Abstract
Campylobacter jejuni is a highly diverse species of bacteria commonly associated with infectious intestinal disease of humans and zoonotic carriage in poultry, cattle, pigs, and other animals. The species contains a large number of distinct clonal complexes that vary from host generalist lineages commonly found in poultry, livestock, and human disease cases to host-adapted specialized lineages primarily associated with livestock or poultry. Here, we present novel data on the ST403 clonal complex of C. jejuni, a lineage that has not been reported in avian hosts. Our data show that the lineage exhibits a distinctive pattern of intralineage recombination that is accompanied by the presence of lineage-specific restriction-modification systems. Furthermore, we show that the ST403 complex has undergone gene decay at a number of loci. Our data provide a putative link between the lack of association with avian hosts of C. jejuni ST403 and both gene gain and gene loss through nonsense mutations in coding sequences of genes, resulting in pseudogene formation.
INTRODUCTION
Campylobacter is a common component of the gut microbiota of many avian and mammalian species, where it is often considered a commensal organism, as it is typically carried without obvious disease symptoms. While diarrheal infections are rarely recorded in animals (1, 2), they are extremely common in humans, where the majority of infections are caused by Campylobacter jejuni (3). Human C. jejuni infection can originate in multiple reservoirs, but it is known that a large proportion of human C. jejuni cases are attributed to chickens (4), typically through handling of raw meat, cross contamination, or direct consumption of undercooked meat. However, this does not account for all cases of campylobacteriosis, and it is clear that isolates from other sources and species can infect humans.
The ubiquity of Campylobacter poses interesting questions about its ecology and infection biology (5). C. jejuni and C. coli have been isolated from numerous avian and mammalian species, including food production animals, such as poultry, pigs, and cattle (6, 7), as well as in companion animals, including cats and dogs (2). Wild birds, fecally contaminated ground and surface waters, and drinking water are also reservoirs for C. coli and C. jejuni (8, 9). While both species are widely distributed, disease-causing C. coli is most commonly associated with food production mammals, especially pigs, but improving knowledge of the population structure and evolution of these organisms is challenging some of the traditional ideas. It is now clear that both C. jejuni and C. coli are frequently isolated from multiple species (10, 11), and understanding lineage distribution across multiple hosts is an important current objective in Campylobacter research.
Multilocus sequence typing (MLST) has been performed on a large number of C. jejuni isolates from clinical samples, veterinary sources, abattoir surveys, and environmental sources (4, 9, 10, 12). These studies have revealed the existence of host-restricted and host generalist lineages (5), with considerable overlap of some lineages (sequence type [ST] complexes) that are found in both animal and clinical samples (4, 12). This host-associated genetic structuring has formed the basis of quantitative attribution studies that estimate the relative contributions of different reservoir hosts to human disease (12, 13). Generalist lineages have also been used to investigate cryptic niche structure (5) and host-specific signals of genetic import in Campylobacter (14). However, less work has focused specifically on host-restricted lineages, such as the ST403 complex (10). A previous MLST study of United Kingdom abattoir isolates found a lack of poultry isolates within this ST complex, with 89% from pigs (16/18; the remaining 2 were associated with cattle).
Lateral gene transfer plays a significant role in bacterial evolution, with the gain of DNA from another lineage potentially conferring novel functions and driving bacterial evolution (15, 16). Campylobacter is usually considered to be a highly recombinogenic organism (17, 18), with homologous recombination introducing as much as eight times more DNA polymorphism than mutation alone. Over time, recombination between lineages has the potential to blur the boundaries between clonal complexes or even between C. jejuni and C. coli (19).
The aim of this study was to investigate C. jejuni ST403 complex isolates at the genome level. We report the lack of any ST403 complex strains isolated from avian host species, the primary reservoir of C. jejuni. The lineage exhibits a specific core genome recombination pattern with little apparent exchange of DNA outside the ST403 complex lineage. This is possibly the result of lineage-specific restriction-modification (R-M) systems. In addition, a number of loci present in a large number of non-ST403 complex C. jejuni isolates were shown to have undergone lineage-specific decay and pseudogenization, a mechanism previously not reported in hypothesized niche restriction events in Campylobacter. Together, our data provide information on evolutionary events that have contributed to the formation of a lineage of C. jejuni that is seemingly not colonizing avian hosts.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
The 13 C. jejuni ST403 complex strains used in this study are listed in Table 1. Campylobacter strains were stored at −80°C in Mueller-Hinton broth containing 20% (vol/vol) glycerol until required. The Campylobacter strains were cultured from −80°C freezer stocks onto modified cefperazone-charcoal-deoxycholate agar (mCCDA) (Oxoid, United Kingdom) and incubated for 48 h microaerobically in a gas jar with the addition of a CampyGen sachet (Oxoid) at 37°C prior to use.
