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Spa32 Regulates a Switch in Substrate Specificity of the Type III Secreton of Shigella flexneri from Needle Components to Ipa Proteins

Juana Magdalena,^1, Abderrahman Hachani,¹ Mustapha Chamekh,¹ Noureddine Jouihri,¹ Pierre Gounon,^2, Ariel Blocker,^3, and Abdelmounaaïm Allaoui^1*

Laboratoire de Bactériologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, 1070 Brussels, Belgium,¹ Station Centrale de Microscopie Electronique,² Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France³

Received 12 November 2002/ Accepted 3 April 2002

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.

Present address: School of Biosciences, Division of Molecular Cell Biology, University of Birmingham, Edgbaston, Birmingham B15-2TT, England.

Present address: INSERM U 452, UFR Médecine, 06107 Nice Cedex 02, France.

Present address: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, England.

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