笹川 千尋

In response to bacterial infection, epithelial cells undergo several types of cell death, including apoptosis, necrosis, pyroptosis, and necroptosis, which serve to expel the infected cells and activate the innate and acquired immune responses. Shigella initially invades macrophages and subsequently surrounding enterocytes the pathogen executes macrophage cell death but prevents epithelial cell death in order to maintain its foothold for replication. To this end, Shigella delivers versatile effector proteins via the type III secretion system (T3SS), allowing it to efficiently colonize the intestinal epithelium. In this article, we review insights into the mechanisms underlying circumvention of the host cell death by Shigella, as an example of bacterial fine-tuning of host cell death pathways.

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

Host cells deploy multiple defences against microbial infection. One prominent host defence mechanism, the death of infected cells, plays a pivotal role in clearing damaged cells, eliminating pathogens, removing replicative niches, exposing intracellular bacterial pathogens to extracellular immune surveillance and presenting bacteria-derived antigens to the adaptive immune system. Although cell death can occur under either physiological or pathophysiological conditions, it acts as an innate defence mechanism against bacterial pathogens by limiting their persistent colonization. However, many bacterial pathogens, including Shigella, have evolved mechanisms that manipulate host cell death for their own benefit.

粘膜上皮は病原細菌に対しさまざまな自然免疫応答の発動により感染を防御する。しかしながら赤痢菌をはじめとする多くの粘膜病原細菌はこの自然免疫による攻撃を回避・抑制する戦略を有しており感染を成立させる。本稿では赤痢菌の自然免疫克服戦略について概略する。(著者抄録)

The gut mucosa acts as a barrier against microbial invaders, whereas resident commensal and foreign invading bacteria interact intimately with the gut epithelium and influence the host cellular and immune systems. The epithelial barrier serves as an infectious foothold for many bacterial pathogens and as an entry port for pathogens to disseminate into deeper tissues. Enteric bacterial pathogens can efficiently infect the gut mucosa using highly sophisticated virulence mechanisms that allow bacteria to circumvent the defense barriers in the gut. We provide an overview of the components of the mucosal barrier and discuss the bacterial stratagems that circumvent these barriers with particular emphasis on the roles of bacterial effector proteins. © 2011 Nature America, Inc. All rights reserved.

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyer's patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naïve, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes-stimulated CD11c+ dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-β, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c+ DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (Muco-Rice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates. Copyright © 2009 by The American Association of Immunologists, Inc.

病原細菌の多くは粘膜上皮細胞を足場として感染し、局所あるいは全身で増殖してさまざまな疾患をひき起こす。赤痢菌などのグラム陰性病原菌はIII型分泌装置からエフェクター蛋白質とよばれる一連の病原因子を分泌して宿主細胞高次機能の調節や修飾を行い、これらの総合作用によって感染を成立させる。一方、宿主の粘膜には上皮バリアーと免疫バリアーを主体とする固有の生体防御機構が構築されており、微生物の侵入を阻止している。しかし、赤痢菌をはじめとする粘膜病原細菌は、これらのバリアー機能を巧みに回避・抑制して、粘膜上皮へ感染・定着することができる。本稿では赤痢菌の分泌するエフェクター蛋白質の機能を概説しながら、赤痢菌と宿主細胞との相互作用に関して紹介する。(著者抄録)

