- Research article
- Open Access
Comparative genomics of Campylobacter concisus isolates reveals genetic diversity and provides insights into disease association
https://doi.org/10.1186/1471-2164-14-585
© Deshpande et al.; licensee BioMed Central Ltd. 2013
- Received: 22 May 2013
- Accepted: 17 August 2013
- Published: 28 August 2013
Abstract
Background
In spite of its association with gastroenteritis and inflammatory bowel diseases, the isolation of Campylobacter concisus from both diseased and healthy individuals has led to controversy regarding its role as an intestinal pathogen. One proposed reason for this is the presence of high genetic diversity among the genomes of C. concisus strains.
Results
In this study the genomes of six C. concisus strains were sequenced, assembled and annotated including two strains isolated from Crohn’s disease patients (UNSW2 and UNSW3), three from gastroenteritis patients (UNSW1, UNSWCS and ATCC 51562) and one from a healthy individual (ATCC 51561). The genomes of C. concisus BAA-1457 and UNSWCD, available from NCBI, were included in subsequent comparative genomic analyses. The Pan and Core genomes for the sequenced C. concisus strains consisted of 3254 and 1556 protein coding genes, respectively.
Conclusion
Genes were identified with specific conservation in C. concisus strains grouped by phenotypes such as invasiveness, adherence, motility and diseased states. Phylogenetic trees based on ribosomal RNA sequences and concatenated host-related pathways for the eight C. concisus strains were generated using the neighbor-joining method, of which the 16S rRNA gene and peptidoglycan biosynthesis grouped the C. concisus strains according to their pathogenic phenotypes. Furthermore, 25 non-synonymous amino acid changes with 14 affecting functional domains, were identified within proteins of conserved host-related pathways, which had possible associations with the pathogenic potential of C. concisus strains. Finally, the genomes of the eight C. concisus strains were compared to the nine available genomes of the well-established pathogen Campylobacter jejuni, which identified several important differences in the respiration pathways of these two species. Our findings indicate that C. concisus strains are genetically diverse, and suggest the genomes of this bacterium contain respiration pathways and modifications in the peptidoglycan layer that may play an important role in its virulence.
Keywords
- Campylobacter concisus
- Comparative genomics
- Pathogenesis
- Phylogeny
- Peptidoglycan
- Respiration
- Campylobacter jejuni
Background
Campylobacter concisus has received increasing attention over the last decade and has been described, in a number of publications, as an emergent pathogen of the human intestinal tract [1, 2]. C. concisus has been isolated from faecal samples of diarrhoeic patients, in some cases contributing to a significant percentage of Campylobacter species cultured [3–5]. Moreover, Hess et al. have reported a case study of gastroenteritis caused by C. concisus[6]. More recently, Nielsen et al. reported a high incidence of C. concisus, almost as high as that of C. jejuni/C. coli, in patients with gastroenteritis from a mixed urban and rural community in Denmark [7]. In a follow-up study [8], the authors found that 80% of C. concisus patients and only 32% of C. jejuni/C. coli patients had diarrhoea for >2 weeks. Significantly, 6 months following diagnosis, 12% of patients infected with C. concisus were diagnosed with microscopic colitis. In contrast, no patient previously diagnosed with C. jejuni/C. coli had microscopic colitis. This is of particular significance as previous studies from our group identified, for the first time, a possible association between C. concisus and newly diagnosed Crohn’s disease (CD) [9]. Based on a C. concisus-specific PCR, a significantly higher prevalence of C. concisus DNA was shown to be present in both biopsy and faecal samples of children with newly diagnosed CD than in controls [9, 10]. In a further study, we identified 31 C. concisus proteins to be immunoreactive in children with CD [11]. Interestingly, a study by Mukhopadhya et al. reported the prevalence of C. concisus DNA in biopsy specimens from adults with UC to be significantly increased (33.3%; 23/69) as compared with controls (10.8%; 7/65), suggesting that C. concisus may also be associated with UC [12].
Investigation of the pathogenic potential of C. concisus strains has shown that the bacterium can adhere to human intestinal epithelial cells, however, only some can invade into the cells through transcellular and paracellular mechanisms [13, 14]. The transcellular invasion of C. concisus strains isolated from chronic intestinal diseases was more than 500-fold higher than that of the other C. concisus strains [13, 14]. Moreover, host cells infected with C. concisus were found to produce high amounts of IL-12, however, only C. concisus strains capable of internalising into host cells induced a significantly increased quantity of IFN-γ with respect to controls [13]. These findings, coupled with the regulation of the proteasome, ubiquitination pathways, the Akt signalling pathway and NF-κB inhibitors, pointed towards the activation of the NF-κB pathway by invasive C. concisus strains [13]. Further investigation of the difference in invasive potential between strains identified a plasmid containing several virulence determinants, including exotoxin 9 [13, 15], which was present in the highly invasive strains but absent in the other strains.
