Extramural Update April 2004

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. Humanand 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. Background Whooping cough, with its prolonged paroxysmal cough and distinctive 'whoop', was well-known by the Middle Ages [1]. Bordetella pertussis was isolated from whooping cough patients in 1906, and the pathogenesis of this disease has been the subject of extensive study. In the 1930s Eldering and Published: 6 September 2006 Genome Biology 2006, 7:R81 (doi:10.1186/gb-2006-7-9-r81) Received: 10 May 2006 Revised: 14 July 2006 Accepted: 6 September 2006 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/9/R81 Genome Biology 2006, 7:R81 R81.2 Genome Biology 2006, Volume 7, Issue 9, Article R81 Brinig et al. http://genomebiology.com/2006/7/9/R81 Kedrick noticed different colony morphology of some strains, which they used as the basis for proposing a new species, B. parapertussis [2]. Later estimates of the percentage of Bordetella-associated cases of cough caused by B. parapertussis range from about 5% to 30% [3-6], depending on case definition and vaccination coverage. This species was considered an obligate human pathogen until 1987, when B. parapertussislike organisms were found in normal and pneumonic lamb lungs [7,8]. B. parapertussis strains from humans appear to be genetically distinct from ovine strains, and while human strains are highly clonal, ovine strains appear more heterogeneous by pulsed-field gel electrophoresis [9], insertion element typing [10], and PCR-based random amplified polymorphic DNA profiles [11]. This species has not been isolated from any other source and is now thought to be composed of two subgroups, one of which infects only humans and the other of which infects only sheep. The unique host specificity of B. parapertussis contrasts with both the obligate human pathogen lifestyle of B. pertussis as well as the broad mammalian host range of the third member of this close family of respiratory pathogens, B. bronchiseptica. Despite considerable genetic similarity among the three species (or subspecies, as some have proposed), together they illustrate a spectrum of host restriction, and may provide insights into the evolution and maintenance of this important phenotype. B. parapertussis affords one of the least-confounded models of host specificity in the bacterial world, because its two subgroups are found in only two hosts, and are known to be very closely related. While one human-associated strain of B. parapertussis has been sequenced completely [12], much less is known about ovine strains, so we undertook a comprehensive investigation of a large collection of such strains, utilizing subtractive hybridization, microarray-based comparative genomic hybridization, and transcript abundance profiling. In addition, we developed an experimental sheep infection model to explore the colonization patterns and resulting pathology caused by an ovine strain of B. parapertussis, compared to a human strain. We found that ovine strains contain a large amount of genetic material not found in human strains, including a unique lipopolysaccharide (LPS) locus and an additional gene encoding a fimbrial subunit, and that the two groups of strains have many differences with respect to genome content and transcript abundance profile. Of particular interest were differences in transcript abundances for several virulence-associated genes and operons, including tracheal colonization factor, dermonecrotic toxin, adenylate cyclase, and the bsc type III secretion system. Inoculation of sheep with either the natural ovine pathogen or a human strain revealed that the ovine strain elicited a less intense granulocyte infiltrate and colonized the nasal turbinate more efficiently and for a longer period of time. Results and discussion Detection of novel sequences and a large island of foreign DNA in ovine B. parapertussis Nearly all previously available genomic information about B. parapertussis has been based on data from the sequenced human strain. In order to discover as yet unrecognized genes and sequences that might be unique to ovine strains, we searched for novel sequences in an ovine strain of B. parapertussis (Bpp5) using suppression subtractive hybridization against a pool of the three sequenced Bordetella genomes (including the sequenced human strain of B. parapertussis). Of the 40 contigs discovered in Bpp5 (GenBank:DQ518927DQ518966), four (comprising 26 of the 100 fragments originally sequenced) had closest BLASTn hits (Additional data file 1) to genes from the anaerobic bacterium Desulfovibrio vulgaris (DVU2019, 2022, 2025, and 2026). To explore the B. parapertussis chromosomal region carrying these sequences, we constructed a fosmid library from Bpp5 DNA and probed it for the D. vulgaris-like sequences. Complete sequencing of a 34 kb fosmid insert (from pBpp5fos495) that hybridized to these probes revealed a large region of foreign DNA immediately downstream of the locus encoding the glycine tRNA (data not shown; GenBank:DQ515909). The foreign DNA shared homology to sequences from several unrelated species, suggesting the presence of an island of mobile horizontally transferred DNA. Partial sequencing of an overlapping fosmid insert (from pBpp5fos353) indicated that this island extended for at least 50 kb in the Bpp5 genome (see Additional data file 2 for a schematic of this region). This discovery is striking given the extremely low rates of gene acquisition in other Bordetella species [12,13], and may indicate that ovine B. parapertussis strains have access to a source of new genetic material to offset the ongoing genome degradation that marks the process of host specificity in this genus. We designed microarray elements for the 40 novel contigs obtained from Bpp5 by subtractive hybridization (see Additional data file 1 for sequences) and added them to our Bordetella microarray to screen other human and ovine strains for these sequences. Of these, 38 array elements yielded data passing the quality filters, and only one of these sequences was detected in any of the 28 human strains. Sixteen array elements were variably detected among the forty-three ovine B. parapertussis strains, while the rest were detected in all ovine strains. All the new array elements were detected in Bpp5, from which the novel sequences were originally identified, as well as in two other strains from sheep in New Zealand (Bpp3 and Bpp4), all three of which cluster tightly based on these data and their total gene contents (see Additional data file 3 for a maximum parsimony tree of all strains). Among the 40 ovine strain-specific contigs were 5 LPS genes that differed from all of the previously sequenced Bordetella LPS genes. Microarray elements for these genes were detected in all 43 ovine strains. Complete sequencing of a Genome Biology 2006, 7:R81 http://genomebiology.com/2006/7/9/R81 Genome Biology 2006, Volume 7, Issue 9, Article R81 Brinig et al. R81.3