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that promotes the development of T-helper lymphocyte 1 (Th1). Chronic gastritis induced by Helicobacter pylori is considered a Th1-mediated process. IL-12 levels in gastric biopsy samples of H. pylori-infected patients are higher than in those of uninfected individuals, but the cellular source of IL-12 remains elusive. IL-12 staining was detected in mucosal epithelial cells, lymphocytes, and macrophages in specimens of patients with H. pylori-positive gastritis. Therefore, we investigated IL-12 p40 mRNA induction by H. pylori in gastric epithelial cells and T cells. Although cag pathogenicity island (PAI)-positive H. pylori induced IL-12 p40 mRNA expression, an isogenic mutant of the cag PAI failed to induce it in both cell types. Supernatants from H. pylori cultures and H. pylori VacA induced IL-12 p40 mRNA expression in T cells but not in epithelial cells. The activation of the IL-12 p40 promoter by H. pylori was mediated through NF-κB. The transfection of IκB kinase and NF-κB- inducing kinase dominant-negative mutants inhibited H. pylori-induced IL-12 p40 activation. Inhibitors of NF-κB, phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase, and Hsp90 suppressed H. pylori- and VacA-induced IL-12 p40 mRNA expression. The results indicate that H. pylori induces IL-12 p40 expression by the activation of NF-κB, phosphatidylinositol 3-kinase, and p38 mitogen-activated protein kinase. Hsp90 is also a crucial regulator of H. pylori-induced IL-12 p40 expression. In addition to the cag PAI, VacA might be relevant in the induction of IL-12 expression and a Th1-polarized response only in T cells. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Background. The inflammatory response in Helicobacter pylori-infected gastric tissue is mediated by cag pathogenicity island (PAI)-dependent activation of nuclear factor-κB (NF-κB). Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is known to play a role in NF-κB activation, but little information is available on the relationship between H. pylori and PI3K/Akt signaling in gastric epithelial cells. We examined whether H. pylori activates Akt in gastric epithelial cells, the role of cag PAI in this process and the role of Akt in regulating H. pylori-induced NF-κB activation. Results. Phosphorylated Akt was detected in epithelial cells of H. pylori-positive gastric tissues. Although Akt was activated in MKN45 and AGS cells by coculture with cag PAI-positive H. pylori strains, a cag PAI-negative mutant showed no activation of Akt. H. pylori also induced p65 phosphorylation. PI3K inhibitor suppressed H. pylori-induced p65 phosphorylation and NF-κB transactivation, as well as interleukin-8 expression. Furthermore, transfection with a dominant-negative Akt inhibited H. pylori-induced NF-κB transactivation. Transfection with small interference RNAs for p65 and Akt also inhibited H. pylori-induced interleukin-8 expression. Conclusion. The results suggest that cag PAI-positive H. pylori activates Akt in gastric epithelial cells and this may contribute to H. pylori-mediated NF-κB activation associated with mucosal inflammation and carcinogenesis.

Bacterial invasion of eukaryotic cells and host recognition and killing of the invading bacteria are a key issue in determining the fate of bacterial infection. Once inside host cells, pathogenic bacteria often modify the phagosomal compartment or enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Cytosolic invaders, however, are monitored by host innate immune systems, such as mediated by Nod/CARD family proteins, which induce inflammatory responses via activation of NF-κB. Furthermore, recent studies indicate that autophagy, a major cytoplasmic degradation system that eliminates cytosolic protein and organelles, also recognizes invading bacteria. Indeed, unless they are able to circumvent entrapping by autophagic membranes, bacteria targeted by autophagy ultimately undergo degradation by delivery into autolysosomes. In this article, we review recent advances in understanding of Shigella strategies to infect epithelial cells, and then focus on recent studies of an intriguing bacterial survival strategy against autophagic degradation. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd.

Bacterial invasion of eukaryotic cells, and host recognition and elimination of the invading bacteria, determines the fate of bacterial infection. Once inside mammalian cells, many pathogenic bacteria enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Recent studies indicate that autophagy also recognizes intracellular bacteria. Although autophagy is a conserved membrane trafficking pathway in eukaryotic cells that sequesters undesirable or recyclable cytoplasmic components or organelles and delivers them to lysosomes, autophagy has recently been described as playing a pivotal role as an intracellular surveillance system for recognition and eradication of the pathogens that have invaded the cytoplasm. Indeed, unless they are able to circumvent entrapping by autophagosomes, bacteria ultimately undergo degradation by delivery into autolysosomes. In this review we discuss recent discoveries regarding Shigella strategies for infecting mammalian cells, and then focus on recent studies of an elegant bacterial survival strategy against autophagic degradation. ©2006 Landes Bioscience.