Although the above studies support the role of C. concisus as an intestinal pathogen, the isolation of C. concisus from healthy individuals, and the failure of some studies to show a significant difference in the prevalence of C. concisus in subjects with diarrhoea and healthy controls [1], has raised contention as to the role of C. concisus in intestinal disease. While these latter findings would to some degree argue against the role of C. concisus in gastroenteritis, the fact that great sequence diversity exists within C. concisus strains [3, 16] raises the possibility that differences may exist in their pathogenic potential. To further examine the importance of C. concisus heterogeneity with respect to disease potential, we sequenced the genomes of six new C. concisus strains and performed comparative analyses of these and two known strains, which allowed us to compare strains isolated from three CD, one chronic gastroenteritis, three acute gastroenteritis patients as well as one from a healthy control.
Results and discussion
Draft genome assemblies and plasmids of six Campylobacter concisus strains
Synteny based similarities and differences across Campylobacter concisus strains. A: Pairwise comparisons between the reference BAA-1457 genome from NCBI and the C. concisus strains sequenced by our group revealed similarities and differences in syntenical placement of genomic blocks. The Mauve alignment tool was used to show paired synteny alignments, which indicate some genome shuffling of C. concisus strains. Inversions are indicated by syntenic blocks placed bellow the main axis. B: Circular layout of seven assembled genomes against the BAA-1457 reference. The CGView tool used with BAA-1457 as the reference (outer 2 rings) highlights that the reference strain contains regions with high diversity (marked by prominent gaps) when compared to the other seven C. concisus strains.
Campylobacter concisus strains used in this study
Strain | UNSW2 | UNSW3 | UNSWCD | UNSW1 | UNSWCS | ATCC 51562 | BAA-1457 | ATCC 51561 |
---|---|---|---|---|---|---|---|---|
Disease | Crohn’s disease | Crohn’s disease | Crohn’s disease | Gastroenteritis | Gastroenteritis | Gastroenteritis | Gastroenteritis | Healthy |
Disease type | Chronic | Chronic | Chronic | Chronic | Acute | Acute | Acute | - |
Invasion | High | High | High | High | Low | Low | No | No |
Adherence | High | High | High | High | High | Low | High | Low |
Motility | High | High | High | High | High | Low | Low | Low |
Reads × 2 | 17,704,213 | 18,593,137 | N/A | 57,904,810 | 19,805,749 | 17,541,124 | N/A | 61,684,406 |
Contigs | 114 | 62 | 96 | 77 | 207 | 28 | 1 | 73 |
N50 | 89,312 | 92,608 | 64,047 | 117,975 | 68,143 | 361,423 | 2,052,007 | 111,029 |
Scaffolds | 25 | 25 | - | 22 | 53 | 9 | 1 | 23 |
Scaffold N50 | 184037 | 229,705 | - | 195,982 | 124,567 | 405,448 | 2,052,007 | 176120 |
Genome size (Mbp) | 2.01 | 1.91 | 1.81 | 1.94 | 2.11 | 1.84 | 2.05 | 1.99 |
NCBI accession | ANNJ00000000 | ANNE00000000 | AENQ00000000 | ANNF00000000 | ANNG00000000 | ANNI00000000 | CP000792.1 | ANNH00000000 |
CP000794.1 | ||||||||
CP000793.1 |
Conservation of genes from UNSWCD and BAA-1457 plasmids across the Campylobacter concisus strains
UNSWCD plasmid | BAA-1457 plasmids | |||||||
---|---|---|---|---|---|---|---|---|
Total genes=26 | pCCON31 Total genes=33 | pCCON16 Total genes=23 | ||||||
C. concisus strain | Orthologs | No-hits | C. concisus strain | Orthologs | No-hits | C. concisus strain | Orthologs | No-hits |
UNSW2 | 9 | 17 | UNSW2 | 8 | 25 | UNSW2 | 6 | 17 |
UNSW3 | 24 | 2 | UNSW3 | 1 | 32 | UNSW3 | 7 | 16 |
UNSW1 | 22 | 4 | UNSW1 | 4 | 29 | UNSW1 | 6 | 17 |
UNSWCS | 5 | 21 | UNSWCS | 24 | 9 | UNSWCS | 4 | 19 |
ATCC 51562 | 7 | 19 | ATCC 51562 | 2 | 31 | ATCC 51562 | 3 | 20 |
ATCC 51561 | 16 | 10 | ATCC 51561 | 4 | 29 | ATCC 51561 | 17 | 6 |
BAA-1457 | 2 | 24 | UNSWCD | 0 | 33 | UNSWCD | 7 | 16 |
The pan and core genomes of Campylobacter concisus
