Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS

Abstract

Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.

Figure 1.. Overall structure of the Dot/Icm T4BSS.

Central tomographic slices through the (A) wild-type and (B) DotF-sfGFP sub-tomogram averages, showing the improved resolution of the DotF-sfGFP structure. Note that because Dot/Icm particles are flexible, all the sub-tomograms going into the average were first aligned on the outer membrane (OM)-associated densities and then on the inner membrane (IM)-associated densities, separately; the image shown is a composite of the two averages concatenated at the yellow line. Number of tomograms and particles used for the subtomogram average are listed in Supplementary Information Table 1. C) Schematic of the major densities in the structure, named for reference. D) Tomographic slices through individual particles showing top views. E) Rotational cross-correlation coefficients of the three top-view particles for symmetries from 8- to 18-fold, showing that 13-fold was the strongest in each case. Y-axis: normalized rotational cross correlation, X-axis: rotational symmetry. F) Applying 13-fold rotational symmetry at the levels of the red and blue lines in the DotF-sfGFP average produced clear structures (other symmetries failed to produce regular density patterns). For scale, the diameters of the red and blue circles are both ~32 nm. Scale bar 10 nm (A,B,F), 20 nm (D).

Figure 2.. Mutant structures, difference maps and architectural model of the Dot/Icm T4BSS.

Top rows (A-F, M-Q): Central slices through the sub-tomogram average structures of each strain imaged. Bottom rows (H-L, R-V): Central slices through the difference maps comparing each average to the wild-type. Yellow represents missing densities and red extra densities. Weak to strong intensities correspond to density differences from one to three standard deviations, respectively, overlaid on the mutant sub-tomogram average. Note missing densities (e.g. panels K and S), additional densities (e.g. panels J and R) or matched yellow/red pairs in an average (e.g. panel L), which likely indicate movement. Number of tomograms and particles used for each of the subtomogram average are listed in Supplementary Information Table 1. W) Based on the difference maps and evidence discussed in the text, known and predicted structures of T4BSS components are superimposed on the central slice of the DotF-sfGFP average (also see Supplementary Movie 1). The DotF-sfGFP average is generated by aligning the outer membrane (OM) and inner membrane (IM) regions separately. Note that the relative orientation of the component structures are not known – the purpose of this schematic is simply to show where in the T4BSS each component is located and how its size and shape compare to the ECT densities. Components whose structures are not known or confidently predictable are depicted as circles (e.g. DotC) or as the shape of densities seen in the sub-tomogram averages or difference maps (e.g. DotH and IcmX). Polypeptide links to the OM are shown as dotted lines. Sequences in DotG with unknown structure are shown as solid lines with speculative path. A large number of transmembrane helices are not shown for inner membrane proteins (DotA, DotE, DotL, DotM, DotP, DotU, DotV, IcmV, and IcmT). In addition, cytoplasmic components of the system are not shown. Currently, organization of the cytoplasmic complex is debated. OM = outer membrane, PG = peptidoglycan cell wall, IM = inner membrane. Lipids are shown in grey and peptidoglycan in brown. X) Three dimensional representation of the Dot/Icm complex showing windowed secretion chamber (salmon:DotH, white:DotD, green:DotK and cyan:DotC), wings (yellow:DotF) and secretion channel (red:DotG). Stoichiometry of DotF is unknown. Similar to (W), the cytoplasmic components are not shown. In this 3D representation, IcmF, IcmX and DotA are not visible. Scale bar 10 nm (A-V and W).

Figure 3.. Dot/Icm-dependent polar targeting of the Legionella core-transmembrane subcomplex.

A) Broth grown L. pneumophila cells were probed with primary antibodies (polyclonal for DotH, DotG, and DotF or HA monoclonal for DotD-HA and DotC-HA), decorated with secondary antibody conjugated with Oregon green and imaged with fluorescence microscopy. Samples assayed include the wild-type strain Lp02 (WT), a strain lacking all 27 dot/icm genes (SΔ), and the SΔ strain expressing individual core-transmembrane subcomplex components. B) DotH localization in wild-type Legionella and in individual dot/icm deletions. DotH was detected by immunofluorescence microscopy in wild-type cells (WT) and individual dot/icm mutant strains. The corresponding deletion strain is boxed in yellow and dot/icm deletions that are affected are boxed in red. Representative images are shown from three independent experiments. Scale bar 2 μm (A,B).

Fig 4.. DotU and IcmF localize to the bacterial poles in the absence of the Legionella T4SS.

