Replacement of Adenylate Cyclase Toxin in a Lineage of Bordetella bronchiseptica

Abstract

Bordetella bronchiseptica is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of B. bronchiseptica strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that cyaA, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the cya locus, which encodes, activates, and secretes CyaA, was replaced by an operon (ptp) predicted to encode peptide transport proteins. Other B. bronchiseptica strains from the same phylogenetic lineage as that of strain 253 also lacked the cya locus, contained the ptp genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the ptp genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of B. bronchiseptica.

Novel, host-restricted genotypes of Bordetella bronchiseptica associated with phocine respiratory tract isolates

“…Only three other RT1 isolates have been identified, one from a pig in the UK and two from guinea pigs, one each from Ireland and Switzerland (Register et al, 1997;Register & Magyar, 1999); none has been typed by MLST. Aside from 22/M63/4/09, six additional B. bronchiseptica ST14 isolates have been reported, three from rabbits, two from guinea pigs and one of unknown origin, collectively representing the USA, Ireland, Australia and South Africa (Diavatopoulos et al, 2005;Buboltz et al, 2008). The DNA sequence of the pertactin repeat region from 22/M63/4/09, allele 1-3a1/2-6a1, is similarly uncommon.…”

“…To compare host-specific diversity quantitatively, we compiled the STs and RTs reported previously for other B. bronchiseptica isolates (Register et al, 1997(Register et al, , 2000(Register et al, , 2012Register & Magyar, 1999;Diavatopoulos et al, 2005;Buboltz et al, 2008;Rath et al, 2008) or documented in the pubMLST database (http://pubmlst.org/bordetella) for all hosts represented by at least seven isolates. Most isolates included in these comparisons were acquired between 1980 and 2005, a period of time similar to that during which the seal isolates evaluated were obtained.…”

Enzymatic Carbon Dioxide Capture

“…RB54 and RB50DbscNDcyaA are previously described derivatives of strain RB50 [33,37]. B. bronchiseptica strain 761, 448, and 308 were obtained from the CDC in Atlanta, Georgia and have been previously described [15,38,39]. B. bronchiseptica strain JC100 has been previously described [15].…”

“…Additionally, dye-swap experiments were performed analogously, in which the fluorescent labels were exchanged to ensure that uneven incorporation did not confound our results. Fluorescently-labeled cDNA copies of the total RNA pool were prepared by direct incorporation of fluorescent nucleotide analogs during a first-strand reverse transcription (RT) reaction [39,[45][46][47]. The two differentially labeled reactions were then combined and directly hybridized to a B. bronchiseptica strain RB50-specific long-oligonucleotide microarray [46].…”

“…Western blots were performed on whole cell extracts of B. bronchiseptica, B. pertussis and B. parapertussis grown to mid-log phase in SS broth as described previously [38,39]. Lysates were prepared by resuspending 1610 9 CFU in 100 ml of Laemmli sample buffer; total cellular protein content were quantitated using the BCA assay to equalize protein content between samples.…”