Horizontally acquired divergent O-antigen contributes to escape from cross-immunity in the classical bordetellae
- Sara E Hester†1, 2,
- Jihye Park†1, 3,
- Laura L Goodfield1, 4,
- Heather A Feaga1, 2,
- Andrew Preston5 and
- Eric T Harvill1Email author
© Hester et al.; licensee BioMed Central Ltd. 2013
Received: 22 April 2013
Accepted: 13 September 2013
Published: 25 September 2013
Horizontal gene transfer (HGT) allows for rapid spread of genetic material between species, increasing genetic and phenotypic diversity. Although HGT contributes to adaptation and is widespread in many bacteria, others show little HGT. This study builds on previous work to analyze the evolutionary mechanisms contributing to variation within the locus encoding a prominent antigen of the classical bordetellae.
We observed amongst classical bordetellae discrete regions of the lipopolysaccharide O-antigen locus with higher sequence diversity than the genome average. Regions of this locus had less than 50% sequence similarity, low dN/dS ratios and lower GC content compared to the genome average. Additionally, phylogenetic tree topologies based on genome-wide SNPs were incongruent with those based on genes within these variable regions, suggesting portions of the O-antigen locus may have been horizontally transferred. Furthermore, several predicted recombination breakpoints correspond with the ends of these variable regions. To examine the evolutionary forces that might have selected for this rare example of HGT in bordetellae, we compared in vitro and in vivo phenotypes associated with different O-antigen types. Antibodies against O1- and O2-serotypes were poorly cross-reactive, and did not efficiently kill or mediate clearance of alternative O-type bacteria, while a distinct and poorly immunogenic O-antigen offered no protection against colonization.
This study suggests that O-antigen variation was introduced to the classical bordetellae via HGT through recombination. Additionally, genetic variation may be maintained within the O-antigen locus because it can provide escape from immunity to different O-antigen types, potentially allowing for the circulation of different Bordetella strains within the same host population.
The classical Bordetella subspecies, B. bronchiseptica, B. parapertussis, and B. pertussis, are very closely related (> 95% DNA sequence identity), but have diverged via large scale DNA loss (up to 25% of genome), or recombination [1, 2]. B. bronchiseptica isolates retain a larger genome, the ability to grow efficiently in environmental reservoirs, such as lake water, and also infect a wide-range of mammals, including immuno-deficient humans [1, 2]. Disease severities can range from asymptomatic carriage to lethal pneumonia , but in general B. bronchiseptica infections are lifelong and benign . B. parapertussis and B. pertussis, the causative agents of Whooping Cough in humans, appear to have independently evolved from a B. bronchiseptica-like progenitor by loss of many genomic regions accompanying their adaptation to a closed life cycle, spreading from human to human without an environmental reservoir [2, 4]. Many genes involved in environmental survival have been lost in the human adapted subspecies, and genes involved in infection are largely retained but differentially expressed . Importantly, there are no known examples of genes acquired horizontally that contribute to the differential infections caused by these organisms . The differences in infection phenotypes of the classical Bordetella subspecies have been related to their differential expression of a largely shared set of virulence factor genes, rather than acquisition of new genes . Intriguingly, our recent comparative analysis of genomes of diverse bordetellae strains revealed that the classical bordetellae pan-genome is open, but with little uptake of new genetic material . Although there are few in depth analyses on individual bordetellae loci to determine mechanisms or the selective pressures contributing to variation, previous analysis has shown some evidence for HGT in several loci shared by most strains, such as the Pertussis Toxin assembly locus . In the previous analysis, one such additional locus predicted to be horizontally transferred was that encoding the O-antigen.
A component of the lipopolysaccharide (LPS), O-antigen is an important Gram-negative factor that protects against innate immunity, blocks antibody binding, and provides protection against environmental stresses, such as antibiotics . There is considerable antigenic variation among O-antigens within and between bacterial species, including differences in sugar composition, chain length, and linkages due to transfer of the entire cluster of O-antigen genes or portions of the locus . For example, Escherichia coli (E. coli) have over 170 antigenically distinct O-antigens, which contribute to evasion of the immune response . Other bacterial species, such as Burkholderia pseudomallei, have less variation with two O-antigen serotypes identified . However, there is still little understanding of why some organisms exchange DNA frequently resulting in increased variation, while others add DNA rarely or never, and of the evolutionary pressures that affect the frequency of HGT in each specific bacterial species.
