笹川 千尋

The gut epithelium self-renews every several days, providing an important innate defense system that limits bacterial colonization. Nevertheless, many bacterial pathogens, including Shigella, efficiently colonize the intestinal epithelium. Here, we show that the Shigella effector IpaB, when delivered into epithelial cells, causes cell-cycle arrest by targeting Mad2L2, an anaphase-promoting complex/cyclosome (APC) inhibitor. Cyclin B1 ubiquitination assays revealed that APC undergoes unscheduled activation due to IpaB interaction with the APC inhibitor Mad2L2. Synchronized HeLa cells infected with Shigella failed to accumulate Cyclin B1, Cdc20, and Plk1, causing cell-cycle arrest at the G2/M phase in an IpaB/Mad2L2-dependent manner. IpaB/Mad2L2-dependent cell-cycle arrest by Shigella infection was also demonstrated in rabbit intestinal crypt progenitors, and the IpaB-mediated arrest contributed to efficient colonization of the host cells. These results strongly indicate that Shigella employ special tactics to influence epithelial renewal in order to promote bacterial colonization of intestinal epithelium.

Live attenuated Shigella vaccines elicit protective immune responses, but involve a potential risk of inducing a strong inflammatory reaction. The bacterial invasiveness that is crucial for Ag delivery causes inflammatory destruction of infected epithelial cells and proinflammatory cell death of infected macrophages. In this study, the noninvasive Shigella mutant Delta ipaB was equipped with Yersinia invasin protein, which has been shown to mediate bacterial invasion and targeting to M cells located in follicle-associated epithelium. Invasin-expressing Delta ipaB (Delta ipaB/inv) was internalized into epithelial. cells and retained in the intraphagosomal space. Delta ipaB/inv did not induce necrotic cell death of infected macrophages nor cause symptomatic damage after intranasal vaccination of mice. Delta ipaB/inv was safer and more effective than the conventional live vaccine, Delta virG. Infection by Delta ipaB/inv caused polymorphonuclear neutrophil infiltration in the lung, but did not induce production of large amounts of proinflammatory cytokines. We concluded that the low experimental morbidity and high vaccine efficacy of Delta ipaB/inv are primarily based on high protective immune responses, which may be enhanced by the polymorphonuclear neutrophil infiltration unaccompanied by tissue injury.

Autophagy is a conserved membrane-traffic pathway in eukaryotic cells that sequesters cytoplasmic components and delivers them to lysosomes. Recent research indicates that the degradation of undesirable or recyclable cytoplasmic components and organelles through autophagy plays a pivotal role as an intracellular surveillance system for recognition and eradication of pathogens that have invaded the cytoplasm. Many invasive bacteria, however, have highly evolved mechanisms to circumvent cellular autophagy. Indeed, recent reports describe intracellular pathogens as being capable of subverting or modifying autophagy activation and persisting within autophagosomes.

The degradation of undesirable cellular components or organelles, including invading microbes, by autophagy is crucial for cell survival. Here, Shigella, an invasive bacteria, was found to be able to escape autophagy by secreting IcsB by means of the type III secretion system. Mutant bacteria lacking IcsB were trapped by autophagy during multiplication within the host cells. IcsB did not directly inhibit autophagy. Rather, Shigella VirG, a protein required for intracellular actin-based motility, induced autophagy by binding to the autophagy protein, Atg5. In nonmutant Shigella, this binding is competitively inhibited by IcsB binding to VirG.

