A 31-kb fragment of the large virulence plasmid of Shigella flexneri is necessary for bacterial entry into epithelial cells in vitro. One locus of this fragment encodes the IpaA, -B, -C, and -D proteins, which are the dominant antigens of the humoral immune response during shigellosis. To address the role of the ipa genes, which are clustered in an operon, we constructed a selectable cassette that does not affect transcription of downstream genes and used this cassette to inactivate the ipaB, ipaC, and ipaD genes. Each of these nonpolar mutants was defective in entIy and lysis of the phagocytic vacuole but was not impaired in adhesion to the cells. We showed that, like IpaB and IpaC, IpaD is secreted into the culture supernatant and that none of these proteins is necessary for secretion of the other two. This result differentiates the Ipa proteins, which direct the entry process, from the Mxi and Spa proteins, which direct secretion of the Ipa proteins. Moreover, lack of either IpaB or IpaD resulted in the release of larger amounts of the other Ipa polypeptides into the culture medium, which indicates that, in addition to their role in invasion, IpaB and IpaD are each involved in the maintenance of the association of the Ipa proteins with the bacterium.

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi–Spa type III secretion apparatus and the secreted IpaA–D proteins, all of which are encoded by a virulence plasmid. In wild‐type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N‐terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.

Shigella species are enteropathogens that invade epithelial cells of the human colon. Entry into epithelial cells is triggered by the IpaB, IpaC and IpaD proteins which are translocated into the medium through the specific Mxi‐Spa machinery. In vitro, Shigella cells secrete only a small fraction of the Ipa proteins, the majority of which remains in the cytoplasm. We show here that upon interaction with cultured epithelial cells or in the presence of fetal bovine serum, S.flexneri release pre‐synthesized Ipa molecules from the cytoplasm into the environment. Evidence is presented that IpaB and IpaD are essential for both blocking secretion through the Mxi‐Spa translocon in the absence of a secretion‐inducing signal and controlling secretion of the Ipa proteins in the presence of a signal. Subcellular localization and analysis of the molecular interactions of the Ipa proteins indicate that IpaB and IpaD associate transiently in the bacterial envelope. We propose that IpaB and IpaD, by interacting in the secretion apparatus, modulate secretion.

Expression of more than 17 virulence genes in Vibrio cholerae is under the coordinate control of the ToxR protein. ToxR is a transmembrane protein that binds to and activates the promoter of the operon encoding cholera toxin. As yet, the ability of ToxR to activate directly other genes in this regulon has not been demonstrated. We have cloned a gene called toxT from V. cholerae 569B; the toxT gene product, like ToxR, can activate the ctx promoter in Escherichia coli. In addition, expression of other genes identified as members of the ToxR regulon (tcpA, tcpI, aldA, and tagA) can be activated in E. coli by the toxT gene product but not by ToxR. When expressed from a constitutive promoter, the toxT gene product partially suppresses the ToxR- phenotype of a toxR deletion mutant of V. cholerae. The level of toxT mRNA is greatly reduced in a toxR mutant of V. cholerae. In addition, growth conditions under which the ToxR regulon is not expressed also repress the synthesis of toxT mRNA. These results suggest that ToxR controls transcription of toxT, whose product in turn is directly responsible for activation of several virulence genes under ToxR control.

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram‐negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the ‘needle complex’ (NC), produced an average image at 17 Å resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2–3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N‐terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

Phosphorylation of histone H3 at Ser10 increases chromatin accessibility to transcription factor NF-kappaB on a subset of genes involved in immune responses. Here we report that a bacterial pathogen abrogated phosphorylation of histone H3 to 'shape' the transcriptional responses of infected host cells. We identify the Shigella flexneri protein effector OspF as a dually specific phosphatase that dephosphorylated mitogen-activated protein kinases in the nucleus, thus preventing histone H3 phosphorylation at Ser10 in a gene-specific way. That activity of OspF enabled shigella to block the activation of a subset of NF-kappaB-responsive genes, leading to compromised recruitment of polymorphonuclear leukocytes to infected tissues. S. flexneri has thus evolved the capacity to precisely modulate host cell epigenetic 'information' as a strategy for repressing innate immunity.

Bacteria of Shigella spp. are responsible for shigellosis in humans. They use a type III secretion system to inject effector proteins into host cells and induce their entry into epithelial cells or trigger apoptosis in macrophages. We present evidence that the effector OspG is a protein kinase that binds various ubiquitinylated ubiquitin-conjugating enzymes, including UbcH5, which belongs to the stem cell factor SCF ␤-TrCP complex promoting ubiquitination of phosphorylated inhibitor of NF-B type ␣ (phospho-I B␣). Transfection experiments indicated that OspG can prevent phospho-I B␣ degradation and NF-B activation induced by TNF-␣ stimulation. Infection of epithelial cells by the S. flexneri wild-type strain, but not an ospG mutant, led to accumulation of phospho-I B␣, consistent with OspG inhibiting SCF ␤-TrCP activity. Upon infection of ileal loops in rabbits, the ospG mutant induced a stronger inflammatory response than the wild-type strain. This finding indicates that OspG negatively controls the host innate response induced by S. flexneri upon invasion of the epithelium.T he intestinal barrier is endowed with detection and defense mechanisms to achieve tolerance to commensal microorganisms and protection against invading microorganisms (1). Invasion by extracellular and intracellular pathogens is sensed by various signaling pathways converging to activate NF-B, a member of the Rel family of transcription factors involved in the activation of a large number of genes in response to pathogens, stress signals and proinflammatory cytokines (2). Under nonstimulating conditions, NF-B is retained in the cytoplasm through its association with inhibitory proteins (I Bs). A variety of signaling pathways activate I B kinases to phosphorylate I Bs, leading to ubiquitination of phospho-I Bs and their degradation by the proteasome (3), which allows translocation of NF-B to the nucleus, activation of NF-B-regulated genes, and establishment of an inflammatory response.Ubiquitination, resulting in the covalent attachment of the 76-residue ubiquitin to target proteins, involves three sequential steps performed by one ubiquitin-activating enzyme (E1), a limited number of ubiquitin-conjugating enzymes (E2s; also known as Ubc in enzyme designations), and a large number of ubiquitin-ligating enzymes (E3s), respectively (4). Each E3 recognizes a set of substrates and cooperates with one or a few E2s. The E3 complex SCF ␤-TrCP , which promotes ubiquitination of phospho-I B␣, consists of five proteins: the scaffold protein Cullin1, the adaptor protein Skp1, the RING domain protein Roc1, the E2 UbcH5b, and the F box protein ␤-TrCP, which interacts with phospho-I B␣ (5).Bacteria of Shigella spp. are the agent of shigellosis in humans, a disease characterized by the destruction of the colonic epithelium that is responsible for 1 million deaths per year (6). These bacteria use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages (7). TTS systems comprise (i) a secretion apparatus that spans the...