The pan genome is the supra-genome defining the entire complement of genes within a species, while the core genome is defined as a set of genes found in all sequenced genomes of a species. In addition to the genes from the two strains available in NCBI, the gene sets defined by the RAST server for the six C. concisus strains and refined by manual curation were used for defining the pan and core genomes for C. concisus. The pan genome was found to consist of 3254 genes with many genes specific for only a sub-set of the strains but dispensable in the other strains. The C. concisus core genome consisted of 1556 genes, with a fraction of the C. concisus core 70/1556 (4.5%) genes encoding hypothetical proteins. Fifty-three KEGG pathways were found to have similar conservation patterns across the eight C. concisus strains (Additional file 1: Table S2). Interestingly, none of the 1556 genes from the core were found to be specific to C. concisus when analysed against all other bacterial genomes in NCBI.
Enrichment of Gene Ontologies for (A) CORE and (B) PAN genes across Campylobacter concisus genomes.
Interestingly, in the pan genome, clearance of foreign intracellular DNA and other DNA-related processes such as DNA integration and DNA restriction-modification were enriched (Figure 2B), suggesting that differences exist in the efficiency of these C. concisus strains to survive phage attacks. This is supported by the enrichment of ‘defence response’ within the pan genome, and would indicate that this process is not conserved among all C. concisus strains.
Phylogenetic analysis based on ribosomal RNA genes
Phylogenetic analysis of (A) 16S and (B) 23S ribosomal RNA genes for the eight Campylobacter concisus strains.
Inter-strain differences and phenotype-based analyses
Gene and metabolic differences within the strains
In order to determine the level of inter-strain diversity within C. concisus, a comparison of the eight strains was performed to identify genes specific to each strain and to strain pairs (Additional file 1: Table S3 and Table S4). Significant differences in the number of specific genes per strain were observed, and many of these genes were found to be syntenic (Additional file 1: Table S3 and Table S4), thus, indicating the possible acquisition of these strain-specific genes by horizontal gene transfer. Interestingly, the relative percentage of genes unique to each strain was found to group the isolates into three categories, low (<1%), medium (1.2 to 2.0%) and high (>2.5%), and this classification corresponded to non-pathogenic, chronic and acute strains, respectively (Additional file 1: Table S5). These findings suggest an optimal level of acquired genes for the pathogenic efficiency of C. concisus, whereby the strain from the healthy subject had a lower number of strain-specific genes relative to its genome size while the strains from acute disease had a higher number when compared to the strains with high pathogenic potential (chronic disease).
Several differences were identified within the metabolic machinery of these eight strains (Additional file 1: Table S6). For example, four of the eight strains contained two genes involved in tetrathionate respiration, a form of respiration which has been shown to give Salmonella strains a competitive advantage in the intestine [20]. Interestingly, the genome of ATCC 51561, isolated from the healthy control, was the only strain to contain an aerotaxis receptor which may increase its ability to monitor oxygen concentrations. Moreover, the genomes of both ATCC 51561 and ATCC 51562 contained the phosphate transport system pstABCS, which has been related to the ability of Shigella strains to translocate from cell to cell [21]. This system may have been acquired by these strains from their interaction with Shigella species within the oral cavity or intestine. The genomes of several strains contained elements of type IV and VI secretion systems, however, these systems appear to be incomplete which suggests that these systems may not be of importance to C. concisus.
In addition, a single gene encoding a DUF2920 superfamily bacterial protein with unknown function (also annotated as a motility accessory factor and a carbonic anhydrase in other Campylobacter species) was found to be well conserved across all strains isolated from patients with intestinal disease, but was absent in the strain from the healthy subject, making it an interesting target for future examination.
