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Supramolecular Structure of the Salmonella typhimurium Type III Protein Secretion System

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Science  24 Apr 1998:
Vol. 280, Issue 5363, pp. 602-605
DOI: 10.1126/science.280.5363.602

Abstract

The type III secretion system of Salmonella typhimuriumdirects the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

Several plant and animal bacterial pathogens have evolved a specialized protein secretion system, termed type III, to interact with host cells [reviewed in (1)]. Characteristics of this system are (i) the absence of a typical, cleavable, sec-dependent signal sequence in secreted substrates; (ii) the requirement of accessory proteins for secretion; (iii) the export of proteins through both the inner and outer bacterial membranes; and (iv) the requirement of activating signals to initiate secretion. Although most of the putative components of this system have been identified, little is known about their function or their organization in the bacterial envelope. Genetic analyses have established that these systems are both structurally and functionally conserved across bacterial species (2).

The human pathogen Salmonella typhimurium encodes two type III secretion systems, although only one of them, located at centisome 63 of its chromosome, appears to be expressed in vitro (3). This system directs the translocation of several bacterial proteins into the host cell (4), which activate host cell signaling pathways, leading to a variety of responses, such as reorganization of the actin cytoskeleton, cytokine production, and the induction of programmed cell death in macrophages (1). This system has also been associated with the assembly of invasomes, appendage-like structures that appear on the bacterial surface upon contact with host cells (5). Some of the putative components of the secretion apparatus share sequence homology with proteins of the flagellar export machinery, suggesting an evolutionary relation.

The similarity between type III secretion components and the flagellar export machinery prompted us to investigate whether the S. typhimurium cell envelope contains structures similar to those involved in flagellar assembly. A mutant strain with a deletion in theflhC gene and therefore lacking all flagellar proteins was osmotically shocked and examined by transmission electron microscopy (TEM). Complex structures resembling a needle (needle complex) were visualized on the cell surface (Fig. 1, A and B). The base of the structure is on the plane of the cytoplasmic membrane and extends to the outer membrane, where it is connected to a thinner structure, or needle, that projects outward. The dense layer around the proximal end of the needle suggests that this complex is attached to the outer membrane through specialized structures. A depression on the outer membrane was often seen in association with the insertion point of the needle complex (Fig. 1B). There were 10 to 100 needle complexes per cell, and these complexes were distinguishable from flagellar basal bodies of osmotically shocked wild-type S. typhimurium cells (Fig. 1D). Although the size of the base is similar to that of the flagellar basal body, the needle itself is much thinner than the flagellar filament. Salmonella typhimuriumstrains carrying mutations in any of 24 different flagellar genes (6) exhibited needle complexes in their envelopes, further demonstrating that these complexes are independent of flagella (7). In contrast, needle structures were absent fromS. typhimurium strains carrying mutations ininvG, prgH, or prgK (Fig. 1C) (7), which encode essential components of the invasion-associated type III secretion system (8,9).

Figure 1

Electron micrographs of osmotically shocked S. typhimurium strains. (A andB) Nonflagellated ΔflhC S. typhimuriumexhibits needle complexes on the bacterial envelope (open arrows). Note the depression at the insertion point of the needle complex (closed arrow). (C) An invasion-defective strain of S. typhimurium carrying a mutation in invG shows no evidence of needle complexes. (D) An S. typhimurium fliK mutant exhibits flagellar polyhook basal bodies that span the inner and outer membranes. TEM samples were prepared as in (20). Samples were negatively stained with 2% phosphotungstic acid (pH 7.0) and observed under a JEM-1200EXII transmission electron microscope (JEOL, Tokyo). Micrographs were taken at an accelerating voltage of 80 kV. Scale bar, 100 nm.

Needle complexes isolated by a CsCl density gradient (10) appear to have cylindrical symmetry because every particle lying on the TEM grid exhibited a similar shape (Fig.2). The base structure resembles the flagellar basal body (11) because it contains two upper (or outer) and two lower (or inner) rings. The lower rings, which interact with the cytoplasmic membrane, are 40 nm in diameter and 20 nm wide and appear to be close together. The upper rings are 20 nm in diameter and 18 nm wide and interact with the outer membrane and the peptidoglycan layer. The two upper rings are more widely separated than the two lower rings. The outermost ring was sometimes observed associated with fragments of the outer membrane, a phenomenon often seen in the L ring of the flagellar basal body (12) (Fig. 2C).

Figure 2

Needle complexes isolated from S. typhimurium ΔflhC. (A and B) Complexes obtained from an enriched fraction of the CsCl density gradient (10). (C) (Top) Needle complexes associated with the bacterial outer membrane through their outer rings. (Bottom) Needle complexes lacking the needle structure aggregating through their outer rings. Scale bar, 100 nm.

The needle structure itself is a stiff, straight tube, 80 nm long and 13 nm wide. The line across its length indicates that the stain solution penetrated into a hollow space in the center of the structure. Occasionally, needle structures were missing from the bases (Fig. 2C), in which case the bases tended to form aggregates through their rings, a phenomenon often observed with flagellar basal body preparations (13).

