Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB - PubMed (original) (raw)
Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB
Helen J Wing et al. J Bacteriol. 2004 Feb.
Abstract
The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread. Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB. In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter. Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF. In contrast, when VirF was induced in the absence of VirB, VirF had variable effects. The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA, which is activated by VirF and not VirB. We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP.
Figures
FIG. 1.
Comparison of IcsP levels in Shigella strains with or without the virulence plasmid. Western blot analysis of the icsP mutant (MBG341) (lane 1) and virulence plasmid-cured Shigella (BS103) (lane 2), each carrying the icsP reporter plasmid (pHJW6), was performed with IcsP antiserum. Equivalent amounts of total cellular protein were loaded onto the gel for the two strains. The experiment was repeated three times, and representative data are shown. Apparent molecular masses are indicated in kilodaltons.
FIG. 2.
IcsP levels in the absence of the transcriptional activators VirF and VirB. Western blot analysis of total cellular protein harvested from wild-type Shigella (2457T), the icsP mutant (MBG341), the virF mutant (MBG338), the virF virB mutant (AWY7), and the virB mutant (AWY3) was performed with IcsP antiserum. Apparent molecular masses are indicated in kilodaltons.
FIG. 3.
Effect of independent expression of VirF or VirB on IcsP levels. Western blot analysis of Shigella strains deficient for both virF and virB but supplied in trans with either virF or virB expressed from an arabinose-inducible promoter was carried out. IcsP expression was measured in (i) virulence plasmid-cured Shigella (BS103) carrying the icsP reporter plasmid (pHJW6) or (ii) the virF virB mutant (AWY7). Cells were grown either with (+) or without (−) induction of virB (A) or virF (B), and data from duplicate experiments are shown. (C) Quantification of bands by integrated densitometry. Approximately fourfold less protein was loaded in the top blot of each panel, due to the presence of icsP on a multicopy plasmid in the virulence plasmid-cured strain.
FIG. 4.
Model of the distinct regulatory pathways that modulate IcsP and IcsA expression in Shigella. VirF positively regulates transcription of icsA and virB. This increases the amount of IcsA on the bacterial surface and leads to increased levels of VirB. VirB positively regulates transcription of icsP, leading to an increase in IcsP from its basal level of expression. VirF may, under certain circumstances, also increase IcsP expression (see the text).
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