Distinctiveness of the genomic sequence of Shiga toxin 2-converting phage isolated from Escherichia coli O157:H7 Okayama strain as compared to other Shiga toxin 2-converting phages

Shiga toxin 2-converting phage was isolated from Escherichia coli O157:H7 associated with an outbreak that occurred in Okayama, Japan in 1996 (M. Watarai, T. Sato, M. Kobayashi, T. Shimizu, S. Yamasaki, T. Tobe, C. Sasakawa and Y. Takeda, Infect. Immun. 61 (1998) 3210-3204). In this study, we analyzed the complete nucleotide sequence of Shiga toxin 2-converting phage, designated Stx2phi-I, and compared it with three recently reported Stx2-phage genomes. Stx2phi-I consisted of 61,765 bp, which included 166 open reading frames. When compared to 933W, VT2-Sakai and VT2-Sa phages, six characteristic regions (regions I-VI) were found in the Stx2 phage genomes although overall homology was more than 95% between these phages. Stx2phi-I exhibited remarkable differences in these regions as compared with VT-2 Sakai and VT2-Sa genes but not with 933W phage. Characteristic repeat sequences were found in regions I-IV where the genes responsible for the construction of head and tail are located. Regions V and VI, which are the most distinct portion in the entire phage genome were located in the upstream and downstream regions of the Stx2 operons that are responsible for the immunity and replication, and host lysis. These data indicated that Stx2phi-I is less homologous to VT2-Sakai and VT2-Sa phages, despite these three phages being found in the strains isolated at the almost same time in the same geographic region but closely related to 933W phage which was found in the E. coli O157 strain 933W isolated 14 years ago in a different geographic area.     

Global changes in cellular gene expression during bacteriophage PRD1 infection

Virus-induced changes in cellular gene expression and host physiology have been studied extensively. Still, there are only a few analyses covering the entire viral replication cycle and whole-host gene pool expression at the resolution of a single gene. Here we report changes in Escherichia coli gene expression during bacteriophage PRD1 infection using microarray technology. Relative mRNA levels were systematically measured for over 99% of the host open reading frames throughout the infection cycle. Although drastic modifications could be detected in the expression of individual genes, global changes at the whole-genome level were moderate. Notably, the majority of virus-induced changes took place only after the synthesis of virion components, indicating that there is no major reprogramming of the host during early infection. The most highly induced genes encoded chaparones and other stress-inducible proteins.     

Genome analysis of a novel Shiga toxin 1 (Stx1)-converting phage which is closely related to Stx2-converting phages but not to other Stx1-converting phages

Two Stx-converting phages, designated Stx1 phi and Stx2 phi-II, were isolated from an Escherichia coli O157:H7 strain, Morioka V526, and their entire nucleotide sequences were determined. The genomes of both phages were similar except for the stx gene-flanking regions. Comparing these phages to other known Stx-converting phages, we concluded that Stx1 phi is a novel Stx1-converting phage closely related to Stx2-converting phages so far reported.     

Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance.     

Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d

Shiga toxin variant type 2d (Stx2d) produced by some strains of Shiga toxin-producing Escherichia coli is composed of an enzymatically active A subunit and a B (binding) pentamer. The cytotoxicity of Stx2d is increased (activated) 10-1000-fold for Vero cells when the toxin is incubated with mucus obtained from the small intestine of mice. In this study we isolated an Stx2d activator and identified it as a mouse elastase with strong homology to human elastase IIIB. Moreover, commercially available porcine pancreatic elastase preparations also activated Stx2d cytotoxicity although with a lower specific activity than isolated mouse elastase. Elastase directly nicked the Stx2d A subunit to A(1) and A(2), an event that did not correlate with activation. However, elastase also reduced the size and changed the isoelectric point of the A(2) peptide, as determined by SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis followed by Western immunoblot analysis. This elastase-mediated size and charge shift in the A(2) peptide of Stx2d occurred concurrently with activation of the toxin. Both the reduction in size of the Stx2d A(2) peptide by incubation with elastase as well as the associated activation of Stx2d cytotoxicity were fully inhibited by elastatinal, an elastase-specific inhibitor.     

Phylogenetic analysis of Shiga toxin 1 and Shiga toxin 2 genes associated with disease outbreaks

Background  

Shiga toxins 1 and 2 (Stx1 and Stx2) are bacteriophage-encoded proteins that have been associated with hemorrhagic colitis, hemolytic uremic syndrome and other severe disease conditions. Stx1 and Stx2 are genetically and immunologically distinct but share the same compound toxin structure, method of entry and enzymatic function.     

Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice

Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell.     

Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli may induce colonic ulceration, bloody diarrhoea and acute renal failure. The A subunit (StxA) is known to inhibit protein synthesis, whereas the B subunits (StxB) bind to Gb3 on the cell surface. However, the mechanisms by which Stxs kill target cells remain unclear. Stx1A or Stx1B genes were introduced into pcDNA3.1 vectors and transfected into NIH3T3 and HeLa cells. The Stx1B gene-transfected cells became apoptotic with accompanying DNA fragmentation, whereas the Stx1A gene-transfected cells were found to be necrotic and no DNA fragmentation occurred. The HeLa/C4 cells integrated with the Stx1B gene with a tetracycline-inducible promoter eventually produced cytoplasmic Stx1B, leading to DNA fragmentation on the addition of doxycycline. These apoptotic changes were abrogated by pretreatment with Z-VAD-fmk. These results suggest that the transfected Stx1B gene induces apoptosis by activating the caspase cascade after Stx1B expression in the cytoplasm.     

Roles of genes 44, 50, and 51 in regulating gene expression and host takeover during infection of Bacillus subtilis by bacteriophage SPO1

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of Bacillus subtilis by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44- single mutant or the 50(-)51(-) double mutant and more so by the 44(-)50(-)51(-) triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44(-)50(-)51(-) triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.     

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.     

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.Key words: adenovirus (HAdV), antiviral mechanism, virus-host interaction, Mdm2, Mdm4, mouse embryonic fibroblast (MEF), DNA-damage response, cell cycle, p21, cyclin D1     

PSK, a polysaccharide from Coriolus vesicolor, enhances oxygen metabolism of murine peritoneal macrophages and the host resistance to listerial infection

PSK, a protein-bound polysaccharide isolated from the basidiomycete Coriolus vesicolor (Fr.) Quél. was examined with regard to its effects of macrophage (M phi) oxygen metabolism in mice, a function important for the expression of M phi antimicrobial activity. The O2(-)-producing ability and chemiluminescence (CL) of host peritoneal M phi s in response to phorbol myristate acetate were markedly elevated by preinjection of PSK (1 or 5 mg per mouse intraperitoneally) around 4-7d before M phi-harvest. The enhanced O2(-)-producing ability due to PSK injection persisted much longer than the enhanced CL, indicating a discrepancy in regulation of generation of active oxygen species such as O2-, H2O2, OH, and 1O2. Daily injections of PSK (1 mg per injection) from 10 to 4d before M phi harvest did not increase the efficacy of PSK over that given by a single 1 mg injection. When PSK (5 mg) was given intraperitoneally to mice in a single injection 10, 7 or 4d before the intravenous Listeria monocytogenes inoculation, a similar increase in the host resistance to the bacteria was noted regardless of the timing of the injection. Multiple PSK injections fron 10 to 4d before the infection also enhanced the host resistance, to the same degree. Therefore, PSK is thought to augment the host resistance to certain intracellular parasites including L. monocytogenes at least to some extent by enhancing oxygen metabolism of the host M phi.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique

There has been no culture method of choice for detecting non-O157 Shiga toxin-producing Escherichia coli strains (STEC) because of their biochemical diversity The aim of this study was the assessment of verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. Stool cultures were performed on Tryptic Soy Broth and sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as Escherichia coli (E. coli) by BBL crystal. Further identifications were performed including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity, specificity of PCR were 100%. In comparative study between verotoxin assessment by Vero cells cytotoxicity and enterohemolytic activity, concordance positive results between both were 53 (91.4%). The most common serogroups of STEC were O157 (33%) and O26 (20%). From this study we can conclude that enterohemolysin production can be used as surrogate marker for STEC. The most rapid and promising approach for detection of STEC is by molecular method.     

Nucleotide sequence of the integration site of the temperate bacteriophage 6220, which carries the Shiga toxin gene stx(1ox3)

The integration site, attR, of the Shiga toxin-encoding phage 6220 (stx(1ox3)) has been determined. The phage integrates into the chromosome of its Escherichia coli host strain, CB6220, within a gene that is homologous to gene Z2577 and encodes an oxidoreductase. This new integration site was found in different Stx1ox3-producing enterohemorrhagic E. coli strains, which were analyzed by PCR.