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1.
Eur J Clin Microbiol Infect Dis ; 34(4): 821-30, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25527446

ABSTRACT

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.


Subject(s)
Bordetella pertussis/classification , Bordetella pertussis/isolation & purification , Genetic Variation , Whooping Cough/epidemiology , Whooping Cough/microbiology , Antigens, Bacterial/genetics , Bordetella pertussis/genetics , Electrophoresis, Gel, Pulsed-Field , Europe/epidemiology , Humans , Minisatellite Repeats , Molecular Epidemiology , Multilocus Sequence Typing , Pertussis Toxin/genetics , Promoter Regions, Genetic , Serotyping
2.
Euro Surveill ; 19(33)2014 Aug 21.
Article in English | MEDLINE | ID: mdl-25166348

ABSTRACT

Pathogen adaptation has been proposed to contribute to the resurgence of pertussis. A striking recent example is the emergence of isolates deficient in the vaccine component pertactin (Prn). This study explores the emergence of such Prn-deficient isolates in six European countries. During 2007 to 2009, 0/83 isolates from the Netherlands, 0/18 from the United Kingdom, 0/17 Finland, 0/23 Denmark, 4/99 Sweden and 5/20 from Norway of the isolates collected were Prn-deficient. In the Netherlands and Sweden, respectively 4/146 and 1/8 were observed in a later period (2010­12). The Prn-deficient isolates were genetically diverse and different mutations were found to inactivate the prn gene. These are indications that Prn-deficiency is subject to positive selective pressure. We hypothesise that the switch from whole cell to acellular pertussis vaccines has affected the balance between 'costs and benefits' of Prn production by Bordetella pertussis to the extent that isolates that do not produce Prn are able to expand. The absence of Prn-deficient isolates in some countries may point to ways to prevent or delay the spread of Prn-deficient strains. In order to substantiate this hypothesis, trends in the European B. pertussis population should be monitored continuously.


Subject(s)
Bacterial Outer Membrane Proteins/analysis , Bacterial Outer Membrane Proteins/genetics , Bordetella pertussis/isolation & purification , Virulence Factors, Bordetella/analysis , Virulence Factors, Bordetella/genetics , Whooping Cough/prevention & control , Amino Acid Sequence , Base Sequence , Bordetella pertussis/genetics , Child , Child, Preschool , Cluster Analysis , Communicable Diseases, Emerging/genetics , DNA, Bacterial/genetics , Europe , Female , Genotype , Humans , Infant , Male , Molecular Typing , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Whooping Cough/epidemiology , Whooping Cough/microbiology
3.
J Microbiol Methods ; 78(3): 297-301, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19577594

ABSTRACT

Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and The Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.


Subject(s)
Bacterial Typing Techniques/methods , Bordetella pertussis/classification , Bordetella pertussis/genetics , DNA Fingerprinting/methods , Whooping Cough/epidemiology , Whooping Cough/microbiology , Alleles , Bordetella pertussis/isolation & purification , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field/methods , Humans , Minisatellite Repeats , Molecular Epidemiology/methods , Molecular Sequence Data , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Sequence Analysis, DNA/methods , Sweden/epidemiology
4.
J Bacteriol ; 188(24): 8385-94, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17041054

ABSTRACT

The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.


Subject(s)
Bordetella pertussis/classification , Bordetella pertussis/genetics , Bordetella/classification , Bordetella/genetics , Genomic Islands/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bordetella/isolation & purification , Bordetella pertussis/isolation & purification , Evolution, Molecular , Genome, Bacterial , Humans , Hydroxamic Acids/metabolism , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Phylogeny , Sequence Analysis, DNA
5.
J Clin Microbiol ; 43(6): 2837-43, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15956406

ABSTRACT

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.


Subject(s)
Bordetella pertussis/isolation & purification , Health Policy , Immunization Programs , Pertussis Vaccine/administration & dosage , Whooping Cough/epidemiology , Adolescent , Adult , Bacterial Proteins/genetics , Bordetella pertussis/classification , Bordetella pertussis/genetics , Child , Child, Preschool , Europe , Fimbriae Proteins , Humans , Infant , Infant, Newborn , Minisatellite Repeats/genetics , Polymorphism, Genetic , Serotyping , Vaccination , Virulence Factors/genetics , Whooping Cough/microbiology , Whooping Cough/prevention & control
6.
J Infect Dis ; 181(4): 1376-87, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10762569

ABSTRACT

Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains. Both methods were found to be highly concordant, generating a dendrogram with 2 main branches. The division of the M. catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences. Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene. In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies. The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains. These findings suggest the existence of subpopulations of M. catarrhalis that differ in virulence, and they may have implications for vaccine development.


