Human infection with the gastrointestinal pathogen Campylobacter jejuni is dependent upon the opportunity for zoo- notic transmission and the ability of strains to colonize the human host. Certain lineages of this diverse organism are more common in human infection but the factors underlying this overrepresentation are not fully understood. We analyzed 601 isolate genomes from agricultural animals and human clinical cases, including isolates from the multihost (ecological generalist) ST-21 and ST-45 clonal complexes (CCs). Combined nucleotide and amino acid sequence analysis identiﬁed 12 human-only amino acid KPAX clusters among polyphyletic lineages within the common disease causing CC21 group isolates, with no such clusters among CC45 isolates. Isolate sequence types within human-only CC21 group KPAX clusters have been sampled from other hosts, including poultry, so rather than representing unsampled reservoir hosts, the increase in relative frequency in human infection potentially reﬂects a genetic bottleneck at the point of human infection. Consistent with this, sequence enrichment analysis identiﬁed nucleotide variation in genes with putative functions related to human colonization and pathogenesis, in human-only clusters. Furthermore, the tight clustering and polyphyly of human-only lineage clusters within a single CC suggest the repeated evolution of human association through acquisition of genetic elements within this complex. Taken together, combined nucleotide and amino acid analysis of large isolate collections may provide clues about human niche tropism and the nature of the forces that promote the emergence of clinically important C. jejuni lineages. Key words: Campylobacter, phylogenetics, adaptation, pathogenesis, human niche. Introduction to infect and survive new selective pressures associated Many bacterial species that are known as causes of gas- with a pathogenic lifestyle. troenteritis are common commensal organisms causing The common gastrointestinal pathogen Campylobacter little or no harm to the host species. For pathogenic strains jejuni is widely distributed among wild and domesticated an- of these species, the pathway to disease can involve a imal species/reservoirs (Sheppard et al. 2011), and the major- series of population bottlenecks. Therefore, clinical iso- ity of the human infections are the result of consumption of lates sampled from patients are a subset of the bacterial contaminated food (Kapperud et al. 2003; Friedman et al. population, representing strains that had the opportunity 2004; Skarp et al. 2016). Campylobacter jejuni populations The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 10(3):763–774. doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 763 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Meric et al. GBE are generally structured by host source (Sheppard et al. 2010, of genotypic and phenotypic plasticity that facilitates rapid 2011), and this has allowed the attribution of the source of host adaptation in a multihost environment (Read et al. human infection based upon comparative multilocus se- 2013; Woodcock et al. 2017; Pascoe et al. 2017) but little is quence typing (MLST) and whole-genome characterization known about the speciﬁc genomic variations that promote of host and clinical isolates (Sheppard, Dallas, MacRae, et al. proliferation of particular STs, within generalist lineages, in 2009; Sheppard, Dallas, Strachan, et al. 2009; Pascoe et al. different niches such as human hosts. 2015; Dearlove et al. 2016; Thepault et al. 2017). These stud- Here we combine nucleotide-based phylogenetic analysis ies revealed chickens as a major source of human campylo- with amino acid sequence-based clustering to characterize bacteriosis (EFSA 2015). On the assumption that all strains are populations of C. jejuni from humans and agricultural animals, equally able to infect humans, the abundance of C. jejuni in and identify candidate genes involved in these possible host farmed chickens (Vidal et al. 2016) and contamination of re- associations. Our hypothesis was that a combined methodo- tail poultry (Wimalarathna et al. 2013) would be enough to logical approach would identify subtle host-associated differ- explain the importance of chickens as a pathogen reservoir. ences between isolates from major generalist groups. These However, recent studies of C. jejuni in poultry have shown analyses identiﬁed sublineages of the ST-21 complex that that some common chicken-associated strains are rare among were overrepresented among isolates sampled from human clinical isolates while others increase in relative frequency disease. The putative functions of genes within human-only (Yahara et al. 2017). This suggests that factors other than amino acid clusters included those important in human path- simple opportunity for transmission are involved in human ogenesis, such as ﬂagella and capsule synthesis. Our study infection. provides a new way of interrogating genomic data sets to In some species, such as Escherichia coli, the emergence of identify candidate genes in a subset of strains that may indi- pathogenic strains can be associated with the acquisition of cate a population bottleneck associated with human speciﬁc attributes which confer increased ability to cause dis- colonization. ease or evade treatment. For example, genetic elements that encode virulence and persistence in humans such as those Materials and Methods carried by phages and plasmids in E. coli or the acquisition of antibiotic resistance in