TABLE 1.
C. jejuni ST403 complex strains used for comparative genomic analysis in this study
DNA extraction and genome sequencing.
Genomic DNA was prepared from overnight agar cultures by harvesting the entire plate growth, resuspension in sterile phosphate-buffered saline (PBS), and then classical phenol-chloroform extraction using phase lock tubes (5Prime). Sequencing was performed on the Illumina Hiseq 2500 platform using 100-bp paired-end sequencing. De novo assemblies were performed using Velvet (20) and improved using the PAGIT suite of programs (21). Genomes were annotated using PROKKA (22).
Phylogenetic inference.
For population structure analyses, the 13 ST403 clonal complex (CC403) genomes were augmented with a data set of 126 genomes of C. jejuni and 60 C. coli genomes previously published and characterized (14). Core genome alignments were produced using MAFFT (23) on 595 genes that were present at 80% nucleotide level identity in every individual genome (5) and concatenated to produce a core genome (24). Trees were reconstructed using an approximation of the maximum-likelihood algorithm implemented in FastTree2 (25).
Comparative genomics.
The 13 ST403 complex genomes were aligned using Mugsy, with a phylogenetic tree constructed from the extracted single nucleotide polymorphisms (SNPs) using FastTree. Comparative genomics of the ST403 complex was performed using EDGAR (26). Iterative BLAST searches were conducted in EDGAR to produce a pangenome for the 13 genomes listed in Table 1 and a further 21 reference genomes of C. jejuni and C. coli (see Table S1 in the supplemental material). The resulting pangenome was subsequently filtered to identify coding sequences unique to the ST403 complex (present in 100% of ST493 genomes and 0% of non-ST403 genomes, with an 80% nucleotide identity cutoff), and coding sequences absent or divergent from the ST403 complex (present in 0% ST403 genomes and >20% non-ST403 genomes, with an 8% nucleotide identity cutoff). The putative functions of these regions were determined by BLASTx against the entire NCBI nonredundant database. Loci identified as ST403 complex unique and ST403 complex absent were validated by searching for their presence within the entire BIGSdb Campylobacter database by BLASTn, using the default parameters in BIGSdb.
Recombination analysis.
To estimate the amount of recombination in the core genome of ST403 complex strains in relation to the remaining C. jejuni population, we used the BratNextGen software (27) on the core genome alignment of all 139 C. jejuni genomes used in our phylogenetic inference. A total of 20 iterations of hidden Markov model (HMM) parameter estimation were performed, and significant (P ≤ 5%) recombinations were obtained with 100 parallel permutation runs executed on a cluster computer. The negligible changes in HMM parameter values already observed after approximately 30% of the iterations indicated sufficient convergence in the estimation procedure.
Nucleotide sequence accession number.
Raw data for the bacterial strains sequenced as part of this study have been deposited in the ENA under accession number ERP006801.
RESULTS
The C. jejuni ST403 clonal complex is a distinct lineage within the species with no catalogued avian isolation.
We examined the host source of ST403 complex isolates in the Campylobacter MLST database (PubMLST) (http://pubmlst.org/campylobacter/). A total of 278 ST403 complex isolates, representing 1.22% of the entire database (accessed 19 August 2014), were composed of 173 from human clinical cases, 82 from pigs, and 23 from cattle, with no isolates from avian sources recorded in the database. Core genome alignments were produced using 13 ST403 complex isolates and 186 previously published genomes of C. jejuni and C. coli (14). The resulting maximum-likelihood phylogeny showed that the ST403 clonal complex is a C. jejuni lineage that clearly sits within the species C. jejuni (Fig. 1), despite the absence of any catalogued isolations from avian hosts. A separate alignment of the ST403 complex genomes identified 2,831 SNPs across the clade, with pig, human, and cattle isolates intermixed (Fig. 2).
FIG 1.
Maximum-likelihood core genome phylogeny of 139 C. jejuni and 60 C. coli isolates. Isolates from the previously identified distinct C. coli clades (5), as well as pig C. coli, and the ST403 complex strains are identified.
FIG 2.
