Prevalence and genetic characterization of pertactin-deficient Bordetella pertussis in Japan

Abstract

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn(-)) B. pertussis was observed in Japan. The Prn(-) isolate was first discovered in 1997, and 33 (27%) Prn(-) isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn(-) isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn(-) isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn(-) isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn(-) isolates belong to MLVA-186, and 6 and 3 Prn(-) isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn(-) clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn(-) isolates have a higher growth potential than the Prn(+) back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn(-) strains have arisen and that Prn expression is not essential for fitness under these conditions.

Figure 1. Prn expression in B. pertussis clinical isolates.

The isolates harboring prn2 allele (BP157, BP159, BP162, BP228, and BP235) and prn1 allele (BP155, BP156, BP232, BP233, and BP243) were cultured on CSM plates. Total protein (10 µg) extracted from the bacterial cells was separated by SDS-PAGE followed by CBB R-250 staining (left panel). Immunoblots (1 µg protein/lane) were incubated with anti-Prn1, anti-PT or anti-FHA antiserum (right panel). Ten ng of purified Prn1, PT, or FHA and total protein (1 µg) from B. pertussis Tohama were run on the gel as positive controls.

Figure 2. Molecular mechanisms of loss of Prn expression.

(A) Deletion of the Prn signal sequence (prn1ΔSS). Prn⁻ isolates (n = 24) have an 84-bp deletion, resulting in a 28-amino acid deletion (Val⁹ to Trp³⁶) in the N-terminal region. (B) IS481 insertion mutation in Prn1 gene (prn1::IS481). Eight Prn⁻ isolates have an IS481 insertion in the forward direction at the 6-bp direct repeats (ACTAGG, 1593–1598 bp) of prn1, and 1 isolate had the insertion in the reverse.

Figure 3. Temporal trend of the occurrence of Prn⁻ isolates in Japan.

The frequencies of Prn⁻ isolates harboring prn1ΔSS and prn1::IS481 were based on 121 B. pertussis isolates collected during 1990–2009. Prn⁺ indicates Prn-expressing isolate.

Figure 4. Geographical distribution of Prn⁻ isolates in Japan during 2001–2009.

Blue and red circles indicate Prn⁻ isolates harboring prn1ΔSS and prn1::IS481, respectively. Numbers of isolates tested are indicated in parentheses.

Figure 5. Minimum spanning tree of MLVA of Prn⁻ and Prn⁺ isolates.

Total 121 B. pertussis isolates, collected during 1990–2009 in Japan, were subjected to MLVA: Prn⁻ isolate harboring prn1ΔSS, 24 isolates; Prn⁻ isolate harboring prn1::IS481, 9 isolates; Prn⁺ isolate, 88 isolates. Each circle in the tree represents a different MLVA type with the MLVA type number. The distance between neighboring genotypes is expressed as the similarity value. Prn⁻ isolates belong to MLVA-186, -194, and -226.

Figure 6. Population dynamics of Prn⁻ strains in in vitro growth competition assay.

Prn⁺ back-mutants and parental Prn⁻ strains were mixed in the ratio 4∶1 (Prn⁺-BP59Sm^r versus BP59Sm^r or Prn⁺-BP202Sm^r versus BP202Sm^r) and cocultured in mSS broth at 36°C. The bacterial cultures were collected at 0, 36, 72 and 144 h, and plated on CSM agar plates. The representation of Prn⁻ strains among 40 colonies was examined by colony-PCR. Data are means and standard deviations from 3 independent experiments.