Staphylococcus(as reviewed in Kaper Bacterial Genomes et al. 2004; Pantosti et al. 2007). In some cases the acquisition A total of 601 C. jejuni genomes were used in this analysis, of small amount of genetic material increases the virulence, as previously published in various studies (Cody et al. 2013; seen in the large scale outbreak of the Shiga-like-toxin pro- Sheppard, Didelot, Jolley, et al. 2013; Sheppard, Didelot, ducing E. coli O104:H4 (Frank et al. 2011). Where speciﬁc Meric, et al. 2013; Pascoe et al. 2017; Yahara et al. 2017) pathogenicity elements can be identiﬁed, it is relatively simple (supplementary table S1, Supplementary Material online). The to identify the agent causing an outbreak and its molecular majority of these came from clinical isolates (n¼ 481) and the cause. However, in C. jejnui, traits associated with clinical rest from agricultural sources, either poultry (n¼ 88) or cattle isolates not only reﬂect virulence but also those that confer (n¼ 32). Most isolates were from the United Kingdom a ﬁtness advantage against the various selective pressures (n¼ 546/601, 90.1%). A total of 134/601 (22.3%) were encountered in the poultry processing chain, such as survival from CC-45 and 467/601 (77.7%) were from CC-21-48- in the nonhost environment (Yahara et al. 2017). 206 (supplementary table S1, Supplementary Material online), The increasing availability of whole-genome data provides which have been shown to form a single sequence cluster in opportunities to investigate the genomic differences underly- previous studies (Sheppard, Didelot, Meric, et al. 2013). These ing variation in proteins and their motifs that may promote constituted all the sequenced genomes available to us when the proliferation of particular pathogenic strains. this study was initiated. CC21-48-206 is henceforth collec- Epidemiological studies of C. jejuni from clinical samples and tively referred to as CC21 group in this study. Sequencing animal reservoirs typically reveal genetically diverse popula- was performed on Illumina platforms, and assemblies were tions. However, isolates belonging to CC21 and CC45 are performed with either Velvet (Zerbino and Birney 2008)or regularly the most common lineages isolated from human Spades (Bankevich et al. 2012). Assembled DNA sequences disease (Karenlampi et al. 2007; Levesque et al. 2008; from various sources (supplementary table S1, Supplementary Mullner et al. 2009; Sheppard, Dallas, MacRae, et al. 2009; Material online) were uploaded to a web-based database Sheppard, Dallas, Strachan, et al. 2009; Sanad et al. 2011; based on the BIGSdb platform (Jolley and Maiden 2010) Mughini Gras et al. 2012; Sahin et al. 2012; Guyard- which allowed archiving, whole-genome gene-by-gene se- Nicodeme et al. 2015). Both of these lineages have been iso- quence alignments and prevalence analyses. In addition, the lated from a variety of sources, including ruminants, poultry, isolation source of all available CC21 group and CC45 isolate wild birds, domesticated companion animals, as well as envi- records (n¼ 17,107) from the pubMLST database (https:// ronmental samples (Sopwith et al. 2008; Sheppard et al. pubmlst.org/campylobacter/; last accessed February 07, 2011, 2014). This ecological generalism may reﬂect a degree 764 Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Convergent Amino Acid Signatures in C. jejuni GBE 2018) were obtained (October 21, 2016) and analyzed to on Tajima and Nei (1984) pairwise distances of protein quantify the numbers of different STs isolated from humans sequences together with the Tamura and Kumar (2002) cor- and agricultural animals and contextualize this study. rection for heterogeneous patterns. The initial number of clusters was chosen by selecting the k associated with the highest log posterior probability under the KPAX2 model. In Phylogenetic Tree Inference total, 100 partitions were then created by applying random Sequence alignments were obtained using a gene-by-gene modiﬁcations to the initial partition obtained by the k- approach (Sheppard et al. 2012). Brieﬂy, the presence of medoids solution to the proposal partition. Split, merge, and 1,668 coding sequences (CDS) from the reference C. jejuni transfer operators were as previously described (Pessia et al. NCTC11168 genome (NCBI accession: NC_002163.1) in all 2015). Each of the 100 partitions was then independently 601 genomes of this study was inferred using BLAST with the used as a starting state for the KPAX2 posterior maximization following parameters: A gene was considered present when a algorithm to ensure that the ﬁnal estimate was as close to the local alignment match with the reference was obtained global posterior mode as possible. The 100 KPAX2 runs were on>50% of the sequence length with>70% sequence iden- done in parallel on a cluster computer, where the individual tity. Using these criteria, 1,058 genes were shared by all 601 runs took approximately 1–2 weeks until convergence. The genomes from our data set, constituting the “core genome.” clustering solution with the highest log posterior probability Gene-by-gene alignments using MAFFT (Katoh and Standley among the 100 independent runs was chosen as the ﬁnal 2013) were concatenated to create a core genome gene-by- estimate. The source of isolates belonging to different KPAX gene alignment that was used subsequently. For protein clusters was indicated for isolates from: human clinical only trees, in-frame translation was performed using custom (clinical); chicken and human clinical sources (chickenþ scripts (supplementary ﬁle 1, Supplementary Material on- clinical); cattle and human clinical sources (cattleþ clinical); line) for each individual gene alignment, which were then and chicken, cattle and human clinical sources concatenated. The resulting concatenations were used as (chickenþ cattleþ clinical) (supplementary table S2, an input for the reconstruction of phylogenetic trees, ei- Supplementary Material online). For each KPAX cluster, char- ther using an approximation of the maximum-likelihood acteristic amino acids were determined (Pessia et al. 2015), as algorithm implemented in FastTree2 (Price et al. 2010) well as corresponding proteins and genes in the C. jejuni (ﬁg. 2)or RAxML (Stamatakis 2014)(supplementary ﬁg. NCTC11168 reference genome (supplementary table S3, S1, Supplementary Material online). For the comparison Supplementary Material online). This allowed for a compari- of nucleotide and in-frame translated phylogenetic trees, son of KPAX clustering results with genome-wide association we used RAxML (Stamatakis 2014)with GTRGAMMA and study (GWAS) results to identify the genes associated with PROTGAMMAGTR models, respectively. For amino acid clinical-only C. jejuni KPAX groups. trees, the analysis used a simple search under the GAMMA model of rate heterogeneity on the protein Prevalence of STs from Human-Only KPAX Clusters among data set using empirical base frequencies and estimating Isolates from Human and Nonhuman Sources a general time reversible model of amino acid substitution. Total prevalence of C. jejuni STs observed to belong to human-only KPAX clusters was quantiﬁed among samples isolated from human and nonhuman sources (mainly poultry KPAX2 Method: Bayesian Clustering Based on and cattle) and was inferred using isolation source informa- Amino Acid Sequence tion speciﬁed in a total of 17,107 CC21, CC48, CC206, and KPAX2 is a new Bayesian method for identifying evolutionary CC45 isolate records, taken from a total of 49,598 archived signals in amino acid sequences that relate to differential evo- isolate records from every CC publicly available in the lution of lineages that may be either monophyletic or poly- pubMLST database (https://pubmlst.org/campylobacter/; phyletic, for example, resulting from the horizontal accessed October 21, 2016). distribution of relevant genomic elements through recombi- nation (Pessia et al. 2015). Earlier analysis of a database of SEER Method: Genome-Wide Association Mapping thousands of inﬂuenza A virus H3N2 subtypes demonstrated that the method could accurately identify antigenic clusters We used a k-mer enrichment method to identify, from the determined by amino acid variation and the sequence posi- nucleotide sequence data, which genomic elements were sig- tions relevant for the antigenic differences (Pessia et al. 2015). niﬁcantly more prevalent in two groups of isolates: The The concatenated set of 601 core genome sequences corre- human-only KPAX clusters (group 1, n¼ 103) compared to sponded to 153,911 amino acid positions, harboring 17,405 the remainder of the C. jejuni population (group 2, n¼ 498) polymorphic sites. KPAX2 was used with the default prior (Weinert et al. 2015; Lees et al. 2016). This binary trait analysis settings, and inference was initialized with a proposal partition was performed to ensure that eventual gene regulatory ele- of the samples obtained using the k-medoids algorithm based ments or accessory genes associated with the clusters would Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 765 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Meric et al. GBE 50 Clinical Agricultural animals 100 CC-21/48/206 CC-45 Sequence type (ST) from this study (with n>10 entries in pubMLST) FIG.1.—Prevalence of clinical and agricultural C. jejuni within ST-21 and ST-45 CCs in a public archive repository. The prevalence of clinical (black) and poultry/livestock (gray) isolation sources in pubMLST for each ST in our data set with more than ten isolate records in the pubMLST database (https://pubmlst. org/campylobacter/; last accessed February 07, 2018). There were a total of 17,107 archived public isolate records. not remain unidentiﬁed, because the KPAX2 method is based Amino Acid Sequence-Based Analysis Reveals Human- only on core protein sequence variation. The input assemblies Only Subclusters contained approximately 31 M unique k-mers with lengths The Bayesian model-based method KPAX2 was used to clas- between 10 and 99 nucleotides. The following ﬁltering steps sify aligned proteins into functionally divergent groups, based were applied to reduce the original k-mer input set by includ- upon amino acid residues of a collection of 601 genomes ing only k-mers that: 1) had>75% frequency in group 1 representing 66 STs belonging to the CC21 group and and<25% frequency in group 2; 2) had a chi-square associ- CC45. A total of 1,058 core CDS used in the nucleotide phy- ation test P-value< 10 ; and 3) had association logeny were in silico translated and a concatenated amino P-value< 10 in a logistic regression model with the three acid alignment produced for each genome-sequenced strain. ﬁrst multidimensional scaling coordinates representing the We then performed Bayesian clustering using the KPAX2 al- population structure correction. The multidimensional scaling gorithm, and the tree was annotated with the 36 KPAX clus- coordinates were calculated from a distance matrix based on ters identiﬁed (ﬁg. 2). KPAX groups could be classiﬁed into 10,000 randomly selected k-mers