Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells

Abstract

A number of pathogenic, Gram-negative bacteria are able to secrete specific proteins across three membranes: the inner and outer bacterial membrane and the eukaryotic plasma membrane. In the pathogen Yersinia enterocolitica, the primary structure of the secreted proteins as well as of the components of the secretion machinery, both plasmid-encoded, is known. However, the mechanism of protein translocation is largely unknown. Here we show that Y. enterocolitica polymerizes a 6-kDa protein of the secretion machinery into needles that are able to puncture the eukaryotic plasma membrane. These needles form a conduit for the transport of specific proteins from the bacterial to the eukaryotic cytoplasm, where they exert their cytotoxic activity. In negatively stained electron micrographs, the isolated needles were 60 -80 nm long and 6 -7 nm wide and contained a hollow center of about 2 nm. Our data indicate that it is the polymerization of the 6-kDa protein into these needles that provides the force to perforate the eukaryotic plasma membrane.electron microscopy ͉ bacterial pathogenicity ͉ cell contact assays ͉ protein translocation

Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis

“…These proteins interact (Neyt & Cornelis, 1999b;Sarker et al, 1998) to presumably form a complex that facilitates pore formation in target cell membranes to allow entry of effector proteins into target cells (Bröms et al, 2003a; Holmström et al, 2001;Neyt & Cornelis, 1999a;Tardy et al, 1999). At odds with this dogma is a report suggesting that the needle component YscF is the sole requirement for effector translocation (Hoiczyk & Blobel, 2001). Clearly, the molecular mechanisms of this process are still unresolved.…”

“…This specialized virulence strategy links protein secretion across the bacterial envelope with translocation of anti-host virulence effectors through the plasma membrane of target eukaryotic cells. Effector proteins localized inside the target cell disable crucial signal transduction pathways, rendering the cell incapable of mounting an effective immune defence (Bliska, 2000; Fällman et al, 2002).The bacterial envelope component of the TTSSs of many plant-and animal-interacting bacteria resembles the flagella basal body, while a hollow needle-like protrusion extends from the bacterial surface through which effectors are believed to traverse (Blocker et al, 1999;Hoiczyk & Blobel, 2001;Jin & He, 2001;Kubori et al, 1998; Sekiya et al, 2001;Tamano et al, 2000). Furthermore, translocation into target cells by Yersinia requires the structural proteins LcrV, YopB and YopD.…”

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

“…The needle has been shown to be composed of a single protein that polymerizes upon secretion (25). Numerous studies have demonstrated that deletion or mutation of the gene encoding the needle results in significant attenuation of virulence (31,53,55,56).…”

“…In addition to its role as an injectisome component, evidence suggests that the needle is also important in host cell detection and thus regulation of T3S (12,30,60). Electron micrographs of the needles from Yersinia, Salmonella Spi-1, and Shigella, comprising the small homologous proteins YscF, PrgI, and MxiH, respectively, reveal straight rigid structures that are very short (approximately 40 to 80 nm) compared to flagella (7,25,34,55). The crystal structure of monomeric MxiH has recently been solved revealing two extended antiparallel helices reminiscent of the D0 portion of flagellin (11,12).…”

The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery

“…It is constructed via the helical polymerization of approximately 150 subunits of the YscF family (Kubori et al, 2000;Hoiczyk and Blobel, 2001). …”