オートファジーは細胞内において恒常的に起きている現象であり,細胞内の不要なタンパク質やオルガネラの定常的な代謝回転に寄与していると考えられている.近年,哺乳類細胞におけるオートファジーの分子機構に関する研究が急速に発展を遂げたことをきっかけに,宿主細胞の感染防御の最前線においてオートファジーが重要な役割を担っているという考え方が認知され始めている(著者抄録)

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is critical for initiation of a bacterial infection. An in vitro infection study previously indicated that EHEC bacteria initially adhere diffusely and then proliferate to develop MC, a process that is mediated by various secreted proteins, such as EspA, EspB, EspD, Tir, and intimin, as well as other putative adherence factors. In the present study, we investigated the role of a large 93-kb plasmid (pO157) in the adherence of O157:H7 (O157Sakai) and found the toxB gene to be involved in the full adherence phenotype. A pO157-cured strain of O157Sakai (O157Cu) developed microcolonies on Caco-2 cells; however, the number of microcolonies was lower than that of O157Sakai, as were the production and secretion levels of EspA, EspB, and Tir. Introduction of a mini-pO157 plasmid (pIC37) composed of the toxB and ori regions restored full adherence capacity to O157Cu, including production and secretion of the proteins. In contrast, introduction of a pO157 mutant possessing toxB::Km into O157Cu could not restore the full adherence phenotype. Expression of truncated versions of His-tagged ToxB also promoted EspB production and/or secretion by O157Cu. These results suggest that ToxB contributes to the adherence of EHEC to epithelial cells through promotion of the production and/or secretion of type III secreted proteins.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH9.8 is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH9.8 is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH9.8 is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH9.8 was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH9.8 does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like β-catenin, IpaH9.8 secreted from intracellular Shigella can be transported into the nucleus.

We investigated the supramolecular structure of the Shigella type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F-1-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures;with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP-depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP bur not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP-actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein-tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-Arp2/3 complex formed on intracellular Shigella.

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immuno-dominant,, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging frost 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [S-35]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wildtype H. pylori bur not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylon CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

The complete nucleotide sequence and organization of the. enteropathogenic Escherichia coli (EPEC) adherence factor (EAP) plasmid of EPEC strain B171 (O111:NM) were determined, The EAF plasmid encodes two known virulence-related operons, the bfp operon, which is composed of genes necessary for biosynthesis of bundle-forming pill, and the bfpTVW (perABC) operon, composed of regulatory genes required for bfp transcription and also for transcriptional activation of the eae gene in the LEE pathogenicity island on the EPEC chromosome, The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the bfp and bfpTVW (perABC) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E. coli, Another ORF, located between the bfp and bfpTVW operons, showed high similarity with trcA, a bfpT-regulated chaperone-like protein gene of EPEC, Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1), In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha(5) beta(1) integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamster ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125(FAK) and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellula signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

The VirG (IcsA) protein of Shigella is required for recruitment of host actin filament (F-actin) by intracellularly motile bacteria. An N-terminal 80-kDa VirG portion (alpha-domain) is exposed on the bacterial surface, while the following C-terminal 37-kDa portion (beta-core) is embedded in the outer membrane. Here, we report that the surface exposed alpha-domain of VirG possesses two distinct functional domains; one is the N-terminal two-thirds portion of the alpha-domain which is required for eliciting F-actin assembly on the bacteria in infected cells, and the other one is the rest of the C-terminal portion of the VirG alpha-domain, which is essential for the asymmetric distribution of VirG on the bacterial surface, Furthermore, we found that vinculin, an actin-binding cytoskeletal protein, accumulates on the surface of bacteria expressing VirG in infected cells, and that the distribution of vinculin coincided with the distribution of VirG and assembled F-actin. The vinculin accumulation depended on the expression of the alpha-domain VirG portion required for F-actin assembly, but the recruitment of vinculin on Shigella appeared prior to the appearance of F-actin in the infected cells. Analysis of proteins interacting with VirG using Xenopus laevis eggs extracts revealed that vinculin was a protein that bound to the alpha-domain portion. This was further confirmed using purified chicken gizzard vinculin, in that the 95-kDa vinculin head part, but not the 30-kDa tail part, directly bound to the alpha-domain portion. These results suggest a possible role for vinculin in recruitment of F-actin to the VirG moiety exposed on Shigella in infected mammalian cells.