Subject(s)
Moraxella catarrhalis/genetics , Moraxella catarrhalis/pathogenicity , Adult , Antigens, Bacterial/genetics , Antigens, Surface/genetics , Bacterial Outer Membrane Proteins/genetics , Base Sequence , Carrier State , Child , Humans , Laryngitis/microbiology , Lung Diseases, Obstructive/genetics , Lung Diseases, Obstructive/microbiology , Molecular Sequence Data , Moraxella catarrhalis/classification , Neisseriaceae Infections/genetics , Neisseriaceae Infections/microbiology , Polymorphism, Restriction Fragment Length , RNA, Ribosomal, 16S/chemistry , Respiratory Tract Infections/microbiology
7.
J Infect Dis ; 179(4): 915-23, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10068587

ABSTRACT

The population structure of Bordetella pertussis in The Netherlands in 5 successive periods, encompassing 1949-1996, was analyzed by DNA typing ("fingerprinting"). In 10 years following the introduction of wide-scale vaccination in 1953, a decrease in genotypic diversity (GD) was observed, suggesting clonal expansion of strains that were adapted to vaccine-induced immunity. In subsequent periods, GD increased to prevaccination levels, probably reflecting a gradual adaptation of the B. pertussis population involving many lineages. In the 1990s, GD decreased again. This decrease coincided with an antigenic shift in the surface protein pertactin. No evidence was found for changes in DNA types or GD in 1996, when a large pertussis epidemic occurred. Thus, gradual changes in the bacterial population previous to 1996 were probably the cause of the 1996 epidemic. The results herein suggest that vaccination has selected for strains that are adapted to a highly vaccinated population. Similar changes may have occurred in other countries, explaining the reemergence of pertussis in vaccinated populations.


Subject(s)
Bacterial Vaccines/immunology , Bordetella pertussis/immunology , Bordetella pertussis/classification , Bordetella pertussis/isolation & purification , DNA, Bacterial/analysis , Genotype , Netherlands/epidemiology , Time Factors , Vaccination
8.
Infect Immun ; 66(2): 670-5, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9453625

ABSTRACT

The Bordetella pertussis proteins P.69 (also designated pertactin) and pertussis toxin are important virulence factors and have been shown to confer protective immunity in animals and humans. Both proteins are used in the new generation of acellular pertussis vaccines (ACVs), and it is therefore important to study the degree of antigenic variation in these proteins. Sequence analysis of the genes for P.69 and the pertussis toxin S1 subunit, using strains collected from Dutch patients in the period 1949 to 1996, revealed three P.69 and three S1 variants which show differences in amino acid sequence. Polymorphism in P.69 was confined to a region comprised of repeats and located proximal to the RGD motif involved in adherence to host tissues. Variation in S1 was observed in two regions previously identified as T-cell epitopes. P.69 and S1 variants, identical to those included in the Dutch whole-cell pertussis vaccine (WCV), were found in 100% of the strains from the 1950s, the period when the WCV was introduced in The Netherlands. However, nonvaccine types of P.69 and S1 gradually replaced the vaccine types in later years and were found in approximately 90% strains from 1990 to 1996. These results suggest that vaccination has selected for strains which are antigenically distinct from vaccine strains. Analysis of strains from vaccinated and nonvaccinated individuals indicated that the WCV protects better against strains with the vaccine type P.69 than against strains with non-vaccine types (P = 0.024). ACVs contain P.69 and S1 types which are found in only 10% of recent Dutch B. pertussis isolates, implying that they do not have an optimal composition. Our findings cast a new light on the reemergence of pertussis in highly vaccinated populations and may have major implications for the long-term efficacy of both WCVs and ACVs.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bordetella pertussis/pathogenicity , Pertussis Toxin , Pertussis Vaccine/immunology , Virulence Factors, Bordetella/genetics , Amino Acid Sequence , Base Sequence , Biological Evolution , Bordetella pertussis/genetics , Humans , Molecular Sequence Data , Polymorphism, Genetic , Vaccination , Virulence
9.
J Bacteriol ; 179(24): 7882-5, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9401052

ABSTRACT

The differential host species specificities of Bordetella pertussis, B. parapertussis, and B. bronchiseptica might be explained by polymorphisms in adherence factor genes. We have found that B. parapertussis and B. bronchiseptica, unlike B. pertussis, contain a full-length gene for the fimbrial subunit FimA. B. bronchiseptica expresses fimA in a BvgAS-dependent fashion.


