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

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.

Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism.

The effectors of enterocyte invasion by Shigella are dependent on a type III secretion system that contains a needle whose length average does not exceed 50 nm. Previously, we reported that Spa32 is required for needle length control as well as to switch substrate specificity from MxiH to Ipa proteins secretion. To identify functional domains of Spa32, 11 truncated variants were constructed and analysed for their capacity (i) to control the needle's length; (ii) to secrete the Ipa proteins; and (iii) to invade HeLa cells. Deletion at either the N-terminus or C-terminus affect Spa32 function in all cases, but Spa32 variants lacking internal residues 37-94 or 130-159 retained full Spa32 function. Similarly, a Spa32 variant obtained by inserting of the YscP's ruler domain retained Spa32 function although it programmed slightly elongated needles. Using the GST pull-down assay, we show that residues 206-246 are required for Spa32 binding to the C-terminus of Spa40, an inner membrane protein required for Ipa proteins secretion. Our data clearly demonstrate that shortening Spa32 affects the length of the needle in a comparable manner to the spa32 mutant, indicating that the control of needle length does not require a molecular ruler mechanism.