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha(5) beta(1) integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha(5) beta(1) integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125(FAK), an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha(5) beta(1) integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha(5) beta(1) integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha(5) beta(1) integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10-flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB. Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.

The ability of Shigella to spread within and between epithelial cells is a prerequisite for causing bacillary dysentery and requires the function encoded by the virG gene on the large plasmid, The outer membrane VirG (IcsA) protein is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells, Recent studies have indicated that an N-terminal 80-kDa VirG portion is exposed on the bacterial cell surface and released into the external medium, while the following 37-kDa C-terminal portion is embedded in the outer membrane, although little is known about the extracellular transport of the VirG protein, In this study, we attempted to elucidate the export pathway of VirG protein across the outer membrane and found that the C-terminal 37-kDa portion, termed VirG beta-core, serves as the self transporter for the secretion of the preceding 80-kDa portion from the periplasmic side of the outer membrane to the external side, Indeed, foreign polypeptides such as MalE or PhoA covalently linked to the N terminus of VirG beta-core were transported to the external side of the outer membrane, and it was further shown that the folding structure of the passenger polypeptide at the periplasmic side of the outer membrane interferes with its translocation, Analysis of the secondary structure of VirG beta-core predicted that the critical structural property was a beta-barrel channel consisting of amphipathic antiparallel transmembrane beta-strands, interspersed by hairpin turns and loops. These results thus strongly suggest that the secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria.

The invasion of colonic epithelial cells by Shigella, an early essential step for causing bacillary dysentery, is mediated by the IpaB, IpaC and IpaD proteins, Secretion of the Ipa proteins from Shigella requires functions encoded by the mxi and spa loci. In this study, we show that contact between the bacteria and epithelial cell triggers release of the Ipa proteins into the external medium, which results in a rapid decrease in levels of Ipa proteins presented on the cell surface. When the bacteria were used to infect polarized Caco-2 cells, release of Ipa proteins occurred efficiently from bacteria interacting with the basolateral surface rather than with the apical surface. Moreover, the interaction of bacteria with components of the extracellular matrix, such as fibronectin, laminin or collagen type IV, also stimulates the release of Ipa proteins. The release of Ipa proteins from Shigella required the surface-located Spa32 protein encoded by one of the spa genes on the large plasmid.

Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S. flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.

Transcription of the virB gene, a transcriptional regulator of invasion genes on the large plasmid of shigella flexneri, is strictly regulated by growth temperature; when bacteria are grown at 37 degrees C, virB transcription is highly activated, while at 30 degrees C the level of virB transcription decreases to less than 5% of that at 37 degrees C. Transcription from the virB promoter is activated by VirF, which is encoded on the same plasmid, in a DNA superhelicity-dependent manner (T. Tobe, M. Yoshikawa, T. Mizuno, and C. Sasakawa, J. Bacteriol. 175:6142-6149, 1993). Here we provide evidence supporting the involvement of negative superhelicity in the thermoregulation of virB transcription. A local negatively supercoiled domain in the virB promoter region was created by activating a divergent transcription from the T7 RNA polymerase dependent promoter, phi 10, which was placed upstream of the virB promoter in the opposite orientation. Transcription from the virB promoter was activated even at 30 degrees C by induction of divergent transcription, Levels of virB transcription correlated with levels of expressed T7 RNA polymerase. Transcriptional activation of virB by the system depended completely upon VirF function, The level of virB transcription achieved by introducing a negatively supercoiled domain was enough to give rise to expression of invasion capacity at 30 degrees C. These results indicated that the repression of virB transcription at 30 degrees C was caused by a reduction in negative superhelicity around the virB promoter region at 30 degrees C.