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

Abstract

SummaryThe 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.

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…and InvJ (336 amino acids) from Salmonella spp. (50). In contrast, a Spa32-YscP hybrid protein containing the central ruler domain of YscP flanked by the N-and C-terminal regions of Spa32 led to a 2-fold increase in needle length in S. flexneri compared with that observed with the native YscP or Spa32 protein (50).…”

“…(Table 6). Furthermore, deletions within Spa32 do not lead to a reduction in needle length (50). Notably, however, Spa32 is functionally interchangeable with YscP from Yersinia spp.…”

“…T3S4 proteins share little amino acid sequence identity with each other but contain a structurally conserved C-terminal domain (termed the T3S4 domain) that is probably essential for the substrate specificity switch and harbors a P-X-L-G amino acid motif (2). T3S4 proteins from both translocation-associated and flagellar T3S systems interact with the C-terminal cytoplasmic domains of members of the conserved YscU/FlhB family of IM proteins, as shown for the T3S4 proteins FliK, Spa32, and HpaC (50,332,381,392) (Tables 2 and 6). Binding of T3S4 proteins to the C-terminal domains of FlhB, YscU, and homologs might induce a conformational change in these domains that alters the substrate specificity of the T3S system.…”

Shigella

“…78 Subsequently, the needle tip forming protein (IpaD) and the translocation pore (IpaB and IpaC) are inserted into the host membrane. IpaD polymerizes at the tip of the needle to form the needle tip structure (light blue part of the diagram in Fig.…”

The blueprint of the type-3 injectisome

“…The helical parameters of the assembled needle vary from 5.6 subunits per turn in Shigella [23] to 6.3 in Salmonella [19]. The length of the growing needle filament is under the control of another component (InvJ (Salmonella), Spa32 (Shigella), or YscP (Yersinia)) [72,75,[78][79][80]; however, the precise mechanism of needle length control is under debate [81]. A recently published structure of truncated PrgI showed that the soluble monomer comprises a helical core and flexible termini (Asp11-Thr18 and Gln61-Asn78) [20].…”