Although the classical Bordetella subspecies share many known antigens that can induce cross-reactive antibodies, their LPS structures differ in ways that may be important to their overall cross-immunity. In B. bronchiseptica, the LPS is comprised of Lipid A, an inner core (Band B), an outer core trisaccharide (Band A) and O-antigen encoded by lpx, waa, wlb, and wbm loci, respectively . The architecture of the LPS amongst the species is similar in its acylated Lipid A and branched-chain core oligosaccharide, although there are marked differences in acylation patterns of the Lipid A between all three subspecies [11, 12]. In addition, several strains of B. parapertussis do not produce the trisaccharide, likely due to a mutation in the wlb locus, while B. pertussis does not produce an O-antigen due to the lack of the wbm locus [10, 13]. The O-antigen locus in most B. bronchiseptica and B. parapertussis strains contains 24 genes, while the recently characterized wbm locus of one B. bronchiseptica strain (MO149) contains only 15 genes, most of which are genetically divergent from the previously characterized loci [1, 4, 10, 14]. The first 14 genes within the O-antigen locus are thought to be responsible for the biosynthesis of the pentasaccharide linker region connecting the O-polysaccharide to the inner core, synthesis of the polymer subunit, and the capping sugar [10, 11, 15]. Specifically, genes within the middle of the O-antigen locus are predicted to be responsible for modifications of the terminal sugar residue [1, 11]. Two sets of modifications have been noted to correlate with O1 and O2-serotypes, suggesting that antibodies against the O-antigen are directed against these terminal modifications [15–17]. Additionally, it has been shown that O1-specific immune serum does not recognize O2-specific O-antigen molecules and vice versa, suggesting that varying antigenicity could allow for evasion of existing immunity within hosts .
This study builds on preliminary evidence of HGT within the O-antigen loci of several newly sequenced Bordetella strains . We observed distinct regions within the locus with lower GC content and greater sequence diversity (<50% sequence similarity), but low dN/dS ratios than the rest of the genomes. In addition, incongruent branching patterns were observed in a phylogenetic tree based on variable genes in the locus compared to a genome-wide SNP tree, suggesting that HGT may have occurred within regions of this locus. Furthermore, there appears to be extensive recombination in regions within the locus. Since HGT appears to be rare amongst bordetellae, we hypothesized that repeated HGT within this locus could be the result of strong selective pressure for escape from immunity to the parental strain’s O-antigen type. B. bronchiseptica strains induced antibodies that efficiently recognized and killed bacteria with the same O-antigen type in vitro, and in vivo protected against infection in mice. However, strains of one O-antigen type were largely unaffected by immunity generated against strains of other O-antigen types. Overall, this work describes evidence of multiple HGT events within regions of the O-antigen locus via recombination, and suggests escape from host immunity as a pressure that could select for these HGT events in the classical bordetellae.
Diversity within the wbm loci correlates with different O-antigen immunogenic types
Horizontal gene transfer of O-antigen loci in the classical bordetellae
Horizontal gene transfer via recombination
Selective advantage of the divergent O-antigens in the bordetellae
The classic examples of HGT involve the acquisition of clusters of new genes to a species, such as plasmids or pathogenicity islands, via mechanisms involving insertion sequences or other mobile elements [20, 21]. While acquiring new genes by HGT is often associated with changes in bacterial pathogenesis, within the classical bordetellae subspecies these differing characteristics have not been attributable to the acquisition of any new genes conferring differential host specificity or increased pathogenesis . Intriguingly, our recent analysis of the genomes of eleven diverse strains of classical Bordetella revealed substantial genome loss in some lineages, but no known acquisition of new genes that could be associated with differing phenotypes . This analysis based on the aforementioned genomes also predicted that the pan-genome of the classical bordetellae was open with limited uptake of new genetic material. The multiple recombination breakpoints within the O-antigen locus appear to represent HGT events within the Bordetella evolutionary history and subsequent exchange between bordetellae subspecies (Figure 7). These organisms therefore represent a model system to understand how pathogens evolve over time, as well as present the opportunity to examine pressures that drive selection for HGT events.
