Establishing a method of vector contamination identification in database sequences.

MOTIVATION: The nucleotide sequence databases are invaluable tools both for the private and the academic research communities, from the retrieval of sequences to homology searching. Several issues related to data quality, such as the existence of sequencing artifacts and errors, are facing the databases. We investigated a major source of these errors, i.e. the presence of vector-contaminated sequences. RESULTS: Using a panel of 180 vector polylinker sequences, we found 0.36% or 3029 vector-matching sequences in GenBank Release 95-96, with an average vector-matching length of 72 nucleotides. The number of vector-contaminated sequences has been growing with the database; however, the percent contamination has remained approximately constant at an average of 0.28% from 1982 to 1996. AVAILABILITY: Access to the database of vector polylinker sequences via sequence similarity searching is available at http://seqsim.ncgr.org/vector/ CONTACT: gas@molinfo.com

The Genome Sequence DataBase (GSDB): improving data quality and data access.

In 1997 the primary focus of the Genome Sequence DataBase (GSDB; www. ncgr.org/gsdb ) located at the National Center for Genome Resources was to improve data quality and accessibility. Efforts to increase the quality of data within the database included two major projects; one to identify and remove all vector contamination from sequences in the database and one to create premier sequence sets (including both alignments and discontiguous sequences). Data accessibility was improved during the course of the last year in several ways. First, a graphical database sequence viewer was made available to researchers. Second, an update process was implemented for the web-based query tool, Maestro. Third, a web-based tool, Excerpt, was developed to retrieve selected regions of any sequence in the database. And lastly, a GSDB flatfile that contains annotation unique to GSDB (e.g., sequence analysis and alignment data) was developed. Additionally, the GSDB web site provides a tool for the detection of matrix attachment regions (MARs), which can be used to identify regions of high coding potential. The ultimate goal of this work is to make GSDB a more useful resource for genomic comparison studies and gene level studies by improving data quality and by providing data access capabilities that are consistent with the needs of both types of studies.

The Genome Sequence DataBase version 1.0 (GSDB): from low pass sequences to complete genomes.

The Genome Sequence DataBase (GSDB) has completed its conversion to an improved relational database. The new database, GSDB 1.0, is fully operational and publicly available. Data contributions, including both original sequence submissions and community annotation, are being accomplished through the use of a graphical client-server interface tool, the GSDB Annotator, and via GIO (GSDB Input/Output) files. Data retrieval services are being provided through a new Web Query Tool and direct SQL. All methods of data contribution and data retrieval fully support the new data types that have been incorporated into GSDB, including discontiguous sequences, multiple sequence alignments, and community annotation.

Pattern of drug usage in bronchiolitis.

Bronchiolitis is a common viral respiratory infection in infants and young children. The objective of this study was to assess the pattern of drug usage in paediatric patients with bronchiolitis. One hundred patients (aged 2 weeks to 22 months) were evaluated. Of these, 64% had a respiratory syncytial virus infection. The drug therapy included albuterol in 99%, corticosteroids in 36%, aminophylline in 12% and ribavirin in 2% of the patients. The drug doses and frequencies, and durations of therapy varied substantially among patients. This emphasizes the need for well-designed research to develop optimal dosage regimens in paediatric patients with bronchiolitis.

Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen.

Bordetella pertussis TOX3201 has a 12-base-pair insertion in the S1 subunit gene of pertussis toxin (PTX), which encodes for a 4-amino-acid insertion between residues 107 and 108 of the mature S1 subunit (Black et al., Science 240:656-659, 1988). This mutant strain has been shown to secrete a holotoxin analog of PTX, designated CRM3201, with reduced ADP-ribosyltransferase activity. In the present study, we evaluated the biochemical, biological, and immunoprotective activities of purified CRM3201. Assay of enzymatic activities showed that CRM3201 had 20 to 30% of the ADP-ribosyltransferase activity and 55 to 60% of the NAD glycohydrolase activity of native PTX. CRM3201, however, had only 2 to 6% of the activity of PTX in clustering CHO cells, promoting leukocytosis, inducing histamine sensitization, and potentiating an anaphylactic response to bovine serum albumin. In contrast, activities associated with the B oligomer (binding to fetuin, hemagglutination of goose erythrocytes, and lymphocyte mitogen activity) were comparable to those of native PTX. Injection of BALB/c mice with CRM3201 mixed with Al(OH)3 elicited high titers of antibody to PTX (as measured by enzyme-linked immunosorbent assay), which neutralized a leukocytosis-promoting dose of PTX in these mice and neutralized PTX in a CHO cell assay. Passive transfer of the anti-CRM3201 antibody protected 20-day-old Swiss-Webster mice against a lethal aerosol challenge with B. pertussis 18323. Active immunization with CRM3201 significantly reduced lung colonization in adult BALB/c mice with a B. pertussis respiratory infection. These results demonstrate (i) that the reduced ADP-ribosyltransferase activity of CRM3201 is associated with reductions in certain biological and toxic activities of PTX (the enzymatic and biological activities are not, however, totally concordant); (ii) that CRM3201 possesses a functional B oligomer; and (iii) that CRM3201 can induce toxin-neutralizing antibodies which protect mice against a respiratory challenge with B. pertussis. Our studies with CRM3201 show that recombinant analogs of PTX have the potential to be developed into safe, protective immunogens for use in new acellular pertussis vaccines.

Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene.

Pseudomonas aeruginosa PAO-E64 is a mutant which produces parental levels of elastase antigen but has no elastolytic activity at 37 degrees C. The lesion (lasA1) in PAO-E64 is not a mutation in the structural gene for P. aeruginosa elastase (P.A. Schad, R.A. Bever, T.I. Nicas, F. Leduce, L.F. Hanne, and B.H. Iglewski, J. Bacteriol. 169: 2691-2696, 1987). A 1.7-kilobase segment of DNA that complements the lasA1 lesion was sequenced. Computer analysis of the DNA sequence showed that it contained an open reading frame which encoded a 41,111-dalton protein. The lasA gene was expressed under an inducible PT-7 promoter, and a 40,000-dalton protein was detected in Escherichia coli lysates. The lasA protein was localized in the outer membrane fraction of E. coli. This lasA protein produced in E. coli activated the extracellular elastase produced by the P. aeruginosa mutant, PAO-E64.

ADP-ribosyltransferase activity of pertussis toxin and immunomodulation by Bordetella pertussis.

Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.

Cloning and characterization of elastase genes from Pseudomonas aeruginosa.

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.

Genetic mapping and characterization of Pseudomonas aeruginosa mutants that hyperproduce exoproteins.

We isolated two mutants of Pseudomonas aeruginosa PAO with defective iron uptake. In contrast to the wild-type strain, the mutants produced extracellular protease activity in media containing high concentrations of salts or iron and hyperproduced elastase, staphylolytic enzyme, and exotoxin A in ordinary media (Xch mutants). The mutations were located in the 55' region of the chromosome, between the markers met-9011 and pyrD.

Species-specific binding of purified pili (AF/R1) from the Escherichia coli RDEC-1 to rabbit intestinal mucosa.

Species-specific colonization of rabbit intestine by RDEC-1 Escherichia coli is an accepted animal model for bacterial mucosal adherence. To determine whether RDEC-1 pili are functional as adherence factors for this organism, we grew the organism under conditions that promoted pilus expression; we isolated the pili, documented their purity, and compared their mucosal adherence properties with those of whole organisms using an indirect immunofluorescence technique. Frozen sections of rabbit, rat, guinea pig, and human small intestine were incubated with either piliated or nonpiliated RDEC-1 organisms or purified RDEC-1 pili and observed for the distribution and intensity (0-4+) of fluorescence. Piliated RDEC-1 organisms fluoresced brightly (4+) and were distributed along the entire mucosal surface of the rabbit ileum. Only a few nonpiliated RDEC-1 attached to rabbit ileum, and they were randomly scattered across the entire section of tissue. Rabbit ileum overlain with pure RDEC-1 pili showed a specific, D-mannose resistant (2-3+) fluorescence on the mucosal surface from the crypts to the villus tips. No fluorescence was seen on the guinea pig, rat, or human mucosal surface overlain with RDEC-1 pili. Purified RDEC-1 pili adhere to the rabbit intestinal mucosa in a species-specific manner and with the same distribution as whole piliated organisms. The data suggest that RDEC-1 produce pili (distinct from type 1 pili) that determine the specificity of the mucosal adherence of RDEC-1 to rabbit ileum.

Genetic transfer of a mucosal adherence factor (R1) from an enteropathogenic Escherichia coli strain into a Shigella flexneri strain and the phenotypic suppression of this adherence factor.