Subject(s)
Bacterial Proteins/genetics , Bordetella bronchiseptica/genetics , Fimbriae Proteins , Fimbriae, Bacterial/genetics , Genes, Bacterial , Amino Acid Sequence , Bordetella bronchiseptica/classification , Gene Expression , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid , Species Specificity
10.
Mol Microbiol ; 11(2): 337-47, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8170396

ABSTRACT

The chromosome of Bordetella pertussis harbours a region of 27 contiguous kb, which contains the bvg, fha and fim genes, involved in the co-ordinate regulation of virulence genes, FHA production and fimbriae production, respectively. The linkage of FHA and fimbrial genes has resulted in some confusion concerning the existence and location of genes required for the production of FHA and the function of the fimbrial genes fimB-D, which were proposed to be involved in both FHA and fimbriae biosynthesis. Through the use of non-polar mutations in each of these genes, we found that fimB-D are required for the production of both serotype 2 and 3 fimbriae, but not for FHA biosynthesis. Furthermore, a large open reading frame, designated fhaC, was identified downstream of fimD. It was shown that fhaC is essential for FHA production but not for fimbriae biogenesis. We propose that insertion mutations in fimB-D affect FHA production because of polar effects on fhaC expression. An insertion in the region downstream of fhaC had only a slight effect on FHA and fimbriae production. The fhaC gene product shows homology with ShIB and HpmB, two outer membrane proteins involved in export and activation of the haemolysins, ShIA and HpmA, of Serratia marcescens and Proteus mirabilis, respectively. Homology is also observed between the N-termini of FHA, ShIA and HpmA. Export of the haemolysins requires the N-termini of these molecules, and when this region was removed from FHA by an in-frame deletion, FHA biosynthesis was abolished. These results suggest that the N-terminus of FHA interacts with FhaC, and that as a result FHA is transported across the outer membrane.


Subject(s)
Adhesins, Bacterial , Bordetella pertussis/genetics , Chromosomes, Bacterial , Genes, Bacterial , Hemagglutinins/genetics , Hemolysin Proteins/genetics , Multigene Family , Virulence Factors, Bordetella , Amino Acid Sequence , Animals , Antigens, Bacterial/genetics , Bordetella/genetics , Bordetella pertussis/metabolism , Bordetella pertussis/pathogenicity , DNA Mutational Analysis , Erythrocytes , Escherichia coli/genetics , Genetic Linkage , Hemagglutination , Hemagglutination Tests , Hemagglutinins/biosynthesis , Molecular Sequence Data , Plasmids , Restriction Mapping , Sequence Homology, Amino Acid , Sheep , Virulence/genetics
11.
Mol Microbiol ; 9(3): 623-34, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8105363

ABSTRACT

We report the purification of a minor Bordetella pertussis fimbrial subunit, designated FimD, and the identification of its gene (fimD). FimD could be purified from the bulk of major fimbrial subunits by exploiting the fact that major subunit-subunit interactions are more stable in the presence of SDS than minor-major subunit interactions. To locate the gene for FimD, internal peptides of FimD were generated, purified and sequenced. Subsequently, an oligonucleotide probe, based on the primary sequence of one peptide, was used to clone fimD. The primary structure of FimD, derived from the DNA sequence of its gene, showed homology with a number of fimbrial adhesins. Most pronounced homology was observed with MrkD, a fimbrial adhesin derived from Klebsiella pneumoniae. These observations suggest that FimD may represent a B. pertussis fimbrial adhesin. With a fimD-specific probe we detected the presence of a fimD homologue in Bordetella parapertussis and Bordetella bronchiseptica but not in Bordetella avium. Cloning and sequencing revealed that the B. parapertussis and B. bronchiseptica fimD product differed from the B. pertussis fimD product in 20 and 1 amino acid residues, respectively. Since B. bronchiseptica is normally not a human pathogen, but causes respiratory disease in a wide range of non-human mammalian species, this may suggest that FimD recognizes a receptor that is well conserved in mammalian species. An in-frame deletion in fimD completely abolished FimD expression and also affected the expression of the major subunits Fim2 and Fim3 suggesting that, in contrast to other adhesins that are minor components of fimbriae, FimD is required for formation of the fimbrial structure.