The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of infections, including diarrhea, and expression of the genes of the locus of enterocyte effacement (LEE) is thought to be crucial for adherence. To identify genes involved in modulating the adherent capacity, bacteria collected from an EHEC O157:H7 strain (O157Sakai) mutagenized by mini-Tn5Km2 were screened for their ability to increase the number of microcolonies (MC) on Caco-2 cells and eight mutants with increased adherence were isolated. Analysis of the mini-Tn5Km2-flanked DNA sequences indicated that one possessed the insertion within an O157 antigen gene cluster, another possessed the insertion within the yhiF gene, and the remaining six mutants had their insertions in the yhiE gene. yhiE and yhiF products share amino acid homology (23% identity) to each other and with members of the LuxR family, which are known as transcriptional regulatory proteins. The mutant having the insertion within the O157 antigen gene cluster did not express the O157 side chain (as determined by agglutination test and immunoblotting with polyclonal O157-specific antiserum), unlike the other seven mutants. Importantly, the other mutants showed enhanced type III secretion. Levels of the related mRNAs of genes of the LEE, but not that of ler mRNA, were also increased compared with those in the wild type. Indeed, when we introduced an in-frame deletion into the yhiE or yhiF gene in O157Sakai, the capacity of the resultant mutants to adhere to Caco-2 cells was greatly increased. When one of the yhiE insertion mutants was orally inoculated into ICR mice, the number of bacteria shed into feces by day 14 was greater than that for the wild type. These results suggest that yhiE and yhiF are involved in the adherence of O157Sakai to epithelial cells as negative regulators for the expression of the genes required for the type III secretion system.

Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhoea in humans. Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells. We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407 or Caco-2. Although EPEC did adhere to some cell lines derived from non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence. In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells. Furthermore, Citrobacter rodentium expressing BFP adhered to Caco-2 cells much more efficiently than to CMT-93 cells. Finally, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence. Following BFP-mediated adherence, secretion of EspB from adherent bacteria and reorganization of F-actin in the host cells was observed. EPEC adhering to CMT-93 cells induced far less secretion of EspB, or reorganization of F-actin in the host CMT-93 cells, than did EPEC adhering to Caco-2 cells. These results indicated that BFP plays an important role in the cell-type-dependent adherence of EPEC and in the progression to the later steps in EPEC adherence.

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.

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% identify 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.

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 47.7min 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 φ21. 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. Copyright (C) 2000 Elsevier Science B.V.

The enterohemorrhagic Escherichia coli (EHEC) 0157: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 0157: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 0157: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.

The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pill (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5 alpha and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.

Although the Cram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium. the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using Tn916 insertion mutagenesis or an isogenic 5. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with 5. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of 5 pyogenes did not permit internalization of the E. coli cells.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) binds to fibronectin via protein P. In this study, we have investigated the binding properties of protein F to various multimeric tissue forms of fibronectin that appear on cell surfaces and in the extracellular matrix, We show that binding of S. pyogenes through protein F is more efficient to an in vitro-derived polymerized form of fibronectin (superfibronectin) than to soluble fibronectin immobilized ins a solid phase, In addition, Chinese hamster ovary cells overexpressing the alpha(5) beta(1) integrin produced an increased amount of a fibronectin matrix and consequently bound a higher number of S. pyogenes cells. Inhibition and direct binding assays using purified Proteins demonstrated that binding to a fibronectin matrix involved both domains of protein F (UR and RD2) that have previously been implicated in interactions with fibronectin, Using intact S. pyogenes bacteria in which various domains of protein F were expressed as hybrids with the surface-exposed region of an unrelated protein, we revealed that, in contrast to the predominantly UR-mediated binding to soluble fibronectin, the maximal binding to the fibronectin matrix required RD2 inn addition to UR. Since in some infections a pyogenes may initially encounter a matrix form of fibronectin, these results suggest that UR and RD2 may be important for the initiation of streptococcal infectious processes.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild-type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus-plaque-forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci, The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.

