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. 2010 Apr 1;6(4):e1000824.
doi: 10.1371/journal.ppat.1000824.

Topology and Organization of the Salmonella Typhimurium Type III Secretion Needle Complex Components

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Free PMC article

Topology and Organization of the Salmonella Typhimurium Type III Secretion Needle Complex Components

Oliver Schraidt et al. PLoS Pathog. .
Free PMC article

Abstract

The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. InvG forms the outer rings and neck region of the needle complex.
(A) Cut-away view and description of individual substructures of the needle complex from S. typhimurium. (OR outer ring; IR inner ring) Bar = 10nm. (B–F) Analysis of complexes obtained from a S. typhimuirum ΔinvG strain: Western blot analysis (B), cryo electron microscopy images (C), and class averages of non-tilted complexes, (D, E) isolated from wild type and ΔinvG mutant S. typhuimurium strains, respectively. (F) Density difference between averaged images of wild type needle complexes and ΔinvG mutant complexes (1F = 1D-1E), indicates the position of the outer ring substructure.
Figure 2
Figure 2. The N- and C-terminus of PrgH are located far away from each other within the needle complex.
(A) N- and C-terminally poly-histidine tagged PrgHs are functional. Culture supernatants of a wild type S. typhimurium (w.t.), PrgH-deficient (ΔprgH), and mutant strains encoding either N- (his-PrgH) or C-terminally (PrgH-his) poly-histidine tagged PrgH were analyzed for the presence of the type III secreted proteins SipB and SptP by Western immunoblot. (C: whole cell lysates; S: culture supernatants). (B, E) Representative class averages obtained by single particle analysis of cryo electron microscopy images of Ni-NTA-labeled needle complexes derived from strains expressing either N-terminally (B) or C-terminally (E) tagged PrgH. (C, F) The total class averages (average of all particles) from the respective data set are shown in (C) (N-terminally labeled PrgH) and (F) (C-terminally labeled PrgH). The diffuse appearance of density at the basal side in (C) indicates that the Ni-NTA-nanogold (Au) label is present in various positions below IR2, but is more restricted above IR1 in C-terminally labeled complexes (F) (IR1 and IR2 = inner ring 1 and 2). (D, G) Density difference between the total averages of the labeled particles and unlabeled wild type complexes (panel D: w.t. needle complexes subtracted from labeled N-terminally tagged complexes (2D = 2C-1D); panel G: w.t. needle complexes subtracted from labeled C-terminally tagged complexes (2G = 2F-1D)).
Figure 3
Figure 3. The C-terminus of PrgK is located at the basal (cytoplasmic) side of the needle complex.
(A) Carboxy terminally poly-histidine tagged PrgK is functional. Culture supernatants of wild type S. typhimurium (w.t.), PrgK-deficient (ΔprgK), and a mutant strain encoding C-terminally (PrgK-his) poly-histidine tagged PrgK were analyzed for the presence of the type III secreted proteins SipB and SptP by Western immunoblot. (C: whole cell lysates; S: culture supernatants). (B) Representative class averages obtained by single particle analysis of cryo electron microscopy images of Ni-NTA-labeled needle complexes derived from strains expressing C-terminally poly-histidine tagged PrgK. (C) Total class average of Ni-NTA-labeled needle complexes obtained from a S. typhimurium strain expressing C-terminally poly-histidine tagged PrgK. The location of the density observed at the basal side (Au) is similar to the diffuse density observed in Ni-NTA-labeled needle complexes obtained from strains expressing N-terminally poly-histidine tagged PrgH (see Fig. 2C ) (IR1 and IR2 = inner ring 1 and 2). (D) Resulting difference in density after subtraction of w.t. needle complexes from labeled C-terminally poly-histidine tagged PrgK complexes (3D = 3C-1D). (E) Subtraction of the total averages of labeled complexes obtained from a strain expressing N-terminally-tagged PrgH from a strain expressing C-terminally-tagged PrgK (3E = 3D-2C).
Figure 4
Figure 4. Organization of PrgH and PrgK within the lower ring of the needle complex.
(A) Insertion of a poly-histidine linker at amino acid 267 of PrgH (267his) or removal of four amino acids from its C-terminus (Δ4) does not alter its function. Culture supernatants of wild type S. typhimurium (w.t.), PrgH-deficient (ΔprgH), and mutant strains encoding a poly-histidine linker at amino acid 267 of PrgH (267his) or a PrgH lacking the terminal four amino acids (Δ4) were analyzed for the presence of the type III secreted proteins SipB and SptP by Western immunoblot. (C: whole cell lysates; S: culture supernatants). (B) Representative class average obtained by single particle analysis of cryo electron microscopy image of Ni-NTA-labeled needle complexes obtained from a S. typhimurium strain expressing PrgH with a poly-histidine tag inserted at amino acid 267. A prominent gold label (Au) is seen at the widest side of the periplasmic face of the IR1 (IR1 and IR2 = inner ring 1 and 2). Bar = 10 nm. (Additional results of the analysis are shown in