Genomic features of Bordetella parapertussis clades with distinct host species specificity

Abstract

Background: The respiratory pathogen Bordetella parapertussis is a valuable model in which to study the complex phenotype of host specificity because of its unique two-species host range. One subset of strains, including the sequenced representative, causes whooping cough in humans, while other strains infect only sheep. The disease process in sheep is not well understood, nor are the genetic and transcriptional differences that might provide the basis for host specificity among ovine and human strains.

Results: We found 40 previously unknown genomic regions in an ovine strain of B. parapertussis using subtractive hybridization, including unique lipopolysaccharide genes. A microarray survey of the gene contents of 71 human and ovine strains revealed further differences, with 47 regions of difference distinguishing the host-restricted subgroups. In addition, sheep and human strains displayed distinct whole-genome transcript abundance profiles. We developed an animal model in which sheep were inoculated with a sheep strain, human strain, or mixture of the two. We found that the ovine strain persisted in the nasal cavity for 12 to 14 days, while the human strain colonized at lower levels and was no longer detected by 7 days post-inoculation. The ovine strain induced less granulocyte infiltration of the nasal mucosa.

Conclusion: Several factors may play a role in determining host range of B. parapertussis. Human- and ovine-associated strains have differences in content and sequence of genes encoding proteins that mediate host-pathogen contact, such as lipopolysaccharide and fimbriae, as well as variation in regulation of toxins, type III secretion genes, and other virulence-associated genes.

Figure 1

Comparative genomic hybridization of 28 human and 43 ovine strains of B. parapertussis. Each column represents a strain, ordered according to a maximum-parsimony tree topology. Each row represents a microarray element (usually the average of duplicates), arranged in order according to the sequenced strain 12822 (represented in the far left column), with sequences not found in that strain below the red horizontal line. Three major clades (which received >95% bootstrap support; see Additional data file 3 for tree) are denoted by colored bars. Missing data are gray (display excludes elements with missing data from more than 35% of strains). RD21 includes chemotaxis and flagella genes that are not detected in ovine strains. In some cases, data for multiple different array elements for the same gene are shown.

Figure 2

Differences in bsc type III secretion locus transcript abundance between human and ovine B. parapertussis. The sequenced strain 12822 gene arrangement is shown. Data are mean-centered for each array element. Transcript abundance of genes in orange was significantly different between the two groups according to significance analysis of microarrays (see text).

Figure 3

Persistence of human and ovine B. parapertussis in the sheep nasal cavity. Bars represent the number of sheep in each of three inoculation groups with positive nasal swab cultures of B. parapertussis (ov, ovine strain; hu, human strains; mix, 200:1 mixture of human and ovine strain). Plain bars indicate animals with low bacterial loads (<100 colonies); hatched bars indicate those with high bacterial loads. Half the animals were sacrificed at day 7 and half at day 14 post-inoculation.

Figure 4

Representative sections of nasal mucosa fromewes 14 days post-inoculation with ovine or human B. parapertussis. (Hematoxylin and eosin stain, 400× magnification.) (a) Ewe inoculated with ovine strain. The epithelium has intact epithelial cells. The section lackssignificant infiltrates of inflammatory cells. (b) Ewe inoculated with human strain. The epithelium is covered by mucinous materialadmixed with neutrophils, eosinophils, necrotic cell debris, and seroproteinaceous fluid. Within the epithelium and lamina propria are moderate infiltrates of neutrophils (open arrowheads)and eosinophils (filled arrowheads). The lamina propria is moderately expanded by edema.