SNP-based phylogeny of the ST403 complex. The isolates are color coded according to their environmental sources. The SNP distance between the 81116-rooted outlier and the ST403 complex is indicated, as is the SNP distance range observed across the ST403 complex.
Identification of lineage-specific restriction-modification systems in the ST403 complex.
We sought to determine the presence of clade-specific genes that may underpin the observed absence of isolation from avian hosts. EDGAR was used to create a pangenome of the 13 ST403 complex strains and 21 reference C. jejuni and C. coli genomes (see Table S1 in the supplemental material). The pangenome was mined to determine loci unique to the ST403 complex strains, and any identified loci were then searched for across the entire Campylobacter BIGSdb genomic database to confirm their restriction to the ST403 complex. From this analysis, a total of 10 ST403 complex-unique loci were identified (Table 2). Of the 10 ST403 complex-unique coding sequences (CDS), 7 putatively encoded hypothetical proteins and 1 encoded a putative recombination F protein. The remaining CDS encoded two putative type II restriction-modification systems, R.HinP1I restriction endonuclease and modification methylase HhaI, and R.Pab1 restriction endonuclease. BLASTx comparisons showed the former R-M system to be orthologous to a system found in Helicobacter cinaedi and the latter R-M system to be orthologous to a system found in Helicobacter pylori.
TABLE 2.
Loci unique to the C. jejuni ST403 complex
| CDSa | Putative functionb | Orthologue(s)c |
|---|---|---|
| cje135_06701 | Hypothetical protein | None outside ST403 complex |
| cje135_06696 | Hypothetical protein | None outside ST403 complex |
| CJ857_00839 | Hypothetical protein | None outside ST403 complex |
| cje135_03870 | R.HinPI restriction endonuclease | H. cinaedi CCug18818 |
| cje135_03865 | Modification methylase Hhal | |
| CJ857_01361 | Hypothetical protein | Cc 317-04/90-3 |
| CJ857_01649 | Hypothetical protein | Weak similarity with Cjj LMG23223 |
| cje135_02353 | Hypothetical protein | Cc LMG23336/Helicobacter bilis ATCC 43879/H. cinaedi PAGU611 |
| cje135_02348 | R.Pab1 restriction endonuclease | H. pylori 51 |
| cje135_02293 | Recombination protein F | Helicobacter pullorum MIT98-5489 |
CDS are annotated according to the genome annotation of the ST403 reference strain ATCC 33560, except for CJ857_00839, CJ857_01361, and CJ857_01649, which are relative to our strain 857 due to ambiguous annotation in ATCC 3560.
The putative function is that ascribed to the CDS by Pfam and BLASTP searches.
Orthologues are as determined by BLASTP against the entire BLAST nrDatabase.
Given the presence of these unique R-M systems across the entire ST403 complex lineage, we sought to determine if there was an accompanying effect on the levels of detectable core genome recombination within ST403 complex strains. BRATNextGen was used to detect recombination events across the C. jejuni core genome alignment constructed for phylogenetic testing (Fig. 3). The resulting recombination profile shows a distinctive pattern of recombination events in the ST403 complex that is composed primarily of intralineage events. Phylogenetic trees were reconstructed on the core genome alignment with all recombination removed (see Fig. S1 in the supplemental material) and on the regions identified as recombination events in the ST403 complex (see Fig. S2 in the supplemental material). Both phylogenies show tight clustering of the ST403 complex strains, with 0.964 bootstrap support for the clustering of the ST403 recombining regions. Combined, these data suggest that the recombination occurring in the ST403 complex is predominantly lineage specific.
FIG 3.
Visualized output of BRATNextGen analysis of the core genome alignment of 139 C. jejuni isolates. On the left, a clustering tree of the 139 isolates is shown based on the proportion of shared ancestry through recombination. The colors of the branches indicate cluster membership, and significant recombinations are indicated by colored rectangles on the right. Shared color in the same column implies that the recombination segments in the respective isolates correspond to a shared origin. A contiguous single-color rectangle along the genome represents a single inferred recombination event. The colors indicate the cluster in which the corresponding recombined genome segment has the highest frequency. For convenience, the clusters corresponding to the darker blue and green hues are indicated by the blue and green boxes, respectively. The ST403 complex genomes are indicated by the red box.
Evidence of gene decay in the C. jejuni ST403 complex.