Subject(s)
Bacterial Adhesion , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins , Bordetella pertussis/genetics , Fimbriae Proteins , Fimbriae, Bacterial , Genes, Bacterial/genetics , Amino Acid Sequence , Base Sequence , Bordetella/genetics , Bordetella/pathogenicity , Bordetella bronchiseptica/genetics , Bordetella bronchiseptica/pathogenicity , Bordetella pertussis/pathogenicity , Cloning, Molecular , Fimbriae, Bacterial/chemistry , Molecular Sequence Data , Mutagenesis, Insertional , Nucleic Acid Conformation , Oligonucleotide Probes , Peptide Fragments/genetics , Restriction Mapping , Sequence Analysis , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Species Specificity , Virulence
12.
Mol Microbiol ; 6(18): 2661-71, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1360139

ABSTRACT

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.


Subject(s)
Adhesins, Bacterial , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Bordetella pertussis/genetics , Fimbriae Proteins , Fimbriae, Bacterial/metabolism , Genes, Bacterial , Hemagglutinins/genetics , Virulence Factors, Bordetella , Amino Acid Sequence , Base Sequence , DNA Transposable Elements , DNA, Bacterial/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Pseudogenes , Sequence Alignment , Sequence Homology, Amino Acid
13.
Microb Pathog ; 12(2): 127-35, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1350044

ABSTRACT

Although the role of fimbriae in bacterial disease has been well established, little is known about the function of Bordetella pertussis fimbriae. To study this function, well-defined fimbrial mutants were constructed. B. pertussis harbours three fimbrial genes, fim2, fim3 and fimX, and strains were constructed in which one or more fimbrial genes were inactivated by means of gene replacement. Analysis of these strains by means of immunoblotting suggested the presence of a fourth fimbrial gene, tentatively designated fimY. A fimbrial mutant was analysed in a mouse respiratory infection model, together with a strain harbouring a deletion in the gene for the filamentous haemagglutinin. Both mutants were affected in their ability to persist in the trachea. Persistence in the nasopharynx was only affected by the mutation in the filamentous haemagglutinin gene. Neither the filamentous haemagglutinin nor the fimbrial mutants were affected in their ability to persist in the lung. Our results suggest that the filamentous haemagglutinin plays a more crucial role than fimbriae in the colonization of the upper respiratory tract of the mouse.


Subject(s)
Bordetella pertussis/genetics , Fimbriae, Bacterial/metabolism , Mutation , Animals , Blotting, Southern , Bordetella pertussis/metabolism , Cloning, Molecular , Male , Mice , Mice, Inbred BALB C , Restriction Mapping , Whooping Cough/microbiology
14.
Infect Immun ; 59(10): 3536-46, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1894360

ABSTRACT

The gene encoding the 35-kDa immunogenic Treponema pallidium subsp. pallidum (T. pallidum) membrane protein C, TmpC, was cloned, sequenced, and expressed in Escherichia coli. The deduced amino acid sequence carries on N-terminal signal sequence with a four-amino-acid motif, which is characteristic for bacterial lipoproteins. Metabolic labeling with radioactive palmitic acid of E. coli expressing TmpC revealed incorporation of the fatty acid into the antigen. The antigen was overproduced, purified to near homogeneity and used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its potential for the serodiagnosis of syphilis. Although all sera from untreated secondary syphilis patients were reactive in this TmpC ELISA, only a minority of the serum samples from untreated patients in the primary or early latent stage of the disease contained significant anti-TmpC antibodies. To study the influence of the lipid moiety on the antigenic properties of the TmpC, TmpA, and TpD lipoproteins, plasmids encoding nonlipidated forms of these antigens were constructed. In addition, a plasmid expressing a lipidated form of the otherwise non-lipid-modified antigen TmpB was constructed. Immunization and absorption experiments with these lipidated and nonlipidated antigens showed that antibodies against the lipid moiety of lipoproteins could not be detected on immunoblots, neither in sera from infected rabbits nor in sera from animals immunized with the lipoproteins. In addition, we were unable to demonstrate cross-reactivity between antibodies against the T. pallidum lipoproteins and those reactive to the Venereal Diseases Research Laboratories test, suggesting that antibodies reactive to the Venereal Diseases Research Laboratories test are unrelated to antilipoprotein antibodies.


