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The V-Antigen of Yersinia Forms a Distinct Structure at the Tip of Injectisome Needles

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Science  28 Oct 2005:
Vol. 310, Issue 5748, pp. 674-676
DOI: 10.1126/science.1118476

Abstract

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.

Type III secretion (T3S) is commonly used by Gram-negative pathogenic bacteria to introduce effector proteins into target host cells (1). Yersinia pestis and Y. enterocolitica, causing bubonic plague and gastroenteritis respectively, share the same T3S system consisting of the Ysc (Yop secretion) injectisome, or “needle complex,” and the secreted Yop (Yersinia outer protein) effector proteins. Three translocator proteins, YopB, YopD, and LcrV, are necessary to deliver the effectors across the target cell membrane (25). LcrV is required for the correct assembly of the translocation pore formed by YopB and YopD in the membrane of the target cell (2, 6). LcrV (also known as V antigen) is a soluble protein important for virulence (7) and is a protective antigen against plague (8). Antibodies against LcrV prevent the formation of the translocation pore (6) and block the delivery of the effector Yops (9). The injectisome is composed of a basal body resembling that of the flagellum and a needle (10). The needle has a helical structure (11) and in Yersinia is formed by the 9.5-kD protein YscF (12, 13).

Transmission electron micrographs of the surface of Y. enterocolitica E40 bacteria suggested that the injectisome needle ends with a well-defined structure (fig. S1). To characterize this structure, we purified needles from multi-effector knockout bacteria (strain ΔHOPEMT) that had been incubated under either secretion-permissive or -nonpermissive conditions (14), then analyzed them by scanning transmission electron microscopy (STEM). A distinct “tip complex” was observed for the wild-type needles, comprising a head, a neck, and a base (Fig. 1A, arrow, and fig. S2A). The tip structure was the same in both cases, but more needles were produced under secretion-permissive conditions (15). The purified needle fraction from secreting bacteria was analyzed to determine the components of the tip complex (fig. S3A). LcrV, YopD, and the needle subunit YscF were found. Other proteins included flagellins, which are usual contaminants of needle preparations (13). Upon cross-linking of purified needles, products formed between YscF and LcrV, suggesting that the latter is a structural component of the needle (fig. S3B).

Fig. 1.

STEM images of negatively stained wild-type needles. (A) Characteristic tip complexes (arrow), comprising a head, a neck, and a base, of wild-type needles isolated from ΔHOPEMT bacteria grown in secretion-permissive (left) and -nonpermissive (right) conditions. (B) Needles formed by lcrV mutant bacteria (ΔHOPEMNVQ, left) and by the complemented mutant (ΔHOPEMNVQ LcrV, right). The needles + of lcrV mutant bacteria are distinctly pointed at one end (asterisk). The tip complex was restored by complementation of the lcrV mutation in trans. Scale bar, 20 nm.

The tip complex observed for wild-type needles was absent from needles prepared from bacteria deprived of LcrV (ΔHOPEMNVQ) (table S1) (16). Instead, this end of the needle was distinctly pointed (Fig. 1B, asterisk, and fig. S2B). The tip complex was restored after the mutation was complemented in trans with lcrV+ (Fig. 1B, right, and fig. S2B). Needles from single yopN or yopQ knockout bacteria were analyzed as controls and displayed the same tip complex as the wild-type needles (fig. S4). Thus, the formation of the tip complex involved LcrV but not YopN or YopQ.

Needles from a yopBD double mutant (15) were analyzed to exclude the possibility that YopD and, although not detected on the gels, the third translocator protein YopB were tip complex components. The appearance of the tip complex was unchanged (fig. S4).

When wild-type needles were incubated with affinity-purified polyclonal antibodies to LcrV, the latter specifically bound to the tip complex, and we observed many examples of two needles joined tip to tip by a single antibody (Fig. 2). No antibodies to LcrV attached to needles purified from the lcrV mutant strain (ΔHOPEMNVQ). Furthermore, antibodies directed against YopB or YopD did not bind to wild-type needles (17). In contrast, affinity-purified polyclonal antibodies against YscF bound to the needle end opposite the tip complex (fig. S5). Together, these results clearly indicate that LcrV forms the observed tip complex.

Fig. 2.

STEM images of wild-type needles incubated with antibodies to LcrV and negatively stained. The antibodies generally attached to the head domain of the tip complex. The small central panels show individual antibodies. Scale bar, 20 nm.

Pseudomonas aeruginosa and Aeromonas salmonicida possess an injectisome closely related to that of Yersinia. Their respective LcrV orthologs, PcrV (32.3 kD) and AcrV (40.2 kD), are different in size to LcrV (37.2 kD). The pcrV+ and acrV+ genes were used to complement the lcrV deletion in Y. enterocolitica E40 (ΔHOPEMNVQ). The recombinant bacteria could assemble translocation pores. Their needles contained proteins with the size of PcrV and AcrV (fig. S6) and exhibited distinct tip complexes (Fig. 3). The head and neck domains of the tip complex formed by PcrV (Fig. 3A, center) were similar to those formed by LcrV, but the base was narrower (fig. S7). The tip complex formed by AcrV was larger (Fig. 3A, right, and fig. S7), more variable in shape, and more fragile, being absent or altered for many needles. This is reflected by the lower resolution of the AcrV average. In all three cases, a central channel seemed to permeate both the needle and the tip complex (Fig. 3B and fig. S7).

Fig. 3.

The tip structures of ΔHOPENMVQ bacteria complemented with LcrV or its orthologs PcrV and AcrV, imaged by STEM. (A) Projection averages (top) and typical single images (bottom) of the tip complexes formed by LcrV (left; resolution 1.5 nm), PcrV (center; resolution 1.5 nm), and AcrV (right; resolution 2.5 nm). A central channel seems to permeate both the needle and the tip complex. The PcrV tip complex is similar to the LcrV tip complex but has a smaller base. Tip structures formed by AcrV (right) were more variable and larger than those made of LcrV. (B) Profiles from the LcrV tip complex average at the locations indicated by white lines in (A), suggesting a central channel. Scale bars, 5 nm in (A) and (B), 10 nm in galleries.

That the needle has a defined tip structure at its distal end, comprising LcrV, is in agreement with previous reports showing that LcrV is surface-exposed (3, 4) and essential for the assembly of a functional translocation pore (6). LcrV may act as an assembly platform for this pore (fig. S8) (6). The IpaD protein from Shigella may function in an analogous fashion (18), although it has no clear sequence homology to LcrV. LcrV can also be compared to the EspA filament of enteropathogenic Escherichia coli, which forms a physical bridge between the needle and the host cell (19). The EspA homolog, SseB of Salmonella SPI-2, forms an undefined sheathlike structure on the distal end of the T3S needle (20).

The localization of LcrV at the tip of the needle and its role in the assembly of the pore may explain the protective action of antibodies to LcrV. Possibly, the antibodies interfere with the function of the tip complex, impairing the translocation process.

Supporting Online Material

www.sciencemag.org/cgi/content/full/310/5748/674/DC1

Materials and Methods

Figs. S1 to S8

Tables S1 and S2

References and Notes

References and Notes

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