from the initial set. The ﬁnal four categories depending on sources of isolates: Human only set of genome-wide signiﬁcant k-mers contained 347 k-mers, (12 KPAX groups, 112 isolates from 20 STs), human and which were mapped to an annotated reference genome to chicken only (10 KPAX groups, 150 isolates from 20 STs), identify their contexts. human and cattle only (4 KPAX groups, 33 isolates from 13 STs), and human, chicken and cattle (10 KPAX groups, 306 isolates from 24 STs). The isolate source within each KPAX Results group is shown in the supplementary table S2, Supplementary STs Vary in Frequency in Human Clinical and Agricultural Material online. Environments KPAX and nucleotide sequence clusters showed incom- Direct comparison of the relative prevalence of sequence plete congruence. Amino acid clustering was polyphyletic types was performed using the entire Campylobacter when superimposed on the nucleotide phylogeny (ﬁg. 2, sup- PubMLST database. This contained a total of 49,598 plementary ﬁg. S1, Supplementary Material online) and in entries on October 21, 2016. Of these 13,095 belonged some cases, divergent lineages shared the same KPAX cluster. to the CCs 21, 48, and 206, previously shown to form a For example, the 138 isolates belonging to ST-21 were found single sequence cluster based upon whole-genome anal- in 7 different KPAX groups containing isolates from various ysis, and 4,012 belonged to CC45 complex. Within the sources. However, particular STs (ST-21, ST-50, ST-47, ST-44, CC21 group there were 8,382 human clinical isolates and ST-861, and ST-190) were assigned KPAX groups encompass- 3,869 originating from agricultural animal sources, while ing only isolates from humans. Examination of isolate records in CC45 there were 1,674 human clinical isolates and in the entire pubMLST database revealed that most isolates 1,685 agricultural isolates. The relative abundance of iso- from STs assigned to human-only KPAX groups (276/283 iso- late STs belonging to CC21-48-206 and CC45 was deter- lates, in 15/20 STs) have also been isolated from humans and mined (ﬁg. 1). In both CCs, there was variation in the other host species, with only ST-6601, ST-6137, ST-5727, and relative frequency of STs isolated from human clinical ST-2355havingbeenisolatedsolely fromhumans(table 1). and agricultural animal samples. Obviously, KPAX clusters were not deﬁned using the whole 766 Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Prevalence in pubMLST (%) 11 Convergent Amino Acid Signatures in C. jejuni GBE 24*+29 8+28* KPAX group sources Clinical + chicken 28* ST-45 complex Clinical + cattle Clinical + chicken + cattle 11* 3* Clinical only 16 3* 17*++ 731+ 28* Polyphyletic KPAX group 11* 24*+17* 0.001 3* 19* 17* 3* 3* 2 14* 5* 34* 3* 17*+20 14*++ 34* 19*+ 27 14*+34* 5* 10+22 ST-21-48-206 complex 23++ 18 30+ 32 FIG.2.—Population structure of 601 C. jejuni ST-21 and ST-45 complex isolates. Isolates are labeled by KPAX group labels (integers) and colored by their source distribution within KPAX groups: Isolates from chicken and clinical sources (yellow), cattle and clinical sources (blue), chicken, cattle and clinical sources (pink), or clinical only (red). Polyphyletic KPAX groups, reﬂecting isolates in the same KPAX group but in multiple lineages on the tree, are indicated with an asterisk. The phylogenetic tree was reconstructed from a whole-genome gene-by-gene amino acid alignment, translated in-frame, using an approximation of the maximum-likelihood algorithm implemented in FastTree2, and using a general time reversible model. genomes of the pubMLST-archived comparative data set; 14 genes to have a role in nonhuman host adaptation however, it is useful to contextualize KPAX-ST correlation (Sheppard, Didelot, Meric, et al. 2013)(supplementary table within a wider data set. It should be noted that the ST desig- S4, Supplementary Material online). Although some of these nation can have poor speciﬁcity in contrast to the lineages associations were sometimes weak in the corresponding stud- determined from whole genomes and therefore an isolate ies, they were nonetheless highlighted and are consistent with from a nonhuman host present in the pubMLST database a general role in transmission and host colonization. may lack the genetic elements identiﬁed in our present To conﬁrm whether these loci were associated with a hu- analysis. man clinical-only sublineage we also performed sequence el- ement enrichment analysis, using SEER (Lees et al. 2016), to identify the genetic basis of human clinical-only sublineage Identiﬁcation of Genes with Human-Associated Amino strains compared with those from other host sources (ﬁg. 3, Acid Signatures within the CC21 Group supplementary tables S5 and S6, Supplementary Material on- We sought to identify the discriminatory amino acids that line). A total of 181 genes (supplementary table S5, resulted in clustering of human clinical-only CC21 group iso- Supplementary Material online), containing 547 enriched k- lates. We identiﬁed a total of 1,213 amino acids sites which mers, were obtained (supplementary table S6, Supplementary mapped to 265 genes (supplementary table S4, Material online). These included genes that have been identi- Supplementary Material online). Mapping the physical loca- ﬁed in previous association studies (supplementary table S5, tion of these against the reference CC21 genome Supplementary Material online), in particular genes with pu- NCTC11168 suggested that these loci were distributed across tative roles in in vitro colonization