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. 2002 Jul;184(13):3433-41.
doi: 10.1128/JB.184.13.3433-3441.2002.

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, Mustapha Chamekh, Noureddine Jouihri, Pierre Gounon, Ariel Blocker, Abdelmounaaïm Allaoui
Affiliations

Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins

Juana Magdalena et al. J Bacteriol. 2002 Jul.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Plasmids used for the inactivation of the spa32 gene. (A) Schematic representation of the 30-kb DNA of the pWR100 plasmid required for the entry of S. flexneri into eukaryotic cells. Arrows indicate positions and orientation of previously described promoters. Open bars, except for the spa32 gene (indicated by a dashed box), show the relative positions of the icsB-ipgABC-ipaBCDA, ipgDEF-mxi, and spa loci. (B) Structure of plasmids used to inactivate the spa32 gene (pMJ2-5), to complement the spa32 mutant (pMJ7-8), and to produce and purify His-tagged recombinant proteins Spa32 (pMJ8), MxiH (pNJH8), and MxiI (pJN54). The black box represents the nonpolar aphA-3 gene that confers resistance to kanamycin. The horizontal arrows indicate the extent and the orientation of the corresponding genes, while small arrows indicate positions of the promoters known to control expression of these genes. A small open box represents the His6 tag motif. Constructs are drawn with the relevant restriction sites: B, BamHI; Ms, MscI; H, HincII; Sf, SfuI; and Sp, SphI.
FIG. 2.
FIG. 2.
Spa32 is required for Ipa secretion upon CR induction. Strains used were: M90T (wild type), mxiD and MJ321 (spa32 mutant), MJ322 (MJ321+pMJ7), and MJ323 (MJ321+pMJ8). Cultures of S. flexneri strains (grown at 37°C) were harvested by centrifugation, suspended in PBS, and incubated in the presence of CR for 15 min at 37°C to induce Ipa secretion. Equal amounts of the culture supernatants and cell lysates were analyzed by SDS-12% PAGE stained with Coomassie blue (A) or were immunoblotted with a monoclonal antibody specific to the IpaC protein (B).
FIG. 3.
FIG. 3.
The spa32 mutant (MJ321) secretes Ipa proteins even in the absence of CR induction. Culture supernatant proteins were prepared from bacteria grown for 3 h at 37°C until the exponential growth phase was reached (OD600 = 0.3). Samples were centrifuged, and equal amounts of precipitated supernatants and cell lysates were analyzed by SDS-12% PAGE and Coomassie blue staining (A) or by immunoblotting with a monoclonal antibody specific for IpaB and IpaC (B) or a polyclonal antibody specific to Spa32 (C and D). The small arrowheads indicate the position of Spa32.
FIG. 4.
FIG. 4.
The spa32 mutant strain (MJ321) exhibits an elongated needle structure. Electron micrographs of Shigella wild-type M90T (left panel), spa32 mutant (middle panel), and spa32 mutant complemented with the pMJ7 plasmid encoding the native Spa32 protein (MJ322) (right panel). Osmotically shocked and negatively stained bacteria (grown at 37°C) were visualized by electron microscopy as previously described by Blocker et al. (7). Bar, 200 nm. The white arrowheads indicate the transmembrane region and the cytoplasmic bulb of type III secretons; the needle (if particularly long) or needle tips are outlined or indicated by black arrowheads.
FIG. 5.
FIG. 5.
The higher secreted amount of MxiH in the spa32 mutant is not due to its overproduction. (A) Wild-type strain (M90T), the spa32 mutant (MJ321), and its derivative complemented with pMJ7 (encoding the native Spa32) (MJ322) were grown to mid-log phase (OD600 = 2). Equal amounts of proteins from the culture supernatants were loaded on 16% Tris-Tricine SDS-PAGE gels, and then stained with Coomassie blue. The positions of the proteins, as well as the sequenced amino acid residues of the N terminus of Spa32n, MxiH, and MxiI are indicated on the right side of each panel. (B) Equal amounts of total culture proteins (supernatant and bacteria; the equivalent of 0.1 OD600) were separated by SDS-16% PAGE and then immunoblotted with the anti-MxiH polyclonal antibody. The strains used were the wild type (M90T), the spa32 mutant, and the mxiD mutant as indicated.
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References

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