Genes associated with adherence potential
Searches for genes present in the strains with high adherence and absent in those with low adherence identified the sodium-hydrogen antiporter NhaC. Sodium-hydrogen antiporters have been linked to regulation of sodium concentrations and pH balance within cells by using H+[22]. These proteins convert the proton motive force to a sodium motive force for efflux of Na+ ions. It is well-known that the flagella of Campylobacter species, including C. concisus[13, 14], play a major role in the adherence to host cells, and bacterial flagella are driven by a proton motive force [23]. Thus, it is conceivable that the absence of NhaC from some C. concisus strains may influence the proton motive force, and thus, influence the strength of flagellar adherence to host microvilli. While this may suggest the involvement of NhaC in the more highly adherent phenotype, other antiporters such as NhaA were identified in the strains with low adherence. Moreover, the fact that strain BAA-1457 has high adherence and low motility would indicate that other factors are involved in this phenotype. Further work is required to establish an association between sodium-hydrogen antiporters and bacterial adherence to host cells.
Genes associated with invasive potential
Syntenic placement of specific conserved plasmid genes in the genomes of invasive strains. All genes were placed on a single scaffold in each species.
The function of the proteins encoded by these plasmid genes has the potential to provide insights into their conservation patterns. For example, the presence of the MobA-like protein and the StbD/E toxin antitoxin system in the other strains most likely relates to their global role in ensuring that daughter cells inherit DNA properly [24]. In contrast, further BLAST searches revealed the exotoxin 9 to be the possible helicase DnaI, and its conserved synteny with the restriction endonuclease, recombinase and DNA methyltransferase suggests a combined function for these proteins within the organism, more likely associated with the ability to survive within host cells than its entry into host cells. Of interest, the sequence of the exotoxin 9 had very high homology (88-92%) to a replicative DNA helicase from Lachnoanaerobaculum saburreum and related species. These organisms have been isolated from the oral cavity and intestinal biopsies of humans, and produce both H2S and NH3[25], which are properties shared with C. concisus (discussed below).
Another single gene on the chromosomal DNA annotated as a hypothetical protein was also found to be specific to the highly invasive C. concisus strains. Following BLAST searches, this protein appears to be a subunit of the molybdopterin synthase enzyme, which synthesises molybdenum cofactor (MoCo). This enzyme is important for respiratory nitrate reductase activity and sulfur metabolism, suggesting that the highly invasive strains may have a competitive advantage over other strains through other forms of respiration.
Phylogeny and SNP analysis of host-related pathways
Analyses of the peptidoglycan biosynthesis pathway within the eight C. concisus strains. A: Phylogenetic analysis of the concatenated sequences of the peptidoglycan biosynthesis pathway within the eight C. concisus strains. B: Genes in the peptidoglycan biosynthesis pathway with SNPs are highlighted in red.
Non-synonymous SNPs across C. concisus strains displaying categorisation based on known phenotypes
Gene ID | aa position | bp position | UNSW2 | UNSW3 | UNSW1 | UNSWCD | UNSWCS | ATCC51562 | ATCC51561 | BAA1457 | Phenotype | Domains | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Name | Type | start | end | ||||||||||||