Further analysis of the pangenome identified a total of 14 loci that were absent or divergent in every ST403 complex genome and present in at least 20% of the non-ST403 complex genomes included in the analysis (Table 3). To allow a more detailed comparison of the nature of absent or divergent loci, the sequence for each was extracted from a relevant reference genome sequence and used to perform pairwise BLAST comparisons against each of the ST403 complex genomes. This confirmed that six of the loci were completely absent from all of the CC403 genomes. More importantly the remaining eight loci all showed patterns of pseudogenization and gene decay across the ST403 complex, with five of those loci containing identical pseudogenization events in every genome (Table 3). Loci C8J_0199 and C8J_0200 had been merged into a single open reading frame (ORF) by a mutation removing the stop codon delineating the two ORFs in the reference genomes, leading to a single polypeptide, followed by a second mutation just downstream introducing a stop codon, while the other four loci contained multiple stop codon mutations that were common across all the ST403 complex genomes and a single locus containing a deletion common across the lineage. The remaining three loci contained multiple insertions, deletions, and SNPs that varied across the ST403 complex but that could result in the loss of gene function of that CDS across the lineage. As the 5 loci showing conserved patterns of pseudogenization represent ST403-unique alleles of those CDS, we searched for matching alleles in the entire BIGSdb Campylobacter database using BLAST. Among the isolates contained in the BIGSdb database, no alleles that matched these 5 loci with >70% nucleotide identity over >50% of the sequence length contained mutations identical to those in the ST403 isolates, further suggesting that these evolutionary events are associated with the ST403 complex.
TABLE 3.
Characterization of loci absent from the C. jejuni ST403 complex
| CDSa | Putative function | ST403 complex mutation |
|---|---|---|
| C8J_0199-200b | Protease/IgA1 protease domain family/serine protease | Genes merged by SNP and pseudogenized by stop codon |
| C8J_0806b | Hypothetical protein (seryl-tRNA synthetase domain; provisional endonuclease subunit domain) | Pseudogenized central deletion in CDS |
| C8J_0815b | Hypothetical protein (cytochrome c oxidase cbb3 subunit) | Pseudogenized by stop codons |
| C8J_0628b | Hypothetical protein (potassium-transporting ATPase subunit) | Pseudogenized by stop codons |
| C8J_0466b | Putative outer membrane protein (assembly complex/hemolysin activation/secretion protein) | Pseudogenized by stop codons |
| CJE0296 | Conserved domain protein (MCP domain signal transduction protein) | Multiple insertions and deletions varying across lineage |
| CJE0392 | Hypothetical protein | Absent |
| CJE0660 | Hypothetical protein | Multiple deletions across lineage |
| CJE0659 | Putative membrane protein/putative dicarboxylate carrier protein MatC/putative integral membrane protein | Absent |
| C8J_0033 | Hypothetical protein (gamma-glutamyltranspeptidase) | Absent |
| C8J_0392 | Hypothetical protein | Entire or central deletion across lineage |
| Cj1158c | Hypothetical protein (putative small hydrophobic protein/small integral membrane protein) | Absent |
| C8J_1559 | Hypothetical protein | Multiple deletions and SNPs across lineage |
| C8J_0239 | Probable methyl-accepting chemotaxis protein signaling domain | Absent |
CDS as annotated in the appropriate reference genome.
Mutations are identical across all ST403 complex isolates.
DISCUSSION
In this study, we investigated the C. jejuni ST403 complex, a lineage of C. jejuni that has never reportedly been isolated from an avian host. In our initial MLST study (10) identifying this lineage, 16 of the 18 ST403 complex isolates were from pigs, with the other 2 from bovine sources, leading to the hypothesis that this was a pig-adapted clone. Subsequent mining of the MLST database has revealed the presence of isolates from other sources, including cattle, and a large number of human isolates within the ST403 complex, the majority of which were isolated from the Dutch Antilles (28). This indicates that isolates from the complex have the capacity to cause human disease. However, the most important observation is that no ST403 complex isolates from poultry have been recorded in PubMLST, suggesting that the ST403 complex may be less well adapted to avian hosts or that it represents a lineage of C. jejuni that has not evolved the ability to colonize avian hosts as well as the many other C. jejuni lineages.
Recent studies of the population structure and ecology of Campylobacter have indicated the presence of generalist lineages, such as the ST21 and ST45 complexes, which contain isolates from multiple sources, as well as specialist lineages, such as the ST61 and ST42 complexes that have been reported to be associated with cattle (9, 10, 29) or the ST354, ST443, ST353, and ST257 complexes that are associated with poultry (9). It is also known that within the generalist lineages there are sublineages with evidence of host association (5, 14), indicating that in some cases adaptation to a particular host might still be occurring. It is possible, therefore, that the ST403 complex represents another specialist lineage of C. jejuni that has evolved to become less suited to colonization of the avian host. This seems more plausible than the possibility that ST403 has not evolved the ability to colonize avian hosts, given its central position in the C. jejuni species phylogeny, as this would require multiple lineages of C. jejuni sharing a most recent common ancestor with ST403 independently evolving to become efficient avian colonizers while ST403 did not.
