Comparative genomics of prevaccination and modern Bordetella pertussis strains
© Bart et al; licensee BioMed Central Ltd. 2010
Received: 9 June 2010
Accepted: 11 November 2010
Published: 11 November 2010
Despite vaccination since the 1950s, pertussis has persisted and resurged. It remains a major cause of infant death worldwide and is the most prevalent vaccine-preventable disease in developed countries. The resurgence of pertussis has been associated with the expansion of Bordetella pertussis strains with a novel allele for the pertussis toxin (Ptx) promoter, ptxP3, which have replaced resident ptxP1 strains. Compared to ptxP1 strains, ptxP3 produce more Ptx resulting in increased virulence and immune suppression. To elucidate how B. pertussis has adapted to vaccination, we compared genome sequences of two ptxP3 strains with four strains isolated before and after the introduction vaccination.
The distribution of SNPs in regions involved in transcription and translation suggested that changes in gene regulation play an important role in adaptation. No evidence was found for acquisition of novel genes. Modern strains differed significantly from prevaccination strains, both phylogenetically and with respect to particular alleles. The ptxP3 strains were found to have diverged recently from modern ptxP1 strains. Differences between ptxP3 and modern ptxP1 strains included SNPs in a number of pathogenicity-associated genes. Further, both gene inactivation and reactivation was observed in ptxP3 strains relative to modern ptxP1 strains.
Our work suggests that B. pertussis adapted by successive accumulation of SNPs and by gene (in)activation. In particular changes in gene regulation may have played a role in adaptation.
The genus Bordetella comprises nine species, of which four are exclusively respiratory pathogens of mammalian hosts: Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis and Bordetella holmesii . The first three species are closely related, while B. holmesii forms a distinct branch . B. bronchiseptica causes chronic and often asymptomatic respiratory tract infections in a wide variety of mammals and is only sporadically isolated from humans. B. parapertussis consists of two distinct lineages, designated B. parapertussis HU and B. parapertussis OV , which infect humans and sheep respectively [3, 4]. B. parapertussis HU and B. pertussis are exclusive human pathogens and the causative agents of pertussis or whooping cough. Both these species have evolved independently from a B. bronchiseptica-like ancestor, a process which has been accompanied by extensive gene loss [4–6].
By far, most cases of whooping cough are caused by B. pertussis. Despite widespread vaccination, pertussis remains a major cause of infant death worldwide . In the 1990s a resurgence of pertussis was observed in several countries with highly vaccinated populations and pertussis has become the most prevalent vaccine-preventable disease in developed countries [8–10]. In the Netherlands, the estimated rate of infection was 6.6% per year for the 3-79-year age group from 1995 through 1996 . Similar percentages have been found in the United States [12–14]. One of the hallmarks of the pertussis resurgence is a shift in disease prevalence towards older persons who have waning vaccine-induced immunity, while recently vaccinated infants are well protected . The reemergence of pertussis has been attributed to various factors including decreased vaccination coverage due to concerns over side effects, suboptimal vaccines, waning vaccine-induced immunity, and adaptation of B. pertussis [1, 9, 10]. The relative contribution of these factors may differ between countries and is the subject of ongoing debate. Pathogen adaptation is supported by several observations. We and others have shown that antigenic divergence has occurred between vaccine strains and clinical isolates with respect to surface proteins which confer protective immunity; pertussis toxin (Ptx), pertactin and fimbriae [1, 16–18]. Further, in a mouse model, pertussis vaccines were less effective against strains carrying non-vaccine type antigens compared to strains with vaccine-type antigens [19–22]. Recently we found evidence that polymorphism in the promoter for Ptx (ptxP) may also be important in adaptation . In the last twenty years two ptxP alleles, ptxP1 and ptxP3, predominated in the Dutch B. pertussis population. The ptxP3 strains were first observed in 1988, gradually increased in frequency, and nearly completely replaced the resident ptxP1 strains in the late 1990s. In the Netherlands, the increase in frequency of ptxP3 strains was associated with the resurgence of pertussis. The ptxP3 strains are found in Asia, Europe, North and South America, and there is evidence that they have spread worldwide in the 1980s and 1990s . The ptxP3 strains produced more Ptx than the ptxP1 strain and epidemiological data suggest that ptxP3 strains are more virulent. Ptx suppresses both the innate and adaptive immune system [24, 25] and we have proposed that increased Ptx production increases pathogen fitness in vaccinated populations by enhancing transmission by hosts in which vaccine immunity has waned. Thus, both antigenic divergence and increased immune suppression in combination with waning immunity are likely to contribute to the pertussis resurgence . Here we extend our studies on adaptation of B. pertussis using comparative genomics. We determined, annotated and compared genome sequences of six Dutch strains, two of which were isolated before vaccination was introduced in 1953 and four modern strains, isolated approximately 50 years later. The modern strains carried either the ptxP1 allele or the ptxP3 allele, while the pre-vaccination strains carried ptxP1 or ptxP2. We identified novel polymorphisms in specific genes and gene categories which may play a role in the persistence and resurgence of pertussis in the face of intensive vaccination.
