Native structure of a type IV secretion system core complex essential for Legionella pathogenesis

Abstract

Bacterial type IV secretion systems are evolutionarily related to conjugation systems and play a pivotal role in infection by delivering numerous virulence factors into host cells. Using transmission electron microscopy, we report the native molecular structure of the core complex of the Dot/Icm type IV secretion system encoded by Legionella pneumophila, an intracellular human pathogen. The biochemically isolated core complex, composed of at least five proteins--DotC, DotD, DotF, DotG, and DotH--has a ring-shaped structure. Intriguingly, morphologically distinct premature complexes are formed in the absence of DotG or DotF. Our data suggest that DotG forms a central channel spanning inner and outer membranes. DotF, a component dispensable for type IV secretion, plays a role in efficient embedment of DotG into the functional core complex. These results highlight a common scheme for the biogenesis of transport machinery.

Keywords: assembly pathway; effector translocation; membrane proteins; nanomachine.

Fig. 1.

Ring-shaped structures on the surface of L. pneumophila strain Lp01. Osmotically shocked L. pneumophila cells were mounted on discharged carbon grids and stained with 1% uranyl acetate. Transmission electron micrographs of a wild-type cell (A) and an isogenic deletion mutant (ΔT4SS) cell lacking all dot/icm genes (B) are shown. Large and small rings are denoted by large and small white arrowheads, respectively. (Scale bar, 200 nm.) (C) Large and small ring structures found on the wild-type L. pneumophila surface. (Scale bar, 40 nm.)

Fig. 2.

Isolation of ring structures with a diameter of ∼40 nm. Ring structures were enriched as described in Materials and Methods, mounted on grids, and stained with 2% (wt/vol) PTA pH 7.0. Transmission electron micrographs of fractions isolated from wild-type cells (A and C) and isogenic deletion mutant (ΔT4SS) cells lacking all dot/icm genes (B) are shown. (Scale bar, 200 nm.) (C) The fraction shown in A was further purified by a size exclusion column chromatography to remove small specks observed. (D) Isolated ring structures from wild-type cells. (Scale bar, 40 nm.) (E) Proteins visualized by silver staining of fractions during the isolation procedure. The procedure involves two consecutive ultracentrifugation processes (Materials and Methods). Starting lysate (whole cell) and pellet fractions after ultracentrifugations (cfg, 1/2 ppt) were analyzed. Five Dot/Icm proteins (arrowheads) were consistently enriched during the procedure. MOMP, the major outer membrane protein of L. pneumophila, is a major contaminant protein in this procedure and is used as an internal control for comparison.

Fig. 3.

Immunoelectron microscopy of the ring structures. (A and B) The ring structures isolated from the wild-type strain were stained with (A) affinity-purified anti-DotF antibody followed by a gold-conjugated secondary antibody or (B) the gold-conjugated secondary antibody only. (C and D) The ring structures isolated from (C) a strain producing DotD–M45 or (D) the isogenic strain producing wild-type DotD were stained with affinity-purified anti-M45 epitope antibody followed by a gold-conjugated secondary antibody. Structures were stained with 2% (wt/vol) PTA pH 7.0 and observed by transmission electron microscope.

Fig. 4.

Analysis of enriched fractions isolated from null mutant strains lacking indicated component protein(s). (A) Fractions isolated from designated L. pneumophila strains were analyzed by SDS–polyacrylamide gels. Core components DotC, DotD, DotH, DotF, and DotG were detected by Western immunoblotting using specific antibodies against indicated core component proteins. MOMP was detected by silver staining and used as an internal control for isolation yield. (B–F) Ring structures visualized by transmission electron microscopy analysis isolated from (B) wild-type, (C) ΔdotG, (D and E) ΔdotF, and (F) ΔdotF ΔdotG strains. In B–D and F, small and large white arrows denote the wild-type and the ΔdotG-type ring structures with small and large pores, respectively. In E, two distinct kinds of complexes isolated from the ΔdotF strain are shown. (G) Schematic cartoons illustrating the observed ring structures isolated from wild-type, ΔdotG, and ΔdotF strains. Internal and external diameters of the complexes are presented as the mean ± SEM. Asterisks denote significant difference (P < 0.001) by z-test or Student t test. ns, not significant.

Fig. 5.

DotF is not an essential component for the type IV secretion. (A) Intracellular growth assay of L. pneumophila wild-type (filled squares), ΔdotF (triangles; dashed line), and ΔdotA (open squares) strains within human U937 monocyte-like cells. (B) Levels of designated effector proteins translocated into Chinese hamster ovary (CHO) cells were measured by the cya reporter assay. CHO cells challenged by L. pneumophila wild-type (black bars), ΔdotF (gray bars), and ΔdotA (shaded bars) strains were extracted and cAMP levels were determined.

Fig. 6.

(A) Relationship between selected components of the type II secretion system (T2SS), the Dot/Icm T4SS, and the pKM101 conjugation system. Sequence-level and higher order similarities are shown by gray and cyan shades, respectively. Core components are boxed. See text for further explanation. (B) A model describing the assembly of the Dot/Icm T4SS core complex. Shaded complexes are active in the type IV secretion. In the absence of DotF, the outer-membrane subcomplex (DotCDH) can form a functionally active subcomplex containing DotG (DotCDHG) at low efficiency. DotF facilitates the integration process of DotG into the outer-membrane subcomplex by acting as an intracomplex chaperone, resulting in robust formation of the active core complex (DotCDHFG).