(A) DotU and IcmF localization was assayed in the wild-type strain Lp02 (WT), ΔdotU ΔicmF mutant strain (JV1181), the super dot/icm deletion strain (SΔ, JV4044) and the SΔ strain expressing dotU and icmF from the chromosome (SΔ(UF)), JV5319). Shown are Dot staining (left) and DNA stained with DAPI (right) for each set. B) Localization of Dot proteins was assayed in the wild-type strain (WT), the SΔ strain encoding dotU and icmF (SΔ(UF)), the SΔ(UF) strain expressing individual components of the core-transmembrane subcomplex (SΔ(UF) + single), and the SΔ(UF) strain expressing all five core components (SΔ(UF) + core). Antibodies used for immunofluorescence are indicated to the left of the panels. Representative images are shown from three independent experiments. Scale bar 2 μm (A,B).

Figure 5.. Reconstitution of the core-transmembrane subcomplex in the SΔ(UF) strain.

(A-H) Combinations of the core-transmembrane subcomplex were expressed in the super dot/icm deletion strain encoding dotU and icmF (SΔ(UF)). Representative images for DotH and DotC-HA localization are shown (A and C, respectively). Proteins expressed is indicated by labels on the left and top of each panel and the protein localized by IFM is shown below the images. The percent of cells with polar localization was determined from three independent experiments (100 cells counted from each experiment) and are shown in panels B and D. All data are representative of 3 independent biological experiments (n=3). In (B) and (D), data are presented as means ± SEM with statistical differences compared to the WT strain by unpaired two-tailed Student’s t-test. Scale bar 2 μm (A,C). (E-H) DotH association with the outer membrane require the targeting factors DotU and IcmF. (E,F) Cells were fractionated by a combination of ultracentrifugation and Triton X-100 solubility, proteins were separated by SDS-PAGE and probed in westerns using DotH specific antibodies. (E) DotH localization was determined in the wild-type strain Lp02 (WT), dotA mutant Lp03, ΔdotC (JV3743), ΔdotD (JV3572), ΔdotU ΔicmF (JV1181), and ΔdotU ΔicmF + complementing clone (JV1199). (F) DotH localization was determined in the SΔ(UF) strain expressing DotH (JV5405), DotC/DotH (JV5458), DotD/DotH (JV5459), DotC/DotD/DotH (JV5460), the core DotC/DotD/DotF/DotG/DotH (JV5443) or the core expressed in the SΔ strain without UF (JV5442). Experiments were done in triplicate and representative images are shown. (G,H) Cells were similarly fractionated and probed in westerns using DotC and DotD-specific antibodies (G and H, respectively). Interaction of the lipoproteins with DotH was determined in the following strains: wild-type Lp02 (WT), SΔ(UF) + DotC (JV5469), SΔ(UF) + DotD (JV5470), SΔ(UF) + DotC/DotD (JV5471), SΔ(UF) strain + DotC/DotD/DotH (JV5460), SΔ(UF) + core (JV5463), and the SΔ strain + core (JV5442). Fractions are indicated at the top of the panels and include total proteins (T), soluble proteins (S), membrane proteins (M), Triton X-100 extractable proteins (E), and Triton X-100 non-extractable proteins (N). Experiments were done in triplicate and representative images are shown.

Figure 6.. Polar targeting and assembly of the Legionella core-transmembrane subcomplex by DotU and IcmF.

The process begins by localization of DotU/IcmF to the poles, which can occur in the absence of any other Dot/Icm protein. This is followed by the recruitment of both DotC and DotH (pathway on the top). At this stage, the lipoprotein DotC is stably inserted in the outer membrane via its lipid domain, whereas DotH remains soluble in the periplasm, associated with DotC and the periplasmic domain of IcmF. Next, DotD arrives at the poles and assists in and/or directly mediates the outer membrane association of DotH (step 2). After formation of the DotC/DotD/DotH subcomplex, DotF and DotG are brought to the poles, thus linking the inner and outer membranes (also see Supplementary Movie 1). DotF localization is strongly dependent on the presence of DotC, DotD, and DotH but improves by the presence of DotG. DotG appears to be able to target to the poles to some level on its own but localizes more efficiently in the presence of DotC/DotD/DotH or DotC/DotD/DotH/DotF. In an alternative pathway (bottom), DotG could arrive at the poles upon arrival of UF and then subsequently DotC, DotH and DotF would join to complete the assembly process.