14. The densities (ρ, g/cm³) of the CsCl fractions are shown in each panel. The average needle lengths were determined by measuring those of 100 (frs. 3, 7, and 11) or 200 (fr. 14) particles. The values represent means ± standard deviations.

FIG. 4.

Localization of EspA and EscC in the EPEC NC. The highly purified NCs in CsCl fraction 14 (Fig. 1) were analyzed by immunoelectron microscopy. (A) EspA was detected with immunogold-labeled anti-EspA antibodies. Sheath-like structures coated with 6-nm gold particles were observed. (B) EscC was detected with immunogold-labeled anti-EscC antibodies. In contrast to EspA localization in the sheath, EscC was localized in the basal body of the EPEC NC. Bar, 100 nm.

FIG. 5.

Interactions between the putative components of the EPEC TTS apparatus. (A) Purified GST-Esc fusion proteins were analyzed by 12% SDS-PAGE followed by CBB staining. Arrowheads indicate presumed bands corresponding to the respective GST fusion proteins. Lane M indicates the size markers. (B) Interactions of Esc proteins in a GST pulldown assay. Whole-cell extracts from E. coli expressing EscD or EscF tagged with the V5 epitope (EscD-V5 or EscF-V5) were incubated with each agarose-immobilized GST-Esc protein. Proteins bound to the beads were resolved by 14% SDS-PAGE and then analyzed by Western blotting using anti-V5 monoclonal antibodies.

FIG. 6.

Effect of esc mutations on secretion of the Esp proteins and Tir effector. (A) Secreted-protein profiles of WT EPEC and various mutant strains. Bacteria were grown in DMEM, and the secreted proteins were resolved by 12% SDS-PAGE and stained with CBB. (B) Immunoblot analysis of Tir and EspA in the culture supernatants (Sup.) and whole-cell extracts (Cell). Anti-EspA and -Tir antibodies were used for immunodetection. Lanes M and WT indicate the size markers and secreted proteins prepared from the EPEC wild type, respectively.