HGT between closely related bacterial species is more frequent, albeit harder to detect, than between distantly related bacteria [20, 22]. This may be because closely related species are in greater contact in that they share overlapping ecological niches or because they have similar genomic characteristics, making transferred DNA less likely to be rejected . Based on low GC content, high SNP densities with low dN/dS ratios, and dissimilar phylogenetic trees across the locus, we identified subsets of genes within the O-antigen locus of B. bronchiseptica strains, including genes encoding O1 and O2-types, that appear to have been acquired via HGT. Although we have not yet identified any associated mobile elements, such as a tRNA  or insertion sequence elements , we have predicted several recombination breakpoints within the locus. In Salmonella enterica, Escherichia coli, and Klebsiella species, genes toward the center of the O-antigen cluster are less conserved compared to the genes at the ends, and this has been suggested to facilitate the exchange of interchangeable modules within the locus to form new O-antigens . The absence of mobile elements near the breakpoints suggests multiple recombination-mediated HGT events involving O-antigen genes contribute to the different O-antigen types of the classical bordetellae subspecies. Previous analysis of classical bordetellae genomes indicates that these bacterial subspecies, particularly B. pertussis, have evolved through genome loss, which is associated with adaptation to a closed lifestyle within its human host . Notably, B. bronchiseptica complex IV strain MO149, isolated from a human, produces the poorly immunogenic O-antigen that shows evidence of recombination based HGT, as well as has lost several genes within the locus. Therefore, it is also possible that variation within the O3-type locus may be indicative of gene acquisition or the genome undergoing reduction as B. bronchiseptica complex IV strains potentially adapt to a new environment.
The functional interdependence of the O-antigen synthesis/assembly pathways appears to limit recombination within the classical bordetellae locus. For example, the segment between wbmA and wbmC, which was predicted to contribute to the linker region synthesis , is confirmed to be conserved as a unit (Figure 2). Since wbmF, wbmG, and wbmH together constitute the pathway for converting UDP-ManNAc3NAcA to the UDP-GalNAc3NAcA for the O PS backbone , they also appear to be conserved as one segment. Similarly, wbmL, wbmM, and wbmN, which are within the same segment (VRB), are known to encode proteins that comprise ATP-binding cassette (ABC) transporter systems to export the O-antigen . The interdependence of genes in these pathways is a critical consideration, since strains recombining in these regions might have a fitness disadvantage and thus be outcompeted. This interdependence also means that an acquired set of genes must be complete and sufficient for a new phenotype, such as an altered O-antigen serotype, in order for there to be selective advantage to its acquisition. Since HGT appears to be quite rare amongst these organisms, it is therefore of great interest that there are potentially at least five different HGT events within this locus in these ten strains. Together, these data suggest that there is strong selection for the newly acquired genes within this locus, and therefore that each set of acquired genes is sufficient to confer a novel and important phenotype.