of surfaces and aggrega- the genome and not under strong linkage disequilibrium tion, host adaptation and clinical disease (Sheppard, Didelot, resulting from physical proximity (ﬁg. 3A). Interestingly, a total Meric, et al. 2013; Pascoe et al. 2015; Yahara et al. 2017). of 24/265 (9.0%) genes were found to be associated with A total of 26 genes were signiﬁcantly associated with previous GWASs (supplementary table S4, Supplementary human-only lineages in both KPAX clustering and SEER asso- Material online). More speciﬁcally, 3 genes were predicted ciation analyses (ﬁg. 3, table 2). Half of these genes have been to have a role in survival from farm to clinical disease described as important for host colonization or pathogenesis, (Yahara et al. 2017), 8 genes to have a role in in vitro coloni- nine in humans or human cell studies, and four in chicken zation of surfaces and aggregation (Pascoe et al. 2015), and colonization studies (table 2), consistent with a broad role for Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 767 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Meric et al. GBE Table 1 Prevalence of isolates from STs found in human-only KPAX groups in human and nonhuman sources KPAX Group ST Total Number of Associated Hosts Prevalence in Prevalence in Isolates in Our Study Human Hosts Nonhuman Hosts in a a in pubMLST (%) pubMLST (%) KPAX-8 ST-21* 138 Human, chicken, cattle 66.5 22.4 KPAX-9 ST-475 5 Human 75.0 19.4 ST-6601# 1 Human 100.0 0.0 KPAX-19 ST-50* 100 Human, chicken 62.8 31.4 ST-5727# 2 Human 100.0 0.0 ST-2355# 1 Human 100.0 0.0 KPAX-20 ST-47* 3 Human 79.2 9.4 ST-5242# 1 Human 100.0 0.0 KPAX-21 ST-572 4 Human 82.7 11.8 ST-5138 1 Human 66.7 33.3 KPAX-26 ST-44* 6 Human 73.2 22.3 KPAX-27 ST-50* 100 Human, chicken 62.8 31.4 KPAX-28 ST-21* 138 Human, chicken, cattle 66.5 22.4 ST-861* 4 Human 86.2 10.3 ST-5018 3 Human 90.5 4.8 ST-190* 2 Human 54.7 43.1 ST-141 1 Human 72.0 24.0 KPAX-30 ST-222 3 Human 78.9 21.1 KPAX-32 ST-122 4 Human 78.2 13.9 KPAX-34 ST-21* 138 Human, chicken, cattle 66.5 22.4 ST-50* 100 Human, chicken 62.8 31.4 ST-3769 1 Human 83.3 16.7 ST-520 1 Human 46.1 51.3 KPAX-35 ST-6137# 2 Human 100.0 0.0 NOTE.—Asterisks indicate STs that also found in other nonhuman-only KPAX groups. Dashes indicate STs that have never been isolated from nonhuman sources in our data setorpubMLST. pubMLST (https://pubmlst.org/campylobacter/) as accessed on October 21, 2016. these genes in host adaptation and/or in multihost ﬁtness. Of et al. 2016; Llarena et al. 2016). Although this has provided a particular note within these genes were the ﬂagellar gene basis for identifying candidate genes with potential functional ﬂgH highlighted in a previous GWAS on nonchicken host signiﬁcance (Morley et al. 2015; Pascoe et al. 2015; Yahara adaptation (Sheppard, Didelot, Meric, et al. 2013), two genes et al. 2017), straight forward genome analysis often ignores (ceuC and ceuE) involved in the enterochelin iron uptake sys- factors relating translation and the production of speciﬁc tem in C. jejuni, a gene (aspB) involved in aspartate metabo- amino acid chains and proteins that may be important in lism, and a gene (fdhD) encoding a formate dehydrogenase, a host adaptation or pathogenicity. For example, although the function that has been highlighted as important for survival four nucleotides can form 64 different triplets they only en- from farm to clinical disease (Yahara et al. 2017). All ﬁve of code 20 amino acids. This means that the same amino acid these genes are known to be important in the invasion of can be encoded by different triplets, typically with variation at mammalian cells and/or human colonization (Palyada et al. the third base, and divergent genomes may have convergent 2004; Guerry 2007; Novik et al. 2010; Sheppard, Didelot, amino acid sequences that are potentially functionally impor- Meric, et al. 2013; Yahara et al. 2017). tant in host adaptation or pathogenesis. Analysis of encoded amino acid sequences in this study identiﬁed polyphyletic nu- cleotide sequence clusters within the CC21 group that clus- Discussion tered together within the same amino acid sequence clusters. An important aim in zoonotic pathogen research is to identify These convergent human-only amino acid KPAX clusters, in genetic and functional variations associated with lineages or divergent genomic backgrounds, may have been overlooked sublineages that cause human infection. Comparative analysis using conventional nucleotide sequence-based approaches. of nucleotide sequence variation across the genome has im- Comparative analysis of the nucleotide sequence of the proved understanding of the epidemiology and evolution of 601 C. jejuni genomes in this study identiﬁed STs belonging Campylobacter (Sheppard, Didelot, Jolley, et al. 2013; Gilbert to the CC21group andCC45that were reportedtohave 768 Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 0.4 1.2 0.2 1.0 Convergent Amino Acid Signatures in C. jejuni GBE Less More prevalent than in reference AB genome COG annotation Not assigned to COGs Function unknown Intracellular traffi cking and secretion Cell wall/membrane biogenesis Transcription Signal transduction mechanisms Cell motility General function prediction only Replication, recombination and repair Secondary metabolites biosynthesis, transport and catabolism Defense mechanisms Translation Nucleotide transport and metabolism Posttranslational modiﬁcation, protein turnover, chaperones C. jejuni Coenzyme transport and metabolism 0.0 NCTC11168 Amino acid transport and metabolism Carbohydrate transport and metabolism Lipid transport and metabolism Energy production and conversion Inorganic ion transport and metabolism -6 -4 -2 0 2 4 6 Prevalence diff erence from reference genome annotation (%) Genes containing KPAX characteristic sites (n=265) Genes containing associated k-mers (SEER) (n=181) Genes containing KPAX characteristic sites (n=265) Genes containing associated k-mers (SEER) (n=181) Overlap (n=26) FIG.3.