Genotypic variation in the Bordetella pertussis virulence factors pertactin and pertussis toxin in historical and recent clinical isolates in the United Kingdom

Abstract

The reemergence of pertussis has been reported in several countries despite high vaccination coverage. Studies in The Netherlands and Finland have investigated polymorphism in the genes coding for two important virulence factors of Bordetella pertussis, pertactin and pertussis toxin, and identified the emergence and subsequent dominance in circulating strains of pertactin and toxin variants not found in the whole-cell vaccine (WCV). The study described here investigated whether such variation had occurred in the United Kingdom, which presently has low levels of pertussis. Sequence analysis of the genes for pertactin (prnA) and the pertussis toxin S1 subunit (ptxA) among isolates of B. pertussis from 285 United Kingdom patients, from 1920 to 1999, revealed three prnA variants, prnA(1), prnA(2), and prnA(3), and two ptxA variants, ptxA(1) and ptxA(2), showing differences in nucleic acid sequence. The proportion of pertactin gene types not included in the United Kingdom WCV, i.e., prnA(2) and prnA(3), has increased in recent years and was found in 21 of 86 (24%) strains from the 1980s and 56 of 105 (53%) strains from the 1990s. To date, the presence of these nonvaccine prnA types has not been associated with a resurgence of pertussis in the United Kingdom. The distribution of prnA and ptxA types in The Netherlands, Finland, and the United Kingdom in the 1990s is distinct. The most striking difference in the United Kingdom isolates is that all 105 of the most recent circulating strains (from 1998 to 1999) are of a pertussis toxin type found in the United Kingdom WCV, i.e., ptxA(1).

FIG. 1

Schematic of the B. pertussis pertactin gene (prnA) coding for the P.69 precursor showing regions of polymorphism. Regions 1 and 2, which code for the repeats GGxxP and PQP, respectively, are shown in black. The region including the mature protein is shown in white. The regions removed from the precursor protein are shown in gray. The arrows show the approximate positions of primers used for PCR and sequencing. The figure was adapted with permission from the work of Mooi et al. (17). Details of primers can be found in Table 1.

FIG. 2

Primary structure of the B. pertussis S1 pertussis toxin gene showing regions of polymorphism. To date, four ptxA variants have been described and designated ptxA(1), ptxA(2), ptxA(3), and ptxA(4). Dots indicate sequence identity with ptxA(3). Numbers indicate the positions of nucleotides relative to the start codon of AJ245366. Nonsilent mutations are shown in bold and associated amino acid changes are indicated beneath the relevant codons. The figure was adapted with permission from the work of Mooi et al. (15).

FIG. 3

Primary structure of the B. pertussis prnA gene showing polymorphism in the eight pertactin types described to date, designated prnA(1) to prnA(8) (6, 15). Nonsilent mutations are shown in bold and associated amino acid changes are indicated beneath the relevant codons. Shaded nucleotides indicate silent mutations. Dots indicate sequence identity and dashes indicate that the sequence is not found. The two main regions of polymorphism comprising repeat units are region 1 near the tripeptide motif RGD and region 2 at the carboxyl-terminal region of the protein. (A) Region 1, comprising repeats coding for the amino acid sequence (GGxxP)_4–6. (B) Region 2, comprising repeats coding for the amino acid sequence (PQP)_4–5.

FIG. 4

Temporal trends in the frequency of pertactin gene (prnA) variants in the United Kingdom B. pertussis population. The number of strains containing distinct prnA variants was determined for each period shown on the x axis. Breaks in the x axis indicate periods for which no strains were available. The United Kingdom WCV contains prnA(1).

FIG. 5

Temporal trends in the frequency of pertussis toxin S1 subunit gene (ptxA) variants in the United Kingdom B. pertussis population. The number of strains containing distinct ptxA variants was determined for each of the six decades shown. The proportion of strains containing ptxA(1) out of the total number of strains is shown for each period. Error bars show the 95% CI for these percentages. The United Kingdom WCV strains contain ptxA(1) and ptxA(2).