Pan-genome analysis indicates that genes associated with diverse phenotypes, antibiotic resistance, and that confer selective advantages are often found within the accessory genome rather than the core genome [26, 27]. O-antigen, which is included in the classical bordetellae accessory genome, may be variable within the subspecies due to its selective advantage of functional interdependence, immunity evasion, or host adaptation. In S. enterica and E. coli, multiple O-antigen serotypes have been identified [28–30] and are hypothesized to contribute to evasion of cross-immunity [31, 32], thereby allowing the circulation of multiple closely related strains with different O-antigens within the same population. Loss of the O-antigen locus by B. pertussis has been suggested to be a result of competition between it and a B. bronchiseptica-like ancestor, which may have circulated within the human population prior to the emergence of B. pertussis. Additionally, previous research investigating cross-protective immunity between O1 and O2-type O-antigens has indicated that lack of protection may allow for the circulation of antigenically distinct B. bronchiseptica isolates within populations . The loss of antigenicity in some B. bronchiseptica complex IV strains, which have been suggested to be more frequently associated with human infections than complex I strains , may in part be due to immune-mediated competition with B. parapertussis in the human population. Additionally, the discovery of the third poorly immunogenic O-antigen type further highlights the significance of the immune response as a likely selective pressure that could be driving HGT amongst classical Bordetella. It is intriguing that the poorly immunogenic O-antigen appears to be specific to B. bronchiseptica complex IV isolates and is not prevalent throughout all B. bronchiseptica and B. parapertussis isolates. This may be indicative of additional functions of the O-antigen to evade host immunity, such as complement deposition , or perhaps evasion of phage, as has been shown in Vibrio cholerae. Poorly immunogenic O-antigens may render B. bronchiseptica isolates more susceptible, whereas O1-and O2-types may be more robust at protecting these bacteria against other environmental factors.
Our study suggests that HGT of portions of the O-antigen locus in Bordetella subspecies, mediated by homologous recombination, is a mechanism to generate divergent O-antigens. The lack of cross-immunity between different O-antigen serotypes may provide an advantage to the acquisition of a new O-antigen type and could explain the high frequency of HGT within this locus, relative to elsewhere in the genomes of Bordetella subspecies. The evidence that O-antigen differences may allow evasion of immunity also leads to the prediction that greater variation will be observed within this locus, relative to others, as more Bordetella genomes are sequenced.
All experiments in this study were carried out in accordance with the National Institute of Health’s recommendations set forth in the Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University at University Park, PA (#31297 Bordetella-Host Interactions). All animals were anesthetized using isoflourane or euthanized using carbon dioxide inhalation to minimize animal suffering.
Bacterial strains and growth
All strains used in this study have been previously described [1, 4, 5, 14]. Bacteria were maintained on Bordet-Gengou agar (Difco, Franklin Lakes, NJ) containing 10% sheep blood (Hema Resources, Aurora OR) and 20 μg/mL streptomycin (Sigma Aldrich, St. Louis, MO). Liquid cultures were grown at 37°C overnight in a shaker to mid-log phase in Stainer-Scholte (SS) broth and heptakis [36, 37].
LPS was purified by a modified Westphal method . Briefly, 500 mL cultures were seeded with mid-log phase (0.5 OD600nm) bordetellae and grown in a shaking incubator at 37°C. Cultures were grown to an OD600nm of 1.0. Bacterial cells were then pelleted at 500 × g and resuspended in 10 mLs of endotoxin free water. An equal volume of 90% w/v phenol was added and the samples were heated to 65°C for 1 hour with stirring. Samples were then chilled followed by centrifugation at 1,000 × g. The aqueous phase was dialyzed using 1000 molecular weight cut off dialysis membrane against ddH2O for 48 hours. After lyophilization, the resulting material was resuspended in Tris buffer (pH7.5) and treated with 25 μg/mL of RNase (Ambion, Austin, TX) and 100 μg/mL of DNase (Mo Bio, Carlsbad, CA). 100 μg/mL of Proteinase K (Ambion, Austin, TX) was then added. Following phenol extraction, the aqueous phase was dialyzed for 12 hours against ddH2O and lyophilized. Resulting LPS was suspended in endotoxin free water.
Purified LPS from the indicated bacterial strains were separated via sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Bedford, MA) as previously described [14, 17, 39]. Membranes were probed with day 28 convalescent serum, at a 1:1000 dilution, from mice inoculated with 104CFU B. bronchiseptica strains RB50, 1289 or MO149. Membranes were then probed with goat anti-mouse (immunoglobulin H+L) horseradish peroxidase-conjugated (1:10,000) antibody (Southern Biotech, Birmingham, AL). All membranes were visualized with ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ).
