Isolation of a repeated DNA sequence from Bordetella pertussis

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.     

A highly conserved 530 base-pair repeated DNA sequence specific for Bordetella pertussis

Abstract The genome of Bordetella pertussis contains a strictly conserved 530 base-pair (bp) repeated sequence present in about 70 to 80 copies and accounting for approximately 1% of the bacterial genome. The repeated element, whose complete nucleotide sequence has been determined, is specific for B. pertussis DNA; it could be detected neither in closely related Bordetella strains nor in other bacterial or eukaryotic DNAs. The repeated sequence is not associated with the control of the expression of virulence determinants.     

Bvg-negative regulation of repeated sequence transferring in Bordetella pertussis cells

A method of monitoring the sequential events of IS481 transposition into the ctag site of bvg operon of Bordetella pertussis has been developed. Reproduction of virulent B. pertussis cells in vitro is accompanied by intrachromosomal site-specific IS481 transposition, which, in turn, results in inactivation of bvg operon of the causative agent and cell avirulent state. Avirulent bvg mutants of B. pertussis are incapable of intramolecular IS481 transposition. The frequency of the transposition increases when MgSO4 and nicotinic acid are present the culture medium. In the absence of these modulating factors. IS481 transposition along B. pertussis chromosome is inhibited but not arrested completely. Negative regulation of the bvg-repressed genes of B. pertussis seems to be a mechanism that controls bvg-dependent IS481 transposition.     

Genetic diversity analysis of isolates belonging to Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species

In this study, Amplified Fragment Length Polymorphism (AFLP) method was used to track differences among human and animal isolates of B. pertussis, B. parapertussis and B. bronchiseptica species. One hundred and sixty representative strains of these species orginated from international and Polish bacterial collections were genotyped according to AFLP involving EcoRI/Msel and SpeI/ApaI restriction/ligation/amplification procedures. This study has confirmed high potential AFLP SpeI/ApaI procedure for intra-species differentiation of B. pertussis and B. bronchiseptica strains. Both AFLP EcoRI/MseI and SpeI/ApaI procedures have been found to be useful for species-specific classification in case of B. pertussis strains. In case of B. bronchiseptica or B. parapertussis species-specific classification, SpeI/ApaI procedure has been found more precise than EcoRI/MseI one.     

Temporal analysis of French Bordetella pertussis isolates by comparative whole-genome hybridization

Bordetella pertussis, a gram-negative beta-proteobacterium, is the agent of whooping cough in humans. Whooping cough remains a public health problem worldwide, despite well-implemented infant/child vaccination programs. It continues to be endemic and is observed cyclically in vaccinated populations. Classical molecular subtyping methods indicate that genome diversity among B. pertussis isolates is limited. Although the whole bacterial genome has been studied by pulsed-field gel electrophoresis, the genes implicated in the diversity have not been identified. We developed a B. pertussis whole-genome DNA microarray representing over 91% of the predicted coding sequences of the sequenced strain Tohama I. Genomic DNA from clinical isolates with various pulsed-field gel electrophoresis profile patterns was competitively hybridized with the DNA microarray and coding sequences were classified as present, absent or duplicated. Our data strongly suggest that the B. pertussis population is dynamic. In France, with highly vaccinated population, the genetic diversity is low and decreasing with time, and clonal expansion correlates with cycles of the disease. This decrease in diversity is essentially due to loss of genes and pseudogenes. The genes deleted are most of the time flanked by insertion sequences.     

A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.     

Evidence for a DNA inversion system in Bordetella pertussis

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosomal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.     

Bordetella pertussis bacteriophage

For the first time Bordetella pertussis bacteriophage was isolated, and its presence was confirmed by electron microscopy and by agar layer titration. The lysogenic strains were activated by their treatment with mitomycin C in a dose of 4.5 mg/ml. The phage system of the Bordetella genus, heretofore unknown, has been revealed: Bordetella pertussis phage lyzed all the tested strains of Bordetella parapertussis (25 strains) and could be passaged in these strains. The phage formed turbid and transparent negative colonies 0.1 mm and 0.15 mm in size. The phage titer (e. g., in strain No. 3865) was 1 X 10(10). The lysogenic variants of Bordetella pertussis, capable of spontaneous release of the phage, were obtained. These variants were characterized by changes in some of their phenotypical properties, e.g., the increased content of certain toxic substances and increased virulence.     

Evidence for a DNA inversion system in Bordetella pertussis

An isolation procedure was developed to provide within one day microcystin-LR, a cyclic heptapeptide toxin from Microcystis aeruginosa PCC 7806. After ODS (octadecylsilyl) solid phase extraction, the crude toxin fraction was chromatographed using a strong anion exchange column. The toxin was eluted with 0.02 M ammonium bicarbonate. An at least 95% purity was revealed on HPLC separation by monitoring at 214 nm. Application of the procedure to the cyclic pentapeptide toxin nodularin from Nodularia spumigena AV2 was examined.     

Failure to convert Bordetella parapertussis to Bordetella pertussis with a pertussis phage

To analyze the described lysogenic conversion of Bordetella parapertussis to a Bordetella pertussis-like form we used the phage 134 to lysogenize a B. parapertussis strain. Southern blot analysis of the isolated ‘lysogens’ showed that they were not true lysogens, but rather chronically infected strains. These pseudo-lysogens did not show any changes in virulence properties compared with the parental strain. The only difference we could show was a change in the LPS-structure: the pseudolysogens had a rough LPS, like B. pertussis, whereas the parental B. parapertussis strain was smooth.     

Virulence factors of Bordetella pertussis

Clearly, B. pertussis has evolved very elaborate mechanisms to maintain itself in the human host. Three different proteins (FHA, pertussis toxin and fimbriae) have been implicated in adherence. Furthermore, a number of toxins are produced (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or suppress the immune response. There is evidence that B. pertussis may survive intracellularly, and the possibility that it is a facultative intracellular parasite should certainly be explored. The availability of a large number of cloned virulence genes, and a system to construct well defined mutants by allelic exchange (Stibbitz et al. 1986) will greatly facilitate the study of Bordetella virulence factors at the molecular level. It opens the possibility to construct avirulent strains, which are still able to colonize and stimulate the local immune response. Such strains may be used as live, oral vaccines, to present (heterologous) antigens to the mucosal immune system of the respiratory tract.     

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [³H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [³H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

Long-Term Preservation of Bordetella pertussis

Viability of Bordetella pertussis was preserved when glycerol broth suspensions were quick frozen and stored at -70 C for as long as 45 months.     

Induction of antibodies to native DNA by components of Bordetella pertussis]

Administration of Bordetella pertussis and some of their components to mice induced an increase of DNA-binding activity of the sera revealed under ionic strength conditions of physiological saline, mostly on the 14th day. It was shown by the inhibition method that interaction between mouse sera and native DNA was specific. Maximum increase in the quantity of mouse sera proteins reacting with DNA under low ionic strength condition of physiological saline (0.05 M NaCl) is revealed on the 7th day. However, in administration of Bordetella pertussis and their cytoplasmic membrane the elevated DNA-binding proteins content persisted up to 14 days.