Genetic control of Bordetella pertussis infection: identification of susceptibility loci using recombinant congenic strains of mice

Abstract

Susceptibility to and severity of Bordetella pertussis infection in infants and children vary widely. The spectrum of clinical symptoms ranges from subclinical infection to mild disease, severe whooping cough, and death. The aims of this study were to examine genetic susceptibilities of mice to B. pertussis and to identify genetic loci in the mouse genome that are involved in susceptibility to B. pertussis infection. For this purpose we screened two sets of recombinant congenic strains (RCS) of mice, HcB and CcS, for differences in the numbers of bacteria in the lung 7 days after inoculation. For both CcS and in HcB mice, a wide range in numbers of bacteria in the lung was found, suggesting that the course of infection is under multigenic control. From both RCS sets of mice, we selected one strain to identify possible susceptibility loci in F(2) hybrid mice. The degree of lung colonization 7 days postinoculation in these F(2) mice was evaluated in relation to genetic markers by linkage analysis. We found three novel loci that are involved in the control of B. pertussis infection. One locus, designated B. pertussis susceptibility locus 1 (Bps-1), was identified on chromosome 12. The presence of the C57BL/10 genome on this locus instead of the C3H genome significantly decreased the number of B. pertussis bacteria in the lung. Bps-1 has a dominant-positive effect on the clearance of B. pertussis from the lung. The function of most genes in this region is unknown. Two other loci, Bps-2 and Bps-3, showed genetic interaction and are located on chromosomes 5 and 11. We aim to identify the gene(s) in these regions which modify susceptibility to B. pertussis.

FIG. 1.

Strain distribution pattern (SDP) of two Recombinant Congenic Strains (RCS). The CcS4 strain (above) was derived from the mouse strains BALB/c as background (displayed in grey) and STS as donor (displayed in black). The HcB28 strain (below) was derived by crossings between mouse strains C3H as background (displayed in grey) and C57BL/10 as donor (displayed in black).

FIG. 2.

Numbers of CFU in the lungs of the CcS RCS (top) and the HcB RCS (bottom). Seven days after inoculation with B. pertussis, lungs were removed and the number of viable B. pertussis was determined. Bars indicate the group average of the number of bacteria in the lung (average of 10 mice per group). Horizontal lines connect groups of mice that are mutually not significantly different according to the Student-Neuman-Keuls test. From these strains Ccs4 and HcB28 were selected for generating F₂ hybrid generations.

FIG. 3.

Numbers of CFU in the lungs of (CcS4 × BALB/c)F₂ and (HcB28 × C57BL/10)F₂ mice 7 days after infection with B. pertussis. Lungs were removed, and the number of viable B. pertussis was determined by counting CFU in the homogenized lungs. Approximately 200 mice per F₂ strain were used. Each dot represents the number of CFU of individual mice.

FIG. 4.

Linkage (LOD score) between lung colonization by B. pertussis (phenotype) and donor chromosomal loci (genotype). The association was calculated by ANOVA with genotypes as a fixed factor and sqrt of number of CFU as a dependent variable. The LOD score is plotted as negative log P against the physical distance of chromosome 12. The region, designated B. pertussis susceptibility locus 1 (Bps-1), has a LOD score of 4.6 (P = 0,0000251; r² = 0,09728).

FIG. 5.

Linkage (LOD score) between lung colonization by B. pertussis (phenotype) and an interaction between two donor chromosomal loci (genotype). The association was calculated by ANOVA with the interaction of two genotypes as a fixed factor, experiment as a random factor, and sqrt of number of CFU as dependent variable. Individual markers showed no linkage with the phenotype, but an interaction was found between chromosome 11 and a locus on chromosome 5 (4.4 cM). The LOD score is plotted as negative log P against the physical distance of chromosome 11. These regions, designated B. pertussis susceptibility loci 2 (chromosome 5) and 3 (chromosome 11) (Bps-2 and Bps-3), have a LOD score of 2.7 (P = 0,002184; r² = 0,070).