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Significant gene order and expression differences in Bordetella pertussis despite limited gene content variation.

ABSTRACT: Bordetella pertussis, an obligate human pathogen and the agent of whooping cough, is a clonal species, despite the dynamic selection pressures imposed by host immunity and vaccine usage. Because the generation of variation is critical for species evolution, we employed a variety of approaches to examine features of B. pertussis genetic variation. We found a high level of conservation of gene content among 137 B. pertussis strains with different geographical, temporal, and epidemiological associations, using comparative genomic hybridization. The limited number of regions of difference were frequently located adjacent to copies of the insertion element IS481, which is present in high numbers in the B. pertussis chromosome. This repeated sequence appears to provide targets for homologous recombination, resulting in deletion of intervening sequences. Using subtractive hybridization, we searched for previously undetected genes in diverse clinical isolates but did not detect any new genes, indicating that gene acquisition is rare in B. pertussis. In contrast, we found evidence of altered gene order in the several strains that were examined and again found an association of IS481 with sites of rearrangement. Finally, we compared whole-genome expression profiles of different strains and found significant changes in transcript abundance, even in the same strain after as few as 12 laboratory passages. This combination of approaches provides a detailed picture of a pathogenic species with little gene loss or gain but with the capacity to generate variation by rearranging its chromosome and altering gene expression. These findings have broad implications for host adaptation by microbial pathogens.

SUBMITTER: Brinig MM

PROVIDER: S-EPMC1428402 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Significant gene order and expression differences in Bordetella pertussis despite limited gene content variation.

Brinig Mary M MM Cummings Craig A CA Sanden Gary N GN Stefanelli Paola P Lawrence Andrew A Relman David A DA

Journal of bacteriology 20060401 7

Bordetella pertussis, an obligate human pathogen and the agent of whooping cough, is a clonal species, despite the dynamic selection pressures imposed by host immunity and vaccine usage. Because the generation of variation is critical for species evolution, we employed a variety of approaches to examine features of B. pertussis genetic variation. We found a high level of conservation of gene content among 137 B. pertussis strains with different geographical, temporal, and epidemiological associa ...[more]

PMID: 16547023

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Project description:In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

Project description:BACKGROUND:Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs). RESULTS:The genomes of B. pertussis and the two closely related species, B. bronchiseptica and B. parapertussis, were screened for homopolymeric tracts longer than expected on the basis of chance, given their nucleotide compositions. Sixty-nine such HPTs were found in total among the three genomes, 74% of which were polymorphic among the three species. Nine HPTs were genotyped in a collection of 90 geographically and temporally diverse B. pertussis strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR) assay. Six HPTs were polymorphic in this collection of B. pertussis strains. Of note, one of these polymorphic HPTs was found in the fimX promoter, where a single base insertion variant was present in seven strains, all of which were isolated prior to introduction of the pertussis vaccine. Transcript abundance of fimX was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, tcfA, bapC, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures. CONCLUSION:Allelic polymorphism at homopolymeric tracts is common within the B. pertussis genome. Phase variability may be an important mechanism in B. pertussis for evasion of the immune system and adaptation to different niches in the human host. High sensitivity and specificity make the PCR/LDR assay a powerful tool for investigating allelic variation at HPTs. Using this method, allelic diversity and phase variation were demonstrated at several B. pertussis loci.

Project description:Resurgence of pertussis has been observed in many countries with high vaccination coverage and clonal expansion of certain Bordetella pertussis strains has been associated with recent epidemics in Europe. It is known that vaccinations have selected strains which are different from those used for vaccine production. However, little is known about the differences in genomic content of strains circulating before the vaccination was introduced. In this study, we compared the genomes of 39 vaccine strains and old clinical isolates collected from Finland (N=5), Poland (N=14), Serbia (N=10) and the UK (N=10). The analysis included genotyping, pulsed-field gel electrophoresis (PFGE) and comparative genomic hybridisation (CGH). Compared to the strain Tohama I, the European strains analyzed have lost three major regions of difference (RD3, 5 and 29). However, difference in frequency of the absent RDs 3, 5 or 29 was observed among the four countries. Of the strains with absent RD5, half had also a duplicated region in the genome. All four RDs (RD22, 23, 24 and 26) absent in Tohama I were present in majority of the tested strains. Results obtained from PFGE analysis correlated well with those of CGH. Recently a novel pertussis toxin promoter allele (ptxP3) was described. Strains with ptxP3 have replaced resident ptxP1 strains. When the recent strains (N=22) selected from the four countries were examined, the ptxP3 allele was found in all countries except Poland. Our result indicates that at least three clusters of B. pertussis circulated in Europe in pre-vaccine era and their genomes were distinct from that of the reference strain Tohama I. Although progressive gene loss occurs in B. pertussis population with time, difference in frequency of the lost genes were observed among the countries. The observed differences in genomic content might be vaccine-driven. The strains were serotyped for the fimbriae, genotyped with LightCycler RT-PCR for pertussis toxin subunit 1 (ptxA), pertussis toxin promoter (ptxP) (Kallonen et al 2011, unpublished data, submitted) and pertactin (prn). The vaccine and old strains were assigned to profiles according to the PFGE results. From every PFGE profile from all countries at least 1 strain was selected and tested for CGH. Altogether 29 strains were analysed with CGH from Poland, Serbia and the United Kingdom, 12, 7 and 10, respectively. In addition 5 old previously published Finnish strains were included in the analysis as well as the reference strain Tohama I (Heikkinen et al., 2007). The hybridisations were performed as previously described (Heikkinen et al., 2007). Microarray data analysis was performed with R/Bioconductor (R Development Core Team, 2008). Print-tip-loess normalization was performed with default parameters and the gains/losses were filtered with Limma package. The regions of difference (RDs) found in this study are presented with the nomenclature defined by Brinig et al. (2006). The RDs which were not described by Brinig et al are indicated by RDs and the consequent unused number.

Project description:BackgroundThe objective of the study was to analyse the evolution of Bordetella pertussis population and the influence of herd immunity in different areas of the world where newborns and infants are highly vaccinated.MethodologyThe analysis was performed using DNA microarray on 15 isolates, PCR on 111 isolates as well as GS-FLX sequencing technology on 3 isolates and the B. pertussis reference strain, Tohama I.Principal findingsOur analyses demonstrate that the current circulating isolates are continuing to lose genetic material as compared to isolates circulating during the pre-vaccine era whatever the area of the world considered. The lost genetic material does not seem to be important for virulence. Our study confirms that the use of whole cell vaccines has led to the control of isolates that were similar to vaccine strains. GS-FLX sequencing technology shows that current isolates did not acquire any additional material when compared with vaccine strains or with isolates of the pre-vaccine era and that the sequenced strain Tohama I is not representative of the isolates. Furthermore, this technology allowed us to observe that the number of Insertion Sequence elements contained in the genome of the isolates is temporally increasing or varying between isolates.ConclusionsB. pertussis adaptation to humans is still in progress by losing genetic material via Insertion Sequence elements. Furthermore, recent isolates did not acquire any additional material when compared with vaccine strains or with isolates of the pre-vaccine era. Herd immunity, following intensive vaccination of infants and children with whole cell vaccines, has controlled isolates similar to the vaccine strains without modifying significantly the virulence of the isolates. With the replacement of whole cell vaccines by subunit vaccines, containing only few bacterial antigens targeting the virulence of the bacterium, one could hypothesize the circulation of isolates expressing less or modified vaccine antigens.

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Significant gene order and expression differences in Bordetella pertussis despite limited gene content variation.

Publications

Significant gene order and expression differences in Bordetella pertussis despite limited gene content variation.

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