Results and Discussion
Isolates used in this study1
Genome size (Mb)
Comparative analysis of Bordetella pertussis genomes
We determined the genome sequences of the six B. pertussis strains through pyrosequencing. As this technology is known to generate errors in homopolymeric nucleotide tracts, SNPs and indels in these regions were filtered out. As a consequence, differences between strains in homopolymeric nucleotide tracts were not identified. However, homopolymeric nucleotide tracts have high mutation rates and may vary during subculturing of a single strain . Thus, genotypes and phenotypes controlled by homopolymeric nucleotide tracts are not stable and changes in these tracts will not represent fixed differences between strains.
We identified 471 SNPs (i.e. bases that were not conserved in one or more of the seven strains), of which 414 and 57 were located in ORFs and intergenic regions, respectively (Additional file 1). Four ORFs were found to contain small insertion or deletions (indels) ranging from eight to 31 bases (Additional file 1). Based on our analyses, the estimated SNP density was 1 SNP per 8,675 bases. Maharjan and coworkers used Microarray-based comparative genome sequencing to detect SNPs in 34% of the Tohama I genome . The Tohama I strain was compared to an Australian isolate from 2006 and a SNP density of 1 SNP per 20,000 bases was found. As we included the whole genome in our comparison and used a larger number of strains, the higher SNP density we found was not unexpected. SNP densities in other monomorphic human pathogens have been found to range from 1 SNP per 2,300 bases in Salmonella enterica serovar Typhi  to 1 SNP per 28,400 bases in Mycobacterium leprae [30, 28]. This places B. pertussis among the most monomorphic human pathogens known. In their analyses, Maharjan and coworkers identified 66 SNPs in 1,229 genes and 4 SNPs in 268 intergenic regions. Of these 70 SNPs, 27 (39%) were also detected in one of the six Dutch strains, while 14 (20%) were specific for the Australian strain.
In addition to the SNPs discussed above, we confirmed 13 large regions of difference (RDs) identified in previous studies using microarrays [31–35] and whole genome sequencing [33, 36]. Further, we found a new polymorphism in RD23 (Additional files 2 and 3).
Genetic relationship based on whole genome sequencing
Evidence for selection in cis regulatory regions
Polymorphisms in pathogenicity-associated genes
Polymorphisms which distinguish strains isolated before and after the introduction of vaccination
Polymorphisms which distinguish ptxP3 strains
We provide the first comprehensive genomic comparison of a bacterial pathogen circulating in a highly vaccinated population. In this study, we included two strains from the prevaccination era and four strains isolated ~50 years later. We confirmed and extended the observation that modern B. pertussis strains differed significantly from prevaccination strains, both phylogenetically and with respect to particular alleles . Further, we identified one highly divergent, possibly ancient, B. pertussis lineage, characterized by the ptxP2 allele. Our work confirmed that B. pertussis strains differ significantly in gene content due to gene loss, a process which may still be ongoing [32, 35]. Further, we found no evidence that acquisition of novel genes has played a role in adaptation, as has been suggested for B. holmesii . In contrast, B. pertussis seems to adapt mainly by the successive accumulation of SNPs. Our work shows that, based on SNP density, B. pertussis is one of the most monomorphic human pathogens. This suggests a recent origin of this species or, more likely, a recent population bottle neck [1, 4].