—Genes associated with clinical-only C. jejuni KPAX groups. (A) GWAS results visualized on a circular reference genome. The outer circle indicates genes from the C. jejuni NCTC1168 reference genome, with core genes shared by all isolates in our data set (black) and accessory genes (gray) indicated. Genes found to contain characteristic amino acid sites deﬁning KPAX groups are represented (red ticks) along with a quantitative visualization of thenumber of these sites per gene (red dots; scale of the quantiﬁcation from 0 to 420). Genes found to contain k-mers associated with clinical-only KPAX groups using SEER are represented (blue ticks) along with a quantitative visualization of the number of these k-mers mapped per gene (blue dots; scale of the quan- tiﬁcation from 0 to 25). Black ticks indicate genes containing both KPAX group characteristic sites and associated k-mers using SEER. (B) Difference in COGs prevalence (%) among genes containing KPAX characteristic sites (red) and genes containing associated k-mers inferred by SEER (blue) with COGs prevalence in the C. jejuni NCTC11168 reference genome annotation. been isolated at different frequencies from agricultural animal asymptomatic carriage of Campylobacter may be underesti- and human sources lineages. This is consistent with other mated and underreported (Calva et al. 1988; Louwen et al. population genomic studies, where the variation in relative 2012; Lee et al. 2013; Islam et al. 2017). These factors could abundance has been explained by the different capacity of inﬂuence the evolution and population structure of symptom- certain strains to survive through the poultry production chain atic bacteria. at atmospheric oxygen concentrations (Yahara et al. 2017). Examination of isolate records in the entire pubMLST data- Asymptomatic carriage of C. jejuni is not thought to be com- base revealed that 97% of the isolates assigned to human- mon in humans in industrialized countries (Lee et al. 2013). only amino acid KPAX clusters are of STs that have been iso- Therefore, under a simple transmission model, amino acid lated from other host species as well as humans (table 1). clusters would be expected to be present in both reservoir Notably, only ﬁve STs from human-only KPAX groups (corre- animal and infected human hosts. For this reason, the exis- sponding to 7/276 isolates in our data set) have never been tence of strongly human-only amino acid KPAX clusters is reported in nonhuman hosts, either in our data set or from unexpected. There are two possible explanations. First, iso- isolate records in pubMLST. On the basis of the known sour- lates assigned to human-only KPAX clusters are derived ces of C. jejuni in human infection—including CC21 group from a source that is not represented in our isolate collection, isolates (Sheppard, Dallas, MacRae, et al. 2009; Sheppard, which has not been captured by the sampling of isolates used Dallas, Strachan, et al. 2009), the close similarity between C. in this study. Second, there are isolates that share amino acid jejuni populations on food and those from clinical samples clusters within CC21 group C. jejuni in our data set that in- (Kittl et al. 2013), and the presence of STs belonging to crease in relative frequency in humans, compared with the human-only amino acid KPAX clusters among agricultural isolates from other hosts. Additionally, it is possible that hosts in pubMLST, it is unlikely that they indicate an unknown Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 769 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 0.8 1.6 0.6 1.4 Meric et al. GBE 770 Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Table 2 List of Genes Associated with Clinical-Only Campylobacter jejuni KPAX Groups Name Alias Operon Predicted Product (COG) COG COG Description Number of Number of Notes References Code Characteristic Mapping Sites (KPAX) k-mers (SEER) cj1346c dxr 500 1-Deoxy-D-xylulose 5-phos- I Lipid transport and metabolism 52 8 phate reductoisomerase genes cj1347c cdsA 500 Phosphatidate I Lipid transport and metabolism 8 1 maf adhesins are included in the (46) cytidylyltransferase genes maf6-Cj1347 genomic region cj1253 pnp 472 Polynucleotide phosphory- J Translation 7 5 lase/polyadenylase cj0762c aspB 285 Aspartate aminotransferase E Amino acid transport and metabo- 61 A aspB mutant is defective for (38) lism genes entry into cultured human ep- ithelial cells cj0810 nadE 301 NAD synthetase H Coenzyme transport and metabo- 61 lism genes cj0006 — 4PutativeNaþ/Hþ antiporter R General function prediction only 5 4 Cj0006 is expressed in vivo when (48) family protein C. jejuni infects chicken cj0389 serS 149 Seryl-tRNA synthetase J Translation 5 1 cj0542 hemA 213 Glutamyl-tRNA reductase H Coenzyme transport and metabo- 33 lism genes cj0767c coaD 286 Phosphopantetheine H Coenzyme transport and metabo- 31 adenylyltransferase lism genes cj1620c mutY 593 A/G-speciﬁc adenine L Replication, recombination and 3 2 An SNP in mutY is associated Dai et al. (2015). glycosylase repair with increase of antibiotic resistance cj0005c — 3 