The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri

Abstract

The type III secretion apparatus (T3SA) is a central virulence factor of many Gram-negative bacteria. Its overall morphology consists of a cytoplasmic region, inner-and outer-membrane sections and an extracellular needle. In Shigella, the length of the needle is regulated by Spa32. To understand better the role of Spa32 we searched for its interacting partners using a two-hybrid screen in yeast. We found that Spa32 interacts with the 33 C-terminal residues (CC*) of Spa40, a member of the conserved FlhB/YscU family. Using a GST pull-down assay we confirmed this interaction and identified additional interactions between Spa40 and the type III secretion components Spa33, Spa47, MxiK, MxiN and MxiA. Inactivation of spa40 abolished protein secretion and led to needleless structures. Genetic and functional analyses were used to investigate the roles of residues L310 and V320, located within the CC* domain of Spa40, in the assembly of the T3SA. Spa40 cleavage, at the conserved NPTH motif, is required for assembly of the T3SA and for its interaction with Spa32, Spa33 and Spa47. In contrast, unprocessed forms of Spa40 interacted only with MxiA, MxiK and MxiN. Our data suggest that the conformation of the cytoplasmic domain of Spa40 defines the multi-step assembly process of the T3SA. INTRODUCTIONShigella flexneri, the causative agent of bacillary dysentery in humans, is able to enter and to disseminate in epithelial cells by using a specialized Mxi-Spa type III secretion apparatus (T3SA) (Parsot, 2009). The T3SA is widespread among Gram-negative bacteria that are pathogenic for animals and plants or are symbionts (Viprey et al., 1998;Dale et al., 2001Dale et al., , 2002 and is devoted to the injection of virulence effectors into target cells. The T3SA is composed of a cytoplasmic region called 'the bulb', a basal body consisting of a pair of rings that span the inner and outer membranes, and an extracellular needle protruding outside the bacterium. This apparatus is evolutionarily and structurally related to the export machinery of flagellar systems (Macnab, 1999;Blocker et al., 2001). Nine T3SA constituents are related to components of the basal bodies of bacterial flagella (Allaoui et al., 1994;Aizawa, 2001;Blocker et al., 2003;Macnab, 2004), including the innermembrane proteins MxiA (FlhA), Spa9 (FliP), Spa24 (FliR), Spa29 (FliQ) and Spa40 (FlhB), and the cytoplasmic proteins Spa47 (FliI), MxiN (FliH) and Spa33 (FliN) (Kane et al., 2002;Allaoui et al., 1992bAllaoui et al., , 1993bAllaoui et al., , 1995Penno et al., 2006;Jouihri et al., 2003;Andrews & Maurelli, 1992; Sasakawa et al., 1993;Schuch & Maurelli, 2001; MoritaIshihara et al., 2005). Additionally, two cytoplasmic proteins, Spa13 and MxiK, which have no obvious counterpart in the flagellar system, are also implicated in Abbreviations: EPEC, enteropathogenic Escherichia coli; GST, glutathione S-transferase; NC, needle complex; T3S, type III secretion; T3SA, type III secretion apparatus; T3SS, type III secretion system; TEM, transmission electron microscopy.3The...

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

“…5). A similar result has previously been described for Spa40 and Spa47 that are the homologues of YscU and YscN in Shigella flexneri (43).…”

Bacterial Type III Secretion Systems: Specialized Nanomachines for Protein Delivery into Target Cells

“…In this case the role of InvJ in needle determination is indirect through its role in inner rod assembly. It has been proposed that SpaS/YscU, a component of the export apparatus, may also be involved in the mechanisms of substrate switching (18; 49; 121; 148). As discussed above, SpaS/YscU protein family possesses a long cytoplasmic domain with autocatalytic protease activity (57).…”

Spa13 of Shigella flexneri has a dual role: chaperone escort and export gate-activator switch of the type III secretion system

“…We reported previously the instability of MxiH in needleless mutants such as spa47, spa40 and mxiA (Jouihri et al, 2003;Botteaux et al, 2010). Since we have shown in this work that Spa13 is able to interact with components required for needle assembly (such as Spa47, MxiA and Spa40), we monitored MxiH production in a spa13 coli producing His-MxiA C were incubated with GST-Spa13 or GST alone bound to glutathione-Sepharose beads and eluted fractions (EF) were analysed as described in Fig.…”