To identify the components of this supramolecular structure, we fractionated purified needle complexes on SDS-polyacrylamide gels and visualized the proteins by silver staining. Although the needle complex was present in several fractions of the CsCl gradient, it was consistently enriched in higher density regions that contained three major protein species of 62, 52, and 31 kD (Fig.3). Minor amounts of other proteins were also present but not consistently. To identify the proteins in the needle complexes, we blotted samples onto membranes, visualized the proteins by Coomassie brilliant blue staining, and determined their NH2-terminal sequence (14).

Figure 3

Components of the needle structure. Needle complexes from S. typhimurium ΔflhC were purified on CsCl density gradients (10), and the proteins were visualized by silver staining of a 12% SDS-polyacrylamide gel. HBB, purified flagellar hook and basal body [isolated from the wild-type strain SJW1103 and included for comparison purposes (20)].

Ten amino acids of the 62-kD polypeptide exactly matched the sequence of the S. typhimurium InvG protein, starting at Ser25 of the predicted sequence (15). InvG is an essential component of the centisome-63 type III secretion system (8) and shares sequence homology with secretins, proteins that organize into homomultimeric structures (16). InvG is also required for the assembly of the appendage-like invasomes (5). The predicted InvG protein contains 564 amino acids, a molecular mass of 62,275 daltons, and a cleavablesec-dependent signal sequence with a signal peptidase recognition sequence located next to Ser25, in close agreement with the observed size and sequence of the 62-kD polypeptide.

The sequence of nine residues from the NH2-terminus of the 52-kD polypeptide (15) was identical to the deduced sequence of PrgH, another component of the type III secretion machinery (9). PrgH has a deduced sequence of 392 amino acids with a predicted molecular mass of 44,459 daltons, and it contains a stretch of nonpolar residues, followed by a canonical lipoprotein-processing site. However, the first amino acid in the sequence of the 52-kD polypeptide corresponds to Met1 of PrgH, indicating that PrgH is not processed as predicted.

Five amino acids from the NH2-terminus of the 31-kD protein (15) exactly matched the predicted amino acid sequence of PrgK, starting at Cys18. This protein, also a component of the type III system, has a predicted molecular mass of 28,210 daltons and a canonical lipoprotein-processing site at position 18, consistent with the size and sequence of the 31-kD polypeptide (9). Proteins of 40 and 39 kD present in low amounts were identified by NH2-terminal sequence analysis as OmpC and OmpF, respectively; these proteins are major components of the outer membrane and therefore likely contaminants of the needle complex preparation.

To confirm that proteins from the type III system are components of the needle complex, we constructed an S. typhimurium strain expressing an epitope-tagged form of PrgH (17). This protein fully complemented a prgH null mutation and allowed bacterial entry into cultured epithelial cells, indicating that the epitope-tagged protein functions similarly to wild-type PrgH. The needle complex was isolated from this strain, labeled with a monoclonal antibody to the epitope tag, and examined by TEM. The antibody specifically decorated the base of the needle complex isolated from the strain expressing the epitope-tagged PrgH, further demonstrating that this protein is a component of this structure (Fig.4B). In contrast, the antibody did not label needle complex structures from S. typhimurium strains expressing wild-type PrgH (Fig. 4A).

Figure 4

Immunoelectron microscopy of needle complexes isolated from an S. typhimurium strain expressing (A) wild-type or (B) M45 epitope–tagged PrgH after staining with a monoclonal antibody to the M45 epitope. Immunoelectron microscopy was carried out as previously described (17). Note the cloud of electron-dense material representing the antibody molecules bound to the base of the structure (B). Scale bar, 100 nm.

Thus, components of the type III secretion machinery are organized in a supramolecular structure that appears to span both the inner and outer membranes of the S. typhimurium envelope. This hypothesis is supported by the following: (i) the structure was observed in the wild type but not in invG, prgH, or prgKmutant strains of S. typhimurium; (ii) InvG, PrgH, and PrgK were the most abundant proteins in highly purified preparations of the needle structure; and (iii) PrgH was identified as one of the components of the needle structure by immunoelectron microscopy. The architecture of the needle complex and the similarity between components of the flagellar export machinery and the type III secretion system provide strong support for the postulated common ancestry of these structures. In this context, the needle complex may be viewed as the functional equivalent of the flagellar basal body, serving as a channel through which the substrate proteins of the secretion apparatus cross the two bacterial membranes. Furthermore, the needle itself may be directly involved in the delivery of effector molecules into the host cell. Contact of S. typhimurium with cultured epithelial cells results in the transient assembly of invasomes on the surface of the bacteria (5), a process that requires the function of the invasion-associated type III secretion machinery. Although the components of the invasomes have not been defined, the needle complex may constitute the base of such structures. Proteins homologous to components of the needle complex are widely distributed among type III secretion systems in plant and animal pathogenic bacteria (1), and appendage-like structures whose assembly requires the function of this secretion system have also been observed in other bacterial pathogens (18). Therefore, it is likely that similar supramolecular structures are a common feature of all type III secretion systems.

  • * Present address: Structural Biology Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.

  • To whom correspondence should be addressed. E-mail: galan{at}asterix.bio.sunysb.edu

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