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Bacterial Injectisomes: Needle Length Does Matter

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Science  25 Feb 2005:
Vol. 307, Issue 5713, pp. 1278
DOI: 10.1126/science.1107679

Abstract

Many pathogenic bacteria use a type III secretion nanomachine (an injectisome) to deliver virulence proteins into the cytosol of their eukaryotic host cells. Most injectisomes possess a stiff needlelike structure of a genetically defined length. We found that a minimal needle length was required for efficient functioning of the Yersinia enterocolitica injectisome. This minimal needle length correlated with the length of the major adhesin at the bacterial surface. The needle may be required for triggering type III secretion, and its length may have evolved to match specific structures at the bacterial and host cell surfaces.

Type III secretion (T3S) allows Gram-negative pathogenic bacteria adhering to the membrane of a eukaryotic cell to paralyze or reprogram this cell by injecting proteins into its cytosol (1). Many T3S nanomachines (injectisomes) possess a stiff needlelike structure of a defined length (2). The needle is thought to function as the conduit for protein translocation. In Yersinia bacteria, including Y. pestis, the needle length is defined by the protein ruler YscP (3), and the proteins injected upon host cell contact, called Yops, are involved in, among other things, caspase activation and macrophage apoptosis (4).

To address the question of why the needle length is controlled, we replaced yscP (515 codons) on the 70-kb virulence plasmid of Y. enterocolitica E40 by either a truncated (388 codons) or an enlarged (680 codons) allele. We incubated the modified bacteria in conditions that artificially induce Yersinia T3S (4) and observed no clear difference in Yop secretion in relation to wild-type bacteria (fig. S1). We compared Yersinia that make short (YscP388, needle length L = 45 ± 13 nm) and long (YscP680, L = 88 ± 23 nm) needles to wild-type bacteria (L = 55 ± 11 nm) (fig. S2) for their capacity to inject the apoptosis-inducing YopP into J774A.1 macrophages, by assaying caspase activation in infected cells. Bacteria making long needles were as efficient as the wild type, but those making short needles were not (Fig. 1A). This suggested that the needle must span a minimal distance, presumably determined by the Y. enterocolitica adhesins YadA and invasin (Inv).

Fig. 1.

(A) Caspase activity (act.) on Y. enterocolitica E40-infected macrophages. Results are the mean ± SEM from five independent experiments, each done in triplicate. The numbers on top indicate how far the needle sticks out (in nm) relative to YadA. NI, not infected; wt, wild-type. (B) YopO143-Cya translocation into macrophages. ΔHOPEMNB is a negative control, not expressing the YopB translocator (4). Data are the mean ± SEM from three independent experiments, each done in triplicate. cAMP, cyclic adenosine monophosphate. (C) Immunoblot of cultured supernatant proteins from Y. enterocolitica E40–infected macrophages. YopN is a positive control; in a yopN mutant, Yop translocation is independent of contact (4).

Although YadA protrudes ∼28 nm from the bacterial outer membrane (5), Inv is only 18 nm long (6). YadA (455 amino acids) has a lollipop-shaped structure with an oval head on top of a coiled-coil rod (5). Variations in coiled-coil domain length result in longer or shorter YadAs (5), modulating the distance between the bacterium and host-cell lipid membranes. We altered YadA length by deletion or duplication of residues 230 to 319 within the coiled-coil rod, yielding shorter (YadA365, estimated length of 15 nm) and longer (YadA545, ∼40 nm) adhesins. We disrupted inv and yadA in Yersinia carrying yscP388, yscPwt, and yscP680 and introduced plasmids encoding YadAwt, YadA365, and YadA545. The YadA variants were as functional as YadAwt in promoting cell attachment (fig. S3). Caspase activity in Yersinia-infected macrophages revealed that shortening YadA suppressed the defect of short needles, whereas lengthening YadA enhanced the deficiency (Fig. 1A). Accordingly, lengthening YadA reduced the efficacy of wild-type needles but not of long needles (Fig. 1A). We monitored the translocation of YopO into J774A.1 by using an adenylate cyclase (Cya) reporter assay (Fig. 1B), confirming that short needles lead to a defect in Yop injection.

Thus, increasing the distance between the needle tip and the host cell by shortening the needle or by lengthening YadA reduces translocation; i.e., the needle needs to have a minimal length to be fully functional. We tested if bacteria with impaired translocation efficiency secreted Yops into the medium when incubated with J774A.1. Regardless of the length of the needle or YadA, we could not detect YopE in this fraction (Fig. 1C), indicating that there was no leakage and suggesting that contact between the needle tip and the host cell membrane is necessary to trigger T3S. It is thus likely that needle length has evolved to match specific structures at the bacterial and host cell surfaces.

Supporting Online Material

www.sciencemag.org/cgi/content/full/307/5713/1278/DC1

Materials and Methods

Figs. S1 to S3

References and Notes

References and Notes

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