Subject(s)
Antibodies, Bacterial/analysis , Antigens, Bacterial/immunology , Lipids/immunology , Lipoproteins/genetics , Membrane Proteins/genetics , Treponema pallidum/immunology , Amino Acid Sequence , Base Sequence , Humans , Lipoproteins/biosynthesis , Lipoproteins/immunology , Molecular Sequence Data , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Syphilis/immunology
15.
EMBO J ; 9(9): 2803-9, 1990 Sep.
Article in English | MEDLINE | ID: mdl-1975238

ABSTRACT

Fimbriae belong to a class of extracellular filamentous proteins which are involved in the attachment of bacteria to host tissues. Bordetella pertussis, the etiological agent of whooping cough, produces two serologically distinct fimbriae. We show that, like a number of other B. pertussis virulence genes, transcription of the fimbrial subunit genes (fim) is positively controlled by trans-acting polypeptides encoded by the bvg locus. In addition to this coordinate control, transcription of the fim genes is regulated at an individual level by phase variation. This process is characterized by a switching between a high and low level of expression of a particular fim gene. We have identified a conserved DNA region, located close to the start of the fim genes, which is likely to be involved in both positive regulation by the bvg locus, and phase variation. This promoter region contains a stretch of approximately 15 C residues and it appears that phase transitions occur by small insertions or deletions in this C-rich region. We propose that these mutations affect transcription of the fim genes by varying the distance between the binding site for an activator and the -10 box. The fim promoter shows homology with the pertussis toxin promoter, which is also positively regulated by the bvg locus.


Subject(s)
Bordetella pertussis/genetics , Fimbriae, Bacterial/physiology , Gene Expression Regulation, Bacterial , Genes, Bacterial , Transcription, Genetic , Base Sequence , Bordetella pertussis/ultrastructure , Genotype , Molecular Sequence Data , Oligonucleotide Probes , Promoter Regions, Genetic , Restriction Mapping , Sequence Homology, Nucleic Acid
16.
FEMS Microbiol Lett ; 54(1-3): 327-31, 1990 Jan 01.
Article in English | MEDLINE | ID: mdl-1969830

ABSTRACT

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.


Subject(s)
Antigens, Bacterial , Bordetella pertussis/genetics , Fimbriae Proteins , Fimbriae, Bacterial/chemistry , Genes, Bacterial , Virulence Factors, Bordetella , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Bordetella pertussis/classification , Cloning, Molecular , DNA, Bacterial/genetics , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Nucleic Acid , Serotyping
17.
Microb Pathog ; 2(6): 473-84, 1987 Jun.
Article in English | MEDLINE | ID: mdl-2907088

ABSTRACT

Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically related B. bronchiseptica polypeptides, were shown to be very similar in amino acid composition and N-terminal amino acid sequence. Homology was observed between the N-termini of these polypeptides, and fimbrial subunits from Escherichia coli, Haemophilus influenzae and Proteus mirabilis. A synthetic oligonucleotide probe, derived from the N-terminal sequence of the B. pertussis serotype 2 fimbrial subunit, was used to identify fimbrial genes in genomic Southern blots. The results suggested the presence of multiple fimbrial subunit genes in B. pertussis, B. bronchiseptica and B. parapertussis. The DNA probe was used to clone one of the three tentative fimbrial subunit genes detected in B. pertussis.


Subject(s)
Bacterial Proteins/analysis , Bordetella/classification , Fimbriae, Bacterial/analysis , Amino Acid Sequence , Bacteria/classification , Bacteria/genetics , Bacterial Proteins/genetics , Bordetella/genetics , Bordetella pertussis/classification , Bordetella pertussis/genetics , Macromolecular Substances , Molecular Sequence Data , Serotyping
19.
Infect Immun ; 42(1): 187-96, 1983 Oct.
Article in English | MEDLINE | ID: mdl-6311744

ABSTRACT

A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.


Subject(s)
Antigens, Bacterial/genetics , Cloning, Molecular , Escherichia coli/genetics , Genes, Bacterial , Treponema pallidum/genetics , Antigens, Bacterial/immunology , Bacteriophage lambda , DNA Restriction Enzymes , Genetic Vectors , Plasmids
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