We investigated clade-specific loci and identified three R-M loci that were unique to the ST403 complex. Strain-specific R-M systems have been reported previously in C. jejuni strains 81116 (30), ATCC 43431 (31), and 81176 (32) and are thought to contribute to the apparent recombination and transformation restriction that has hindered genetic manipulation of the organism for some time. Mutagenesis of the type IIG R-M enzyme Cj1051c in NCTC11168 increased the strain's ability to take up plasmid DNA, including that from Escherichia coli (33). Single nucleotide polymorphisms in known R-M systems in the Japanese ST4526 clone are thought to be responsible for the reduced uptake of plasmid DNA compared to NCTC11168 (34), as well as contributing to the ability of the clone to thrive in Japan. In other organisms, such as Neisseria meningitidis, R-M systems have also been reported to play key roles in the formation of structured phylogenetic clades and patterns of recombination (35). The distinct recombination pattern of the ST403 complex isolates showed within lineage recombination, as evidenced by the tight phylogenetic clustering of the recombinant regions. Recombination is thought to play an important role in niche adaptation and acquisition of a host signature (36). However, this recombination appears to be restricted, as it was recently reported that two major generalist lineages (the ST21 and ST45 complexes) have limited recombination with each other but readily recombine with other specialist, host-adapted lineages (5).
Besides the presence of ST403 complex-specific R-M systems, there are a number of coding sequences that are absent from isolates within the complex or that have degraded compared with homologues in other C. jejuni strains. It is not possible here to determine if the genes were present in the common ST403 complex ancestor and were deleted through time or were never present in the ancestral lineage. However, the high prevalence of these absent genes across C. jejuni and some C. coli clades suggests that they have most likely been lost in the CC403 lineage through time, a hypothesis supported by the presence of a central deletion in locus C8J_0806 that is identical across the ST403 complex. What is clear is that the ST403 complex shows signs of lineage-specific mutations in distinct loci. There are several possible explanations for these findings, but one possible evolutionary scenario is that the selection pressure at these loci changed with a move away from an avian host reservoir and that mutations resulting in loss of function have increased in the ST403 complex because they do not influence fitness. Three loci appear to be undergoing similar processes, with multiple independent deletions and mutations across the ST403 complex, possibly suggesting that the process is ongoing. Interestingly, none of these loci have clearly identifiable functions that one may associate with avian colonization or, indeed, niche adaptation, such as those described for cattle-associated C. jejuni lineages (14), but predominantly encode hypothetical proteins.
Functional investigation may improve our understanding of the possible role of the ST403 complex pseudogenized genes in adaptation away from avian hosts. Rather than a simple case of no longer being able to colonize birds, it may be that loss of these loci results in reduced competition with other lineages, low colonization numbers, or reduced ability to survive transmission outside the host. Furthermore, evidence of acquisition of specific R-M systems and the pseudogenization and loss of several loci suggests that both the loss and gain of specific loci may be associated with adaptation to a restricted host set in C. jejuni. The combination of both gene gain and adaptive gene loss is known to have played a role in niche adaptation in other enteric bacterial pathogens (37, 38). Further work will be necessary to quantify the influences of host, pathogen, and environmental factors on colonization of different host species by C. jejuni, and the increasing availability of bacterial genomes and understanding of gene function will provide a basis for future investigation.
Supplementary Material
ACKNOWLEDGMENTS
L.M. was funded by a Nottingham Trent University Ph.D. studentship. A.M. is supported by the Royal Society (IE121459). S.K.S. is supported by a Wellcome Trust Career Development Fellowship, with additional funding from the BBSRC (BB/I02464X/1) and MRC-CLIMB (MR/L015080/1). J.C. is supported by ERC grant 239784 and AoF grant 251170. DNA sequencing was carried out at the Exeter Sequencing Service, which is supported by the following grants: Wellcome Trust Institutional Strategic Support Fund WT097835MF, Wellcome Trust Multi User Equipment Award WT101650MA, and BBSRC LOLA award BB/K003240/1.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00546-15.
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