Escherichia coli strain RDEC-1 avidly adheres to rabbit ileal brush borders. Two separate experiments were designed to determine whether pili promote this adherence. (1) Adherence of strain RDEC-1 was phenotypically suppressed by changing the culture medium. Loss of adherence was correlated with the absence of pili. Thus, growth of strain RDEC-1 in Penassay broth (Difco Laboratories, Detroit, Mich.) promoted both adherence and expression of pili on greater than or equal to 90% of organisms, whereas growth in brain-heart infusion medium suppressed adherence and reduced the percentage of piliated organisms to less than or equal to 13%. (2) The adherence ability of strain RDEC-1 was genetically transferred to previously nonadherent and nonpiliated Shigella flexneri. The Shigella exconjugants that inherited the adherence ability were uniformly piliated, while all nonadherent Shigella exconjugants were nonpiliated. Finally, the pili on both RDEC-1 and the Shigella exconjugant strains were shown to be distinct from type 1 pili. Therefore, unique pili confer upon strain RDEC-1 the ability to adhere to rabbit intestinal brush borders.

Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli.

The 140-megadalton plasmids of Shigella flexneri serotypes 1, 3, and 5, in addition to the 120-megadalton plasmid of Shigella sonnei, are associated with virulence. The present study showed that a 140-megadalton plasmid is also associated with virulence in Escherichia coli. When these plasmids were cleaved with EcoRI or BamHI restriction endonucleases, considerable homology was evident in plasmids from S. sonnei strains, whereas only a few common fragments were observed among the S. flexneri and enteroinvasive E. coli plasmids. Nitrocellulose filter hybridization demonstrated that, despite variations in restriction sites, all these plasmids shared a considerable complement of homologous sequences. Minicell-producing strains were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Transmission electron microscopy of infected HeLa cells showed that minicells from invasive strains retained the invasive phenotype. Sixteen polypeptides were labeled when S. flexneri 5 minicells were incubated with [35S]methionine. Fourteen of these plasmid-coded polypeptides were associated with the outer membrane in invasive strains of S. flexneri 5, and nine polypeptides of similar molecular weight were labeled in the outer membrane of invasive strains of S. flexneri 3, S. sonnei, and E. coli. Seven of the S. flexneri 5 polypeptides were not labeled in a noninvasive strain which had sustained a large deletion in the virulence-associated plasmid, and none were labeled in minicells which no longer harbored this plasmid.

Species specificity of in vitro Escherichia coli adherence to host intestinal cell membranes and its correlation with in vivo colonization and infectivity.

We have previously described an in vitro assay for examining the mucosal adherence of a rabbit diarrheagenic Escherichia coli, RDEC-1. That assay defined the in vitro characteristics of RDEC-1 adherence to brush borders isolated from rabbit ileal epithelial cells. The present study was conducted to examine the species specificity of both in vitro RDEC-1 adherence and in vivo infectivity of RDEC-1 and to compare these specificities. Species specificity in vitro adherence was examined by using brush borders prepared from intestinal epithelial cells of rats, guinea pigs, and rabbits, as well as from a surgically resected specimen of human ileum. Strain RDEC-1 adherence to rabbit brush borders in vitro was significantly greater (P < 0.001) than its adherence to brush borders from any of the other species. Regional specificity of in vitro adherence of RDEC-1 to ileal segments of rabbit intestinal mucosa was also demonstrated. There was significantly greater adherence of RDEC-1 to rabbit ileal brush borders as compared to rabbit jejunal brush borders (P < 0.05). In vivo infectivity was assessed by inoculating RDEC-1 into rats, guinea pigs, and rabbits. RDEC-1 elicited diarrhea in all inoculated rabbits with the mean onset of illness occurring 5 days after inoculation. In contrast, none of the RDEC-1-inoculated rats or guinea pigs developed diarrhea. Furthermore, colonization studies in these animals revealed that RDEC-1 heavily colonized the ileum and cecum (10(9) RDEC-1 colony-forming units/g of tissue) of rabbits; however, only minimal colonization was observed in guinea pigs and rats. In conclusion, the correlation between in vitro adherence and in vivo infectivity that we have observed suggests that the presence of receptors, specific for bacteria, on the surface of the host intestinal mucosa determines species susceptibility to enteric colonization and infectivity by certain strains of enteropathogenic E. coli.