Structure and composition of the Shigella flexneri‘needle complex’, a part of its type III secreton

Abstract

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram‐negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the ‘needle complex’ (NC), produced an average image at 17 Å resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2–3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N‐terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

“…In fact, the 9-kDa band of the type III secretion complexes partially purified from M94, the ⌬spa32 mutant, ⌬spa32/pTB101-mxiH, and ⌬spa32/pMW119Tp-spa32 was confirmed to be MxiH by direct N-terminal amino acid sequencing (data not shown). It is worth noting that the 9-kDa protein band also contained MxiI, because the sequencing also yielded an MNYIY sequence corresponding to the N-terminal amino acids of MxiI, even though it was detected as a minor fraction, as reported previously (4).…”

“…The basal part of the Shigella type III complex is composed of MxiD, MxiG, and MxiJ (45), while that of Salmonella is composed of InvG, PrgH, and PrgK (24). The needle is composed of MxiH and MxiI in S. flexneri and PrgI and PrgJ in Salmonella, in which MxiI and PrgJ are indicated to be minor essential components (4,22,25,45). The needle portion is essential, not only as the molecular bridge connecting the pathogen to the host cell but also in mediating the delivery of a set of effector proteins (25,45).…”

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

“…3 In all cases, the T3SA is anchored in the bacterial envelope by a basal body, which supports an external needle composed of many copies of a small, polymerized needle protein terminating at a needle tip complex that contributes to secretion control. 5 Shigella is currently the only pathogen for which each step in the T3SA needle tip complex maturation can be artificially induced to mimic natural maturation. [6][7][8] As a first step of Shigella T3SA maturation, IpaD forms a pentameric structure at the tip of the nascent MxiH needle.…”

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

“…Single particles of NCs from S. enterica SPI-1 (Marlovits et al, 2004(Marlovits et al, , 2006 and from S. flexneri (Blocker et al, 2001;Sani et al, 2007;Hodgkinson et al, 2009) were characterized by cryo-electron microscopy (cryo-EM). These reconstitutions show that an inner rod with a central channel of approximately 2-3 nm is anchored at the basis of the chamber by a socket-like structure and traverses the chamber of the NC.…”

“…It consists of a 12-14-mer of a protein belonging to the YscC family of secretins ( Figure 1) (Koster et al, 1997;Kubori et al, 2000;Tamano et al, 2000;Blocker et al, 2001;Burghout et al, 2004b). Secretins are found not only in injectisomes but also in the type II secretion apparatus, in type IV pili (Collins et al, 2004;Chami et al, 2005) as well as in filamentous phages (Russel, 1994).…”