rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells.

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 alpha5beta1 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 hamister 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 pp125FAK 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 cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into 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 alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, 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, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

Rho family GTPase Cdc42 is essential for the actin-based motility of Shigella in mammalian cells.

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 but 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.

Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

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 immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 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 [(35)S]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 wild-type H. pylori but 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. pylori 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.

Kinetics of v-src-induced epithelial-mesenchymal transition in developing glandular stomach.

The oncogene function in primary epithelial cells is largely unclear. Recombination organ cultures in combination with the stable and transient gene transfer techniques by retrovirus and electroporation, respectively, enable us to transfer oncogenes specifically into primary epithelial cells of the developing avian glandular stomach (proventriculus). In this system, the epithelium and mesenchyme are mutually dependent on each other for their growth and differentiation. We report here that either stable or transient expression of v-src in the epithelium causes budding and migration of epithelial cells into mesenchyme. In response to the transient expression of v-Src or a constitutive active mutant of MEK, we observed immediate downregulation of the Sonic hedgehog gene and subsequent elimination of E-cadherine expression in migrating cells, suggesting the involvement of MAP kinase signaling pathway in these processes. v-src-expressing cells that were retained in the epithelium underwent apoptosis (anoikis) and detached from the culture. Continuous expression of v-src by, for example, Rous sarcoma virus (RSV) was required for the epithelial cells to acquire the ability to express type I collagen and fibronectin genes (mesenchymal markers), and finally to establish the epithelial-mesenchymal transition. These observations would partly explain why RSV does not apparently cause carcinoma formation, but induces sarcomas exclusively.

Sequences essential for IS50 transposition. The first base-pair.

Sequences near the ends of the insertion element IS50 are essential for its transposition, probably because they serve as sites upon which the IS50-encoded transposase protein acts. To determine if these essential sequences include the first base-pair at each end of IS50 we generated 5'C to 5'G transversions at these positions. Each mutation reduced the transposition frequency to 1% to 2% of wild-type. DNA sequence analyses showed that the mutant 5'G is preserved during transposition.

Localization of action of the IS50-encoded transposase protein.

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp+), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp- IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules.

Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12.

Escherichia coli O157:H7 is a major food-borne infectious pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Here we report the complete chromosome sequence of an O157:H7 strain isolated from the Sakai outbreak, and the results of genomic comparison with a benign laboratory strain, K-12 MG1655. The chromosome is 5.5 Mb in size, 859 Kb larger than that of K-12. We identified a 4.1-Mb sequence highly conserved between the two strains, which may represent the fundamental backbone of the E. coli chromosome. The remaining 1.4-Mb sequence comprises of O157:H7-specific sequences, most of which are horizontally transferred foreign DNAs. The predominant roles of bacteriophages in the emergence of O157:H7 is evident by the presence of 24 prophages and prophage-like elements that occupy more than half of the O157:H7-specific sequences. The O157:H7 chromosome encodes 1632 proteins and 20 tRNAs that are not present in K-12. Among these, at least 131 proteins are assumed to have virulence-related functions. Genome-wide codon usage analysis suggested that the O157:H7-specific tRNAs are involved in the efficient expression of the strain-specific genes. A complete set of the genes specific to O157:H7 presented here sheds new insight into the pathogenicity and the physiology of O157:H7, and will open a way to fully understand the molecular mechanisms underlying the O157:H7 infection.

A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis.

A series of variants of transposon Tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. A thermosensitive replication mutant of the broad-host-range transmissible plasmid R388 was also constructed for use as a suicide vector for the delivery of transposable elements.

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans.

The structural gene (pag gene) for a 210 kDa protein antigen of Streptococcus sobrinus serotype g was cloned and compared with that (pac gene) of a 190 kDa protein antigen of Streptococcus mutans serotype c. Immunodiffusion analysis revealed that the product of the pag gene immunologically cross-reacted with that of the pac gene. Southern blot and nucleotide sequence analyses revealed that a significant homology existed between the middle regions of the two structural genes.

Evolutionary perspective on a composite Shigella flexneri 2a virulence plasmid-borne locus comprising three distinct genetic elements.

Nucleotide sequence analysis of a Shigella flexneri 2a virulence plasmid-borne locus revealed that it comprised three distinct genetic elements: a stretch of colicin 1a/1b-linked sequence, a truncated IS911 element, and a third element containing two ORFs that shared a high level of similarity to a Salmonella-specific chromosomal sequence. Examination of other known IS911-like sequences showed that these sequences also were frequently associated with other accessory elements and appeared to be prone to partial deletion events. Analysis of the data led to a model of the evolution of this unusual composite locus.

Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937.

Shigella flexneri, but not a non-invasive mutant derivative rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-gamma (IFN gamma) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFN gamma-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RA-differentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFN gamma-pretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state.

Diversity of DNA sequences among Vibrio cholerae O139 Bengal detected by PCR-based DNA fingerprinting.

Vibrio cholerae O139, a causative agent of a large epidemic of cholera-like illness, has suddenly emerged and spread widely over several months. To investigate the characteristics unique to O139, traditional typing techniques for V. cholerae, such as biochemical characteristics, antibiotic susceptibility and detection of toxin production, were performed, with the result that 145 O139 strains, except for two O139 strains isolated from Argentina and Germany, were indistinguishable from O1 strains. Thus, in order to clarify the genetical relatedness among O139 strains, and between O139 and O1 strains, the RAPD (random amplified polymorphic DNA) DNA fingerprinting method was undertaken. Although the RAPD arrays in five O139 isolates from Vellore with one arbitrary primer were slightly different from the other O139 strains, the RAPD patterns of the 145 forty-five O139 strains except for two O139 strains from Argentina and Germany were quite similar to each other, but were different from those of O1 strains, indicating that those O139 epidemic strains are closely related to each other regardless of their place of isolation. Furthermore, the RAPD patterns of the O139 strains resembled those of E1 Tor strains rather than classical strain, and a small change in the RAPD pattern of O139 strains occurred during subculture for 200 generations. These results taken together suggested that O139 V. cholerae have emerged from a common origin associated with the E1 Tor strain.

