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...

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.

The 0 antigen of the ShigeUla flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. flexneri mutants which produce a semi-rough LPS by using an 0-antigenspecific phage, Sf6c. Western immunoblotting was used to show that the LPS produced by the semi-rough mutants contained only one 0-antigen repeat unit. Thus, the mutants are deficient in production of the 0-antigen polymerase and were termed rfc mutants. Complementation experiments were used to locate the r*c adjacent to the rjb genes on plasmid clones previously isolated and containing this region (D. F. Macpherson, R. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mol. Microbiol 5:1491-1499 downstream of the rfc gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several minor codons within the first 25 amino acids. This is in contrast to the upstream gene rJbG, which also has a high percentage of rare codons but whose gene product could be detected.The positioning of the rare codons in the rfc gene may restrict translation and suggests that minor isoaccepting tRNA species may be involved in translational regulation of rfc expression. The low percentage of G+C content of rfc genes may be a consequence of the selection pressure to maintain this form of control.The lipopolysaccharide (LPS) of gram-negative bacteria constitutes a major protective barrier against dyes, detergents, and hydrophobic agents. The 0 antigen of the LPS of Shigella flexneri is a virulence determinant (27,32), and immune responses against this antigen correlate with immunity to shigellosis (4,8,9). As with many other members of the family Enterobacteriaceae, the 0 antigen is encoded by the rfb locus near his (45, 49) on the chromosome. This locus encodes enzymes which synthesize the nucleotide-activated sugar precursor (dTDP-rhamnose) (30) and nucleotide sugar transferases, which assemble onto the lipid carrier bactoprenol, a tetrasaccharide repeat unit with the following structure: rhamnose-rhamnose-rhamnose-N-acetylglucosamine. This tetrasaccharide repeat unit is then polymerized by the 0-antigen polymerase into long chains. An 0-antigen ligase covalently attaches these units onto incomplete LPS molecules composed of lipid A and core sugars (21).Several phenotypes related to the structure of LPS molecules are recognized. The wild-type LPS pattern observed after silver staining of LPS separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) is termed smooth LPS (S-LPS distribution of 0-antigen chain lengths with an average of about 12 to 17 0-antigen tetrasaccharide repeat units. A variation of this has been observed in Escherichia coli K-12 cells harboring the cloned S. flexneri rfb genes but lacking the rol (31) gene. In this case, the 0-antigen chain lengths are more or less randomly distributed. LPS molecules which lack 0 antigen are termed rough (R-LPS) and can be visualized as multiple bands migrating near the dye front by silver s...

SummaryBacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of ª ª ª ª 15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an antiactivator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.

SummaryWe have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri Oantigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3 kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int ) and attachment site (attP ), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the Oantigen specifically. We determined that gtrX-mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K-12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA-derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [ 35 S]-methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34 kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP-glucose onto bactoprenol and GtrII then transfers the glucose onto the O-antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype-converting genes, when compared with their homologues in E. coli K-12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin.

Bacteria of Shigella spp. use a virulence plasmid-encoded type III secretion (TTS) system to invade the colonic epithelium in humans. The activity of the TTS apparatus is tightly regulated in the wild-type strain and is induced upon contact of bacteria with epithelial cells, whereas it is deregulated, i.e., constitutively active, in some mutants. Under conditions of deregulated secretion, approximately 20 proteins are secreted, including VirA, OspB to OspG, and at least three members of the IpaH family, all of which are encoded by the virulence plasmid. Conditions inducing or deregulating the activity of secretion also induce the transcription of virA and four ipaH genes. The transcription of virA and ipaH9.8 requires both MxiE, a transcriptional activator of the AraC family, and IpgC, the chaperone of IpaB and IpaC, acting as a coactivator. Using reporter plasmids containing lacZ transcriptional fusions, we showed that the ipaH7.8, ipa4.5, ospC1, and ospF promoters are activated under conditions of deregulated secretion and that both MxiE and IpgC are necessary and sufficient for their activation in both Shigella flexneri and Escherichia coli. Promoter mapping and deletion analysis of the ipaH9.8, virA, and ospC1 promoters identified a 17-bp motif, the MxiE box, which overlaps the ؊35 region and is essential for the activation of these promoters. The presence of eight MxiE boxes on the virulence plasmid suggests that 11 genes encoding secreted proteins may be regulated by the activity of secretion. We also present evidence that at least one ipaH gene that is carried by the chromosome is controlled by MxiE and IpgC.