The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement

Abstract

Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology.IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology.

Keywords: Bordetella pertussis; evolution; genomics; pertussis; rearrangement; whooping cough.

FIG 1

Genome structure variability among isolates of B. pertussis. (A) The order and orientation of genome content in recent circulating isolates (H866, J018, H346, and H559) varied, primarily due to large inversions, and differed greatly from those in vaccine reference strains, such as Tohama I (E476). (B) Genomes analyzed were derived primarily from isolates of the ptxP3 lineage, with only a few isolates from the ptxP1 lineage or vaccine reference strains, and comprised 62 unique chromosomal structures. Vaccine reference strains included 10536 (B203), CS (C393), and Tohama I (E476 and J169). Black bars indicate total numbers of genomes, and white bars indicate numbers of unique structures in each group. (C) The abundance distribution of structures observed in the 247 genomes of the ptxP3 lineage included 35 structures that were observed in only one isolate (singletons). Structures are named according to their associated PFGE profile. Minor variants with shared PGFE profiles are named CDC046-2 and CDC046-3, for example.

FIG 2

Global structure relationships among 257 B. pertussis genomes. Genome structure alignments were clustered according to relative positional changes in homologous sequence blocks. Genomes of the ptxP1 and ptxP3 lineages and vaccine reference strains, which have diverged through accumulation of SNPs, were structurally distinct. Common structures shared by colinear groups of ptxP3 isolates are color coded and labeled according to their associated PFGE profile. Colinear groups of minor variants that share a PFGE profile with a more common structure but differ by rearrangement (e.g., CDC046-2) are colored the same and indicated with circles.

FIG 3

Phylogenetic reconstruction of 257 B. pertussis isolates using maximum parsimony. SNP profiles across 1,473 core variable positions discretely separated ptxP1 and ptxP3 lineages and vaccine reference strains (inset). The phylogenetic distribution of Prn production, prn alleles, and fimH alleles is color coded according to the key. Abundant genomic structures are color coded and labeled according to their associated PFGE profile. Scale bars indicate substitutions per site.

FIG 4

Genomic rearrangement in a phylogenetic context. (A) A common inversion was detected in multiple genetic backgrounds within the ptxP3 lineage, between structures CDC046 and CDC013 (fimH2), CDC002 and CDC010 (fimH1), and CDC300 and CDC237 (fimH1). (B) Emergence of CDC237, the PFGE profile most commonly recovered in the United States every year since 2012, can be inferred from the phylogeny as a series of sequential inversions, which ultimately create novel, local structural conformations (red boxes) detailed in Fig. 5.

FIG 5

Novel structural conformation in CDC237. A single inversion between IS481 insertions (black) differentiates structures CDC217 and CDC237, creating local conformations (highlighted in Fig. 4) not present in any other structures, except CDC300. Neighboring genes located at the boundaries encoded proteins with various functions, such as transporters, proteases, and proteins involved in Bps polysaccharide biosynthesis and Fe-S cluster assembly. Additional IS481 insertions are shaded gray. Indicated coordinates and locus tags correspond to positions in clinical isolate J010 (GenBank accession no. CP012085). A full list of annotated genes at these loci is available in Data Set S1.

FIG 6

Geographic origin of U.S. B. pertussis isolates studied and their genomic structures. (A) Sequenced isolates analyzed in this study were recovered primarily in 30 states in 2000 to 2014, and some states contributed more isolates through participation in the Enhanced Pertussis Surveillance/Emerging Infection Program Network. The geographic distribution of these isolates does not reflect national patterns of disease incidence. See Materials and Methods for selection criteria and Table S1 for specific isolate information. Pie chart diameter represents numbers of isolates and colors indicate genomic structures, named according to their associated PFGE profile, as detailed in the key. (B) Relative frequencies of predominant PFGE profiles (bottom) and total numbers (top) of B. pertussis isolates recovered in the United States and submitted to the Centers for Disease Control and Prevention from 2000 to 2015.