Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri

Abstract

Proteins directly involved in entry and dissemination of Shigella flexneri into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 6C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an ipaB mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of virB, whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 6C and in virF, virB, mxiE, ipaB, ipaB mxiE and orf81 mutants grown at 37 6C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the orf81 mutant. Expression of 13 genes was increased in the secretion-deregulated ipaB mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells. INTRODUCTIONBacteria of Shigella species are responsible for shigellosis, a human disease characterized by the destruction of the colonic epithelium. Shigellae and enteroinvasive Escherichia coli both contain a virulence plasmid (VP) of approximately 200 kb that encodes most proteins directly involved in entry and dissemination of bacteria into epithelial cells. Sequence analysis of the VP pWR100 (Buchrieser et al., 2000) and its derivative pWR501 (Venkatesan et al., 2001) from a Shigella flexneri strain of serotype 5 and the VP pCP301 (Jin et al., 2002) from an S. flexneri strain of serotype 2a indicated that the VP is composed of a mosaic of approximately 100 genes and numerous insertion sequences. The VP genes exhibit a G+C content from 30 to 60 mol%, suggesting that they were acquired from different sources. The current model of the TTS pathway prop...

TheShigella flexnerieffector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes

“…I B␣ degradation occurred after 1 h of infection of HeLa cells by the wild-type strain, probably because the increase in the number of intracellular bacteria overwhelmed the OspG protective effect, because there is no sustained production and delivery of OspG when bacteria are intracellular. Indeed, OspG production is tightly regulated by the TTS apparatus activity and controlled by MxiE (14), and transcription of MxiE-regulated genes is induced upon entry of bacteria into cells but repressed during multiplication of bacteria within epithelial cells (11). A role of OspG in interfering with activation of NF-B in vivo is supported by the observation that the inflammatory response induced upon infection of ileal loops by the ospG mutant was more severe than with the wild-type strain.…”

“…We present the functional analysis of the effector OspG, a 196-residue protein whose production is regulated by secretion activity (9,14). A two-hybrid screen in yeast and in vitro studies indicated that OspG binds ubiquitinylated E2s, including UbcH5.…”

Control of gene expression by type III secretory activity

“…Contact of Shigella with host cells activates secretion by the injectisome and triggers transcription of ∼15 effector genes [22]. Expression of the effector genes is controlled by MxiE, an AraC-like transcriptional activator.…”

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

“…Activation of transcription involves a regulatory cascade in which the product of the virF gene activates the downstream virB promoter . The VirB regulatory protein in turn alleviates H-NSmediated repression of the operons coding for the structural virulence genes (Adler et al, 1989;Le Gall et al, 2005;McKenna et al, 2003). VirB shows strong amino acid sequence homology to plasmid partitioning proteins such as ParB from plasmid/phage P1 and SopB from the F plasmid (Beloin et al, 2002).…”