Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-κB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-κB induction. Dominant-negative versions of CARD4 block activation of NF-κB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.

Summary The virulence plasmid‐encoded type III secretion system of Shigella flexneri consists of the Mxi–Spa secretion apparatus, secreted proteins IpaA–D and IpgD involved in entry of bacteria into epithelial cells, cytoplasmic chaperones IpgC and IpgE and 15 other secreted proteins of unknown function, including VirA and members of the IpaH family. The activity of the Mxi–Spa apparatus is regulated by external signals, and transcription of virA and ipaH genes is specifically induced in conditions of active secretion. We present genetic evidence that regulation of these genes involves both MxiE, the transcriptional activator of the AraC family encoded by the mxi operon, and IpgC, the chaperone for IpaB and IpaC. We also show that together MxiE and IpgC are sufficient to activate virA and ipaH9.8 promoters in Escherichia coli. In S. flexneri, increasing the expression of IpgC led to a concomitant increase in IpaH production in conditions of non‐secretion. This suggests that the activity of secretion is sensed by the presence of free IpgC, which acts as a coactivator to allow MxiE to activate transcription at its target promoters.

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...