The 33 Carboxyl-Terminal Residues of Spa40 Orchestrate the Multi-Step Assembly Process of the Type III Secretion Needle Complex in Shigella Flexneri

Microbiology. 2010 Sep;156(Pt 9):2807-2817. doi: 10.1099/mic.0.039651-0. Epub 2010 May 27.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Bacterial Secretion Systems*
  • Gene Expression Regulation, Bacterial
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Transport
  • Shigella flexneri / chemistry
  • Shigella flexneri / genetics
  • Shigella flexneri / physiology*
  • Virulence Factors / genetics
  • Virulence Factors / metabolism


  • Bacterial Proteins
  • Bacterial Secretion Systems
  • Membrane Proteins
  • Spa32 protein, Shigella flexneri
  • Virulence Factors