Our results provide evidence that regions involved in binding of regulatory proteins are subject to diversifying selection suggesting a role in adaptation. Indeed, this is exemplified by the rapid emergence of ptxP3 strains with increased pertussis toxin production . A number of recent studies have highlighted the importance of changes in gene regulation in adaptation of pathogens [48–51]. It is noteworthy that for a number of virulence factors (Ptx, Prn and TcfA), SNPs were found in both the protein-encoding genes and the (putative) promoter regions, suggesting adaptation at both the structural and regulatory level for the same phenotype. Of interest is a SNP in the promoter region for the type III secretion toxin (BteA) and chaperone genes (BtcA). For B. bronchiseptica it has been shown that BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells [43, 44]. The ancestral allele was found in all strains from before 1977, but subsequently replaced by a novel allele which increased in frequency to ~90% in 2000-2008. This suggests that the novel allele may significantly affect strain fitness, although it is also possible that its increase in frequency is due to hitchhiking with other loci which affect fitness. In any case, this allele may be an important marker for successful lineages. The ptxP3 lineage, associated with the resurgence of pertussis in the Netherlands, has emerged recently and spread worldwide . We found that the ptxP3 strains comprised a young branch which diverged recently from modern ptxP1 strains. Several alleles were identified, which were uniquely associated with the ptxP3 lineage and may thus have contributed to its success. Two ptxP3-specific SNPs were in known virulence genes, fim3 and bscI, coding for the serotype 3 fimbriae and a component of the type III toxin secretion system, respectively. We also observed both reactivation and inactivation of genes in the ptxP3 lineage. In conclusion, this work has identified a number of genetic loci which are associated with highly successful strains. Further analyses of these loci can contribute to our understanding of the evolution of bacterial pathogens.
Strain, culture conditions and DNA isolation
The six clinical isolates used in this study are described in Table 1. Strains were grown on Bordet Gengou (BG) agar supplemented with 15% sheep blood and incubated for 3 days at 35°C. DNA was isolated using QIAGEN Genomic-tip 100/G kit, according to the manufacturer's instructions.
Genome sequencing and detection of polymorphisms
B1831 was sequenced using the 454 GS-G20 sequencer (Roche) and the other five isolates were sequenced using the 454 GS-FLX sequencer (Roche), according to the manufacturer's instructions. The generated reads were assembled de novo into contigs using the Newbler assembler (Roche).
SNPs, insertions and deletions were detected by mapping the contigs to the previously sequenced and annotated B. pertussis Tohama I genome  using BLAST. We filtered SNP calls as described in . Briefly, SNPs with low base quality, SNPs within 15 base of the end of a contig, SNPs in repetitive sequences, such as insertion sequence elements, and SNPs in homopolymeric tracts were removed. Single base insertions or deletions in homopolymeric tracts were ignored as these are often a result of 454 sequencing errors . To determine the accuracy of these SNPs we verified 88 SNPs in the sequenced strains by combining PCR with mass spectrometry (Sequenom). All 88 SNPs were correct. Only for a subset of small indels (≥ 6 b), which distinguished sets of strains, were checked by resequencing and included in the analyses. Genome sequences have been submitted to the NCBI Nucleotide Sequence data base (accessions numbers; strain B0558, ADKR00000000; strain B1193, ADKS00000000; strain B1831, ADKT00000000; strain B1834, ADKU00000000; strain B1917, ADKV00000000; strain B1920, ADKW00000000).
Only SNPs for which the allele at the orthologous nucleotide was determined in all strains were included in the phylogenetic analysis. A Maximum Likelihood tree was derived using PhyML  with the following parameters: model, HKY; transition/transversion ratio; estimated; proportion of invariable sites, estimated; number of relative substitution rate categories, 4; gamma distribution parameter, estimated; starting tree, BIONJ; optimize tree topology, yes; optimize branch lengths and rate parameters, yes.
To determine if there is evidence for selection in cis-regulatory loci, we compared the expected distribution of the distance of SNPs to the start of the gene with the actual distribution of these distances using the Chi square test. This was done for all genes.
This project was supported by the RIVM (SOR project S/230446).
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