C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were bred in our Bordetella- and pathogen-free breeding rooms at The Pennsylvania State University, and all animal experiments were performed with approval and in accordance to institutional guidelines. 4 to 6 week old mice were vaccinated intraperitoneally with 200 μl of LPS (100 ng) on days 28 and 14 prior to challenge as previously defined . Mice were lightly sedated with 5% isoflurane (IsoFlo, Abbott Laboratories) in oxygen, and 104CFU B. bronchiseptica strains RB50, 1289 or MO149 were pipetted in 50 ul of phosphate-buffered saline (PBS) (Omnipur, Gibbstown, NJ) onto the external nares. This method reliably distributes the bacteria throughout the respiratory tract [39, 41]. To quantify bacterial numbers, mice were sacrificed at the indicated time points and the lungs, trachea and nasal cavities were excised. Organs were then homogenized in PBS, the appropriate dilution plated on BG agar with antibiotics, and CFU determined by counting colonies. For collection of convalescent or vaccination-induced serum from mice, inoculated animals were sacrificed 28 days post-inoculation or vaccination with 100 ng of purified LPS and bled orbitally as previously described . To obtain serum, blood was incubated at room temperature for 30 minutes and then spun for 5 minutes at 250 × g. Serum was collected and stored at −80°C. For all appropriate data the average +/− the standard deviation (error bars) are presented. Results were analyzed using the Student’s t test with a P value of <0.05 considered significant.
Sequence analysis and GC content
Sequence percent similarity of the O-antigen locus of all eight previously published strains  and flanking regions based on B. parapertussis strain 12822 was plotted between 0% and 100% using zPicture . GC content was calculated using the sliding window method (window size 1,000 base pairs) across the genome of B. bronchiseptica strain RB50 or the O-antigen locus of all eight strains using R . Average and standard deviation for the genome-wide GC content were also calculated by R.
Phylogenetic analysis and dN/dS ratios
Multiple alignments of individual genes in the O-antigen locus were generated by the MEGA5 software, and maximum likelihood trees were constructed with a Tamura-Nei model and 1,000 bootstrap replicates . dN and dS values were computed using PAML package  with the Nei-Gojobori method . In Hyphy, PARRIS was used to detect site-specific selection in wbmF and wbmC, while taking recombination and synonymous rate variation into account .
The O-antigen locus nucleotide sequences were aligned based on B. parapertussis hu strain 12822, using Ssaha v2.2.1 . Then, both Recombination Detection Program (RDP3)  and Genetic Algorithm for Recombination Detection (GARD)  were used to detect recombination breakpoints in the O-antigen locus. In RDP3, default setting (with RDP, GENECONV, MaxChi, BootScan, and SiScan) was used, except adding two more detection methods, including Chimaera and 3Seq, and listing all recombination events. Once the analysis was complete, B. parapertussis ov strain Bpp5 was corrected to be the recombinant strain based on parsimony and all the other recombination events were accepted, and then rescanned. All the predicted recombination events were detected by at least six methods in RDP3, except the last small fragment in B. parapertussis ov strain Bpp5, which was detected by four methods. The same alignment was analyzed by GARD program available at the datamonkey server using HKY85 substitution model.
As previously described , bacteria were grown to mid-log phase, and 103CFU were incubated in serum containing antibodies generated against either O1-type LPS, O2-type LPS or O3-type LPS at the indicated concentrations, with naïve serum, or PBS for 1hour at 37°C. Bacteria were serially diluted and plated on BG containing 25 μg/mL of streptomycin. CFU were counted and compared to the initial inoculums in order to determine percent survival of bacteria. For all appropriate data the average +/− the standard error (error bars) are presented. Results were analyzed using the Student’s t test with a P value of <0.05 considered significant.
Availability of supporting data
The data sets supporting the results of this article are included within the article (and its Additional files).
We acknowledge Laura Weyrich for critical review of this manuscript, and are also grateful to all members of the Harvill lab for support and helpful discussion. We acknowledge Bob Ernst and Mark Pelletier for LPS extraction training. This work was supported by National Institutes of Health (grant number GM083113 to ETH) and by the Agriculture and Food Research Initiative Competitive Grants Program Grant (grant number 2010-65110-20488) from the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (SEH and JP).
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