A spontaneous 99-kb chromosomal deletion results in multi-antibiotic susceptibility and an attenuation of contact haemolysis in Shigella flexneri 2a.

A Tn5-generated mutant (strain S2430) of Shigella flexneri 2a (strain YSH6000) exhibited attenuated virulence and, in addition to the Tn5 insertion in the SalI K fragment of its virulence plasmid, had a 99-kb deletion within its chromosome. Unlike its wild-type parent, strain S2430 was susceptible to ampicillin, streptomycin, tetracycline and chloramphenicol. An independent multi-antibiotic susceptible variant of strain YSH6000 had a similar deletion. Southern blot analysis of pulsed field electrophoresis gels enabled the sizing of this deletion and its mapping to a region of the chromosome on NotI fragment D bounded by the S. flexneri homologues of ompA and pyrC. Hybridisation experiments with a probe specific to the multi-antibiotic resistance region indicated that this large deletion was responsible for antibiotic susceptibility. Both strain S2430 and a derivative of the antibiotic-susceptible variant, with a Tn5 insertion in its SalI K fragment, exhibited an equal reduction in contact haemolysis compared with the Tn5-bearing derivative of strain YSH6000. However, strain S2430 alone clearly displayed delayed plaque forming ability in LLC-MK2 monolayers, suggesting that the two examples of this deletion may not be identical.

Uptake pathways of clinical and healthy animal isolates of Campylobacter jejuni into INT-407 cells.

Campylobacter jejuni isolates obtained from human and animal sources showed different invasion levels into human embryonic intestinal (INT-407) cells. There was no significant relation between the degree of invasion and cytotoxins production. The depolymerization of both microfilaments by cytochalasin-D and microtubules by colchicine, demecolcine and nocodazole or stabilization of microtubules by paclitaxel reduced the invasiveness of C. jejuni, although microfilament depolymerization showed greater inhibition than microtubule depolymerization. Interference with receptor-mediated endocytosis by G-strophanthin and monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular C. jejuni cells. Furthermore inhibition of only host protein kinases by staurosporine, but not phosphoinositide 3-kinase by wortmannin or protein kinase-C by calphostin-C, significantly reduced invasion of epithelial cells by C. jejuni. These data suggest that the internalization mechanism triggered by C. jejuni is strikingly different from the microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria such as enteroinvasive strains of Escherichia coli, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica and Yersinia pseudotuberculosis.

Enhanced anti-Shigella activity of erythromycin supplemented with sulfadiazine.

Sulfadiazine enhanced the anti-Shigella activity of erythromycin. Erythromycin passes through the type III secretion apparatus and suppresses the growth of invasive Shigella organisms. Sulfadiazine enhanced this effect at the concentration under minimum inhibitory concentration and it came from not only the folate-inhibiting activity but also from a new function. It has proved that sulfadiazine stimulated type III secretion in Shigella as determined from the secretion of the pathogenic protein IpaB. As Congo red induced secretion of Ipa proteins and uptake of erythromycin through the type III secretion gate, sulfadiazine which is similar to Congo red in chemical structure may induce the uptake in the same way.

Effect of erythromycin on Shigella infection of Caco-2 cells.

Erythromycin (EM), one of the macrolides, shows a dose-dependent effect on Shigella flexneri invasion of Caco-2 cells even at concentrations less than the minimum inhibitory concentration (subMIC). LS13, a strain of S. flexneri 1b, invaded Caco-2 cells in vitro. When the strain was treated with subMIC of EM, the invasion efficiency decreased. The carrier rate of the invasion plasmid containing virulence genes was reduced by EM treatment, as determined by the colony pigmentation test on Congo red agar plates. Presence of the invasion plasmid was found to increase susceptibility of the organisms to EM. The growth of virulent organisms carrying the invasion plasmid was inhibited at 25 microg ml(-1) of EM, whereas the growth of organisms without the plasmid was inhibited at 100 microg ml(-1) of EM. This was supported by the finding that the MIC of EM for a virulent isolate of S. flexneri 2a YSH6000 (6.25 microg ml(-1)) and for the mutant strain del-17 (50 microg ml(-1)), carrying the type III apparatus, impaired plasmid. These findings suggested that EM passed through the type III apparatus and suppressed the growth of invasive organisms selectively. This mechanism may account for the clinical effect of EM on shigellosis.

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak.

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.

Comparison of bacteriological, genetic and pathological characters between Escherichia coli O115a,c:K(B) and Citrobacter rodentium.

Murine pathogenic Escherichia coli O115a,c:K(B) (MPEC) is the causative agent of mouse megaenteron, the pathology of which resembles that of transmissible murine colonic hyperplasia caused by Citrobacter rodentium. We compared their genetic and pathological features to reveal the relationship between these two bacteria. To evaluate the genetic distances, 16S rDNA genes were sequenced and biochemical reactions were tested. Mouse strain susceptibility tests, using CF1 MPEC-susceptible germfree mice and BALB/cA(Jic) resistant mice were performed. MPEC strains and C. rodentium showed more than 99.6% identity by comparison of 16S rDNA gene sequences. All results from biochemical reactions and the mouse strain susceptibility tests were identical. It is proposed that MPEC should be reclassified as C. rodentium.