Molydopterin containing R General function prediction only 2 2 Infection of and adherence to (47) oxidoreductase human Caco2 cells in vitro was strongly reduced in a cj0005c mutant cj0069 — 38 Hypothetical protein Cj0069 J Translation 2 1 Involved in the proximal re- Asakura et al. (2007). sponse to cell adhesion and bioﬁlm formation cj0598 — 231 Hypothetical protein Cj0598 S Function unknown genes 2 5 cj0689 ackA 259 Acetate kinase C Energy production and conversion 2 2 Involved in nutrient acquisition, genes acetate metabolism cj1076 proC 404 Pyrroline-5-carboxylate E Amino acid transport and metabo- 21 reductase lism genes cj1157 dnaX 426 DNA polymerase III subunits L Replication, recombination and 2 2 Highlighted in a study as a puta- (52) gamma and tau repair tive Guillain–Barre syndrome marker Convergent Amino Acid Signatures in C. jejuni GBE Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 771 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 cj1508c fdhD 555 Formate dehydrogenase ac- C Energy production and conversion 2 3 Formate metabolism is involved (12) cessory protein genes in host association and survival in the food chain fromfarmto human disease cj0498 trpC 200 Indole-3-glycerol-phosphate E Amino acid transport and metabo- 1 2 In a genomic region identiﬁed as (53) synthase lism genes important for cell hyperinva- siveness in a transposon assay cj0518 htpG 206 Heat shock protein 90 O Posttranslational modiﬁcation, pro- 1 1 Associated in GWAS on bioﬁlm tein turnover, chaperones genes formation (heatshock protein); Pascoe et al. (2017) cj0543 proS 213 Prolyl-tRNA synthetase J Translation 1 3 cj0687c ﬂgH 258 Flagellar basal body L-ring N Cell motility genes 1 3 Flagellar assembly cluster; ﬂa- (23, 37) protein gellar motility is important for human and chicken coloniza- tion, and possible secretion of virulence factors/Associated with cattle adaptation in GWAS cj1056c — 398 Putative carbon–nitrogen R General function prediction only 1 1 Expression of cj1056c is modu- Reid et al. (2008). hydrolase family protein lated at low pH in vitro cj1261 racR 477 Two-component regulator K Transcription 1 6 The Campylobacter RacRS system (50, 51) regulates fumarate utilization in a low oxygen environment, andracRmutants show re- duced colonization of chicken cj1271c tyrS 479 Tyrosyl-tRNA synthetase J Translation 1 1 TyrS was overexpressed in a poor (23, 49) colonizer of chicken/ Associated with cattle adapta- tion in GWAS cj1353 ceuC 502 Enterochelin uptake P Inorganic ion transport and metab- 1 5 Uptake of siderophores is a de- (45) permease olism genes scribed virulence/host coloni- zation trait cj1355 ceuE 502 Enterochelin uptake peri- P Inorganic ion transport and metab- 1 5 ceuE mutant shows decreased (39) plasmic binding protein olism genes chicken colonization NOTE.—Genes are overlapping between the two analyses (KPAX and SEER). As predicted by OperonPredictor (http://biocomputo2.ibt.unam.mx/OperonPredictor/; last accessed February 07, 2018). Meric et al. GBE host source population, although this cannot be ruled out in abilities (Palyada et al. 2004). Additionally, the cdsA gene is this study. located in the genomic region of known maf adhesins, in- Our results are therefore consistent with the increase in volved in survival and host colonization (Karlyshev et al. relative frequency of particular amino acid sequence subclus- 2002). Knockout mutants of cj0005c, an uncharacterized ox- ters that are uncommon in animal hosts, among isolates from idoreductase, have been shown to be strongly impaired in humans. Host colonization potential is inﬂuenced by the infection abilities and adherence to human Caco2 cells adaptive genomic variations that exist before and after trans- in vitro (Tareen et al. 2011), whereas a neighboring gene, mission to the new host species (Geoghegan et al. 2016). In cj0006, encoding a putative transporter, has been shown in both cases, population bottlenecks reduce the genetic vari- global transcriptomic studies to be overexpressed in vivo ance in the population at interhost transmission which would when C. jejuni infects chicken (Hu et al. 2014). Finally, the account for the increased relative frequency of human-only tyrS gene, predicted to encode a tyrosyl-tRNA synthetase, has amino acid KPAX clusters. It remains difﬁcult to differentiate been observed to be overexpressed in a poor chicken colo- genetic changes associated with bottlenecking and drift from nizer strain of C. jejuni (Seal et al. 2007). Additionally, it has adaptive physiological changes that directly impact pathogen- been associated with mammalian (cattle) adaptation in a pre- esis, such as human tissue tropism and virulence. vious GWAS from our laboratory (Sheppard, Didelot, Meric, Furthermore, human passage can induce genetic variation et al. 2013). in contingency genes coding surface structure through frame Genes predicted to have a role in metabolism were also shifts and phase variation (Bayliss et al. 2012; Revez et al. highlighted. The ackA and aspB genes are involved in acetate 2013; Thomas et al. 2014). However, the sharing of amino and aspartate metabolism, respectively, and have been shown acid sequence clusters by polyphyletic lineages is evidence of in mutagenesis studies to be important for entry into human homoplasy and investigating the putative function of these epithelial cells in vitro (Novik et al. 2010). The fdhD gene genes may provide clues about their potential role in human encoding a formate dehydrogenase was also associated colonization. Human-only