Fig. S4). (C) Resulting difference in density after subtraction of unlabeled w.t. needle complexes from labeled complexes isolated from strains expressing a poly-histidine insertion following amino acid 267 in PrgH. (D) Mutant needle complexes carrying truncated PrgH can be selectively disassembled into larger and smaller rings by shifting pH to 10.5 and subsequent negative staining, whereas wild type (w.t.) needle complexes maintain the integrity of the base. (E) En face class-average derived from single particle analysis from negatively stained electron microscopy images of inner rings substructures. The substructures were obtained by selective disassembly of needle complexes isolated from a mutant strain encoding for a C-terminally, four amino acid truncated PrgH. The ring substructure is organized in two larger concentric rings with different diameters (∼180Å and ∼250Å). Bar = 10nm. Rotational cross-correlation analysis revealed that the maximum of the cross-correlation peak is repeatedly obtained every 15°, demonstrating that the larger concentric rings of the inner ring structure from the prgHΔ4 mutant strain exhibit 24 fold symmetry. (F) Surface accessibility of lysine K168 is increased in complexes obtained from a S. typhimurium ΔinvG strain. Primary amines of wild type and ΔinvG mutant complexes were acetylated with Sulfo-NHS-Acetate and the ratio of modified to non-modified peptides was determined by mass spectrometry. Each bar represents the ratio of acetylation of lysines between specific PrgK peptides obtained from the ΔinvG mutant and the same peptides obtained from a wild type complex (Table S1). The ratio of peptide acetylation is an average of two independent mass spectrometry measurements. (G) Lysine 168 is surface exposed in modeled PrgK rings. A monomeric PrgK structure was modeled using EscJ as a template and protein-protein contacts present in the EscJ crystal structure were used to form a PrgK ring (top and side view). Lysine 168 is located on top of the ring and its side group nitrogen is surface exposed as highlighted in blue in the surface view of the cut-out segments of individual monomers. The cut-out segment shows two PrgK molecules in surface representation (yellow and light-yellow) followed by one neighboring molecule on each side but displayed in ribbon style.
Figure 5
Figure 5. Domain interactions and relative orientations of needle complex components.
(A) Proximity of specific domains of the base proteins, InvG, PrgH, and PrgK within the needle complex. The block diagrams shows the three major base proteins, InvG, PrgH, and PrgK and covalent cross-links of peptides obtained from chemically derivatized needle complexes at primary amino groups. Amino acid position are indicated for the full length proteins prior signal peptide cleavage (processed InvG starts at Ser-25, and processed PrgK starts at Cys-18 [6]) protein-cross-links found are indicated with amino acid position and with crossing lines between proteins. While the position of non-derivatized lysines (presumably due to lack of surface exposure) is shown as vertical lines within the block diagram, the positions of derivatized lysines (surface accessible) is indicated as vertical lines extending from the block diagram (Table S2). Note that lysines within the N-terminal domain of PrgK are not derivatized, suggesting that the majority of PrgK within fully assembled needle complexes is not surface exposed. (B–E) Topographic model of the needle complex: Localization of InvG, PrgH, and PrgH within the base of the needle complex. The N-terminal domain of InvG (blue-grey) reaches far down into the neck region and is in close contact with the C-terminal domain of PrgH (white and grey), which resembles the larger of the two concentric rings. Insertion of a poly-histidine tag between amino acid 267 (yellow) and 268 (orange) and subsequent Ni-NTA nanogold labeling further determines the position of this domain within the complex. Sites of interaction found by cross-linking and mass spectrometry, for which in the case of PrgH and EscJ/PrgK no atomic structure is available, are labeled as red dots. The N-terminal domain of PrgH is pointing to the cytoplasmic side of the complex, and interacts with the C-terminal domain of PrgK. For both, no high resolution structure is available as of yet. The N-terminal domain of PrgK is located within the complex and is therefore packed into its position by PrgH from the side and InvG from the top. (C) Top and side view of the modeled PrgH (white/grey alternating) and PrgK (yellow/bright yellow alternating), as well as sites accessible for nanogold labeling (267/268) and chemical derivatization (K168). (D) Proposed relative position of protein domains from PrgH, InvG, and PrgK. The side group nitrogen of K38 (InvG) and K168 (PrgK) are highlighted in blue. (E) Top-view of three PrgK and PrgH monomers extracted from the modeled inner ring structure highlighting sites for chemical derivatization (PrgK (K168, blue)) and Ni-NTA nanogold labeling of a poly-histidine insertion at position 267/268 (yellow/orange) within PrgH.

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