A virG thyA double mutant of shigella flexneri 2a was constructed as a candidate live-attenuated oral vaccine. In the keratoconjunctivitis model it did not provoke any adverse reaction by itself on guinea pigs' eyes and completely protected them from provoking keratoconjunctivitis. When (2.7-4.8) x 10(10) of the vaccine was inoculated intragastrically after 1 day fasting in cynomologus monkeys three times at weekly intervals, a watery stool was observed at 40% as a side-effect. Upon intragastric challenge after 1 day fasting with 7.5 x 10(9) of the virulent strain four weeks after the last vaccination, a statistically, significant difference was obtained in the mortality rate but not in the morbidity, rate between the vaccine and the control group, although the clinical findings were less severe in the vaccine group than in the control. These results together with the histopathological and immunological findings indicate that the vaccine deserve further detailed studies.

N1308, a chromosomal Tn5 mutant of Shigella flexneri 2a, was described previously as a lipopolysaccharide (LPS) mutant with a short O side chain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers and was negative in the Sereny test. In this study, the wild-type locus inactivated in N1308 was cloned and further defined by means of complementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a YSH6200 DNA was sufficient to restore both normal LPS and virulence phenotype to the mutant. DNA sequencing of this region revealed four genes, rfbA, rfbB, rfbC, and rfbD, encoding the enzymes required for the biosynthesis of activated rhamnose. The four genes were expressed in Escherichia coli, and the expected protein products were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was shown to have normal levels of surface IpaC and IpaD, while a Western blot (immunoblot) of whole-cell lysates or outer membrane fractions indicated an elevated level of appropriately localized VirG. An in vitro invasion assay revealed that N1308 had normal primary invasive capacity and was able to multiply and move normally within the initial infected cell. However, it exhibited a significant reduction in its ability to spread from cell to cell in the monolayer. A double immunofluorescence assay revealed differences between LLC-MK2 cells infected with the wild-type YSH6000 and those infected with N1308. The wild-type bacteria elicited the formation of the characteristic F actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing deposition of F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of the cytoskeletal protein.

Expression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature-regulated. virB transcription is under the control of VirF and H-NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30 degrees C and 37 degrees C was isolated using miniTn 10-kan (miniKAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of II amino acids to the C-terminus of the Rho protein. Consequently, decreased transcription termination activity at a rho-dependent terminator, lambda t(L1), was observed. In the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30 degrees C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoter, since the effects of the mutation on virB transcription were abolished when its promoter region was replaced by the tao promoter. Temperature-responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30 degrees C as well as at 37 degrees C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature-dependent changes in DNA structure, thus contributing to thermoregulation of virB transcription.

The 7-kb region 5 on the large 230-kb plasmid pMYSH6000 in Shigella flexneri 2a YSH6000 is one of the virulence-associated DNA segments required for the invasion of epithelial cells (C. Sasakawa, K. Kamata, T. Sakai, S. Makino, M. Yamada, N. Okada, and M. Yoshikawa, J. Bacteriol. 170:2480-2484, 1988). To elucidate the functional organization of region 5 and to determine the virulence-associated genes encoded by region 5, we performed insertion and deletion mutagenesis, DNA subcloning, and complete nucleotide sequencing of region 5 and found that region 5 contained 11 open reading frames (ORFs) named ORF-1 through ORF-11 which could be translated into proteins with molecular masses of 15.1, 47.5, 13.2, 33.0, 33.4, 24.2, 9.4, 28.5, 39.9, 9. 1, and 10.4 kDa, respectively. Complementation tests of the 14 Tn5-induced noninvasive mutants of region 5 with the above plasmid constructs have indicated that region 5 consists of an operon and that ORF-2 through ORF-9, but not ORF-1, ORF-10, and ORF-11, are essential for invasion, and 7 of 8 ORFs (ORF-2 and ORF-4 through ORF-9) and presumably the remaining ORF (ORF-3) are required for secretion of the Ipa proteins. The transcriptional organization, as determined by a promoter-proving vector, S1 nuclease protection, and primer extension RNA sequencing analysis revealed that region 5 is transcribed from a promoter located 47 bp upstream of the 5' end of ORF-2 for the 47.5-kDa protein and that the promoter activity identified was regulated by the virB gene, the transcriptional activator on the 230-kb plasmid.

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulse-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.