KPAX clusters are present in every with isolates belonging to human-only amino acid clusters. major lineage within the CC21 group (ﬁg. 2)and are notably Formate metabolism has been previously implicated in host absent among CC45 isolates. This asymmetry cannot be association and survival in the food production chain from explained by an insufﬁcient sample size from the CC45 pop- farm to human disease (Yahara et al. 2017). The racR gene ulation in our data set and may suggest that, despite being an which regulates fumarate utilization in a low-oxygen environ- efﬁcient human colonizer, CC45 strains may lack the suitable ment also displayed human-associated variation and racR-de- genetic background for acquisition of genomic elements that ﬁcient mutants have shown reduced chicken colonization are associated with elevated human colonization that we ob- in vivo (Bras et al. 1999; van der Stel et al. 2015). Other genes serve in the CC21 group. Further analysis of larger sample with variation associated with the CC21 human amino acid sets, potentially including phenotypic analyses, is needed to clusters included the dnaX gene that encodes a DNA poly- conﬁrm this. merase and is a marker for the campylobacteriosis sequelae Genome-wide association methods that have recently Guillain–Barre syndrome (Godschalk et al. 2006)and trpC that been applied to bacteria (Sheppard, Didelot, Meric, et al. encodes an indole-3-glycerol-phosphate synthase in a geno- 2013) allow the investigation of genetic variation that under- mic region important for human cell hyperinvasiveness (Javed lies important phenotypes. By quantifying the nucleotide se- et al. 2010). quence that was enriched in isolates from humans (Lees et al. Genomic variation associated with clinical C. jejuni isolates 2016) across the genomes, we were able to investigate the includes elements associated with the primary host putative function of genes with human-only amino acid KPAX (Sheppard, Didelot, Meric, et al. 2013) and the food produc- clusters. A total of 26 genes were identiﬁed (table 2), half of tion chain (Yahara et al. 2017), as well as variation which which have been previously linked to host colonization or confers an adaptive advantage to human colonization and pathogenesis, nine in humans or human cells, four in chicken. may directly impact pathogenesis (Thompson and Gaynor For example, ﬂgH, a gene associated with ﬂagellar assembly 2008). Evidence of genetic bottlenecks and selection fostered (table 2) and otherwise associated with adaptation in a mam- by this complex ﬁtness landscape will not only be reﬂected in malian host (Sheppard, Didelot, Meric, et al. 2013). Flagellar nucleotide sequence variation but also in features, such as motility has been shown to be important for human and gene order, distribution of CDS on leading and lagging chicken colonization, and possibly for the secretion of viru- strands, GC skew, and codon usage (Bentley and Parkhill lence factors into host cells (Guerry 2007). Genes directly in- 2004; Rocha 2004). By combining analysis of nucleotide se- volved in host colonization also included ceuCE, involved in quence and amino acid variation we were able to identify a enterochelin uptake (table 2). The uptake of siderophore has subset of human-associated C. jejuni. As these isolates are been described as a virulence/host colonization trait in found in nonhuman hosts, we interpret this as evidence of Campylobacter (Richardson and Park 1995), and a ceuE mu- a genetic bottleneck that increases the relative frequency of tant has been shown to be altered in chicken colonization certain strains in the infected individuals. Although larger scale 772 Genome Biol. Evol. 10(3):763–774 doi:10.1093/gbe/evy026 Advance Access publication February 14, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/763/4857209 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Convergent Amino Acid Signatures in C. jejuni GBE Frank C, et al. 2011. Epidemic proﬁle of Shiga-toxin-producing Escherichia studies are necessary to conﬁrm a potential adaptive role for coli O104:H4 outbreak in Germany. N Engl J Med. the human-associated variation, our analysis has identiﬁed a 365(19):1771–1780. group of human-pathogenic C. jejuni that do not exhibit typ- Friedman CR, et al.; Emerging Infections Program FoodNet Working ical source-sink epidemiology, potentially reﬂecting human Group. 2004. Risk factors for sporadic Campylobacter infection in tissue tropism or virulence. the United States: a case-control study in FoodNet sites. Clin Infect Dis. 38(s3):S285–S296. Geoghegan JL, Senior AM, Holmes EC. 2016. Pathogen population bottle- Supplementary Material necks and adaptive landscapes: overcoming the barriers to disease emergence. Proc Biol Sci. 283(1837):20160727. Supplementary data areavailableat Genome Biology and Gilbert MJ, et al. 2016. Comparative genomics of Campylobacter fetus Evolution online. from reptiles and mammals reveals divergent evolution in host- associated lineages. Genome Biol Evol. 8(6):2006–2019. Godschalk PC, et al. 2006. Identiﬁcation of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barre syn- drome by high-throughput AFLP analysis. BMC Microbiol. 6:32. Acknowledgments Guerry P. 2007. Campylobacter ﬂagella: not just for motility. Trends Microbiol. 15(10):456–461. G.M. was supported by an National Institute for Social Care Guyard-Nicodeme M, et al. 2015. Prevalence and characterization of andHealthResearchFellowship(HF-14-13). 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