Bile efflux (Cme) pathway | |||||||||||||||
CmeB | 848 | 2544 | GAC:D | GAC:D | GAT:D | GAT:D | GAC:D | GAG:E | GAG:E | GAT:D | Adherence | ACR_tran | PFAM | 2 | 1024 |
CmeB | 519 | 1557 | AGG:R | AGG:R | AGG:R | AGG:R | AGG:R | AAG:K | AAG:K | AAG:K | Motility | ACR_tran | PFAM | 2 | 1024 |
CmeD | 227 | 681 | AAG:K | AAG:K | AAG:K | AAG:K | AGG:R | AGA:R | AGG:R | AGG:R | Invasive | NA | |||
CmeD | 229 | 687 | AGT:S | AGT:S | AGT:S | AGT:S | AAT:N | AAT:N | AAT:N | AAT:N | Invasive | NA | |||
CmeD | 233 | 699 | TCT:S | TCT:S | TCT:S | TCT:S | TCT:S | TTT:F | TTT:F | TTT:F | Motility | NA | |||
CmeD | 234 | 702 | TTA:L | TTA:L | TTA:L | TTA:L | TTA:L | ATA:I | ATA:I | ATA:I | Motility | NA | |||
CmeD | 163 | 489 | CCA:P | CCA:P | CCA:P | CCA:P | CCA:P | GCG:A | GCA:A | CCA:P | Adherence | OEP | PFAM | 32 | 201 |
CmeD | 362 | 1086 | AGA:R | AGA:R | AGA:R | AGA:R | AGA:R | AAA:K | AAA:K | AGA:R | Adherence | NA | |||
00540|Lipopolysaccharide biosynthesis | |||||||||||||||
CCC13826_0577 | 161 | 483 | AAC:N | AAC:N | AAC:N | AAC:N | AAC:N | AGC:S | AGC:S | AAC:N | Adherence | NA | |||
CCC13826_0577 | 161 | 483 | AAC:N | AAC:N | AAC:N | AAC:N | AAC:N | AGC:S | AGC:S | AAC:N | Adherence | PfkB | PFAM | 6 | 298 |
CCC13826_0470 | 104 | 312 | ATT:I | ATT:I | ATT:I | ATT:I | ATT:I | ACT:T | ACT:T | ATT:I | Adherence | Glyco_transf_9 | PFAM | 75 | 290 |
00550|Peptidoglycan biosynthesis | |||||||||||||||
CCC13826_1265 | 202 | 606 | AGA:R | AGA:R | AGA:R | AGA:R | AGA:R | AAG:K | AAA:K | AAA:K | Motility | EPSP_synthase | PFAM | 8 | 409 |
CCC13826_1740 | 184 | 552 | GTA:V | GTA:V | GTA:V | GTA:V | GTA:V | ATG:M | ATG:M | GTA:V | Adherence | NA | |||
CCC13826_1740 | 184 | 552 | GTA:V | GTA:V | GTA:V | GTA:V | GTA:V | ATG:M | ATG:M | GTA:V | Adherence | Mur_ligase_M | PFAM | 64 | 257 |
CCC13826_1242 | 26 | 78 | GGC:G | GGC:G | GGC:G | GGC:G | GGC:G | GAT:D | GAC:D | GGT:G | Adherence | Glyco_transf_28 | PFAM | 2 | 139 |
CCC13826_1242 | 26 | 78 | GGC:G | GGC:G | GGC:G | GGC:G | GGC:G | GAT:D | GAC:D | GGT:G | Adherence | NA | |||
CCC13826_1254 | 361 | 1083 | CTG:L | CTA:L | CTA:L | CTA:L | CTG:L | ATA:I | ATA:I | ATA:I | Motility | NA | |||
CCC13826_1254 | 361 | 1083 | CTG:L | CTA:L | CTA:L | CTA:L | CTG:L | ATA:I | ATA:I | ATA:I | Motility | Transpeptidase | PFAM | 295 | 532 |
CCC13826_1254 | 636 | 1908 | GTC:V | GTT:V | GTT:V | GTC:V | GTT:V | ATT:I | ATT:I | GTT:V | Adherence | NA | |||
CCC13826_1254 | 636 | 1908 | GTC:V | GTT:V | GTT:V | GTC:V | GTT:V | ATT:I | ATT:I | GTT:V | Adherence | NA | |||
Sulfur relay system | |||||||||||||||
CCC13826_1163 | 132 | 396 | GAT:D | GAT:D | GAT:D | GAT:D | GAC:D | GAA:E | GAG:E | GAC:D | Adherence | Aminotran_5 | PFAM | 3 | 366 |
N-glycosylation pathway | |||||||||||||||
PglB_699 | 20 | 60 | ATC:I | ATC:I | ATC:I | ATC:I | ATC:I | CTT:L | CTT:L | CTT:L | Motility | STT3 | PFAM | 10 | 503 |
PglI | 19 | 57 | AAA:K | AAA:K | AAA:K | AAA:K | AAA:K | CAA:Q | CAA:Q | AAA:K | Adherence | Glycos_transf_2 | PFAM | 7 | 123 |
PglI | 36 | 108 | GTT:V | GTT:V | GTT:V | GTT:V | GTT:V | ATA:I | ATA:I | ATA:I | Motility | Glycos_transf_2 | PFAM | 7 | 123 |
PglJ | 312 | 936 | GAT:D | GAT:D | GAT:D | GAT:D | GAG:E | GAG:E | GAG:E | GAG:E | Invasive | Glycos_transf_1 | PFAM | 195 | 351 |
Analysis of N-linked glycans has revealed that C. concisus, along with Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter lanienae, Campylobacter sputorum, display differing arrangements of Hex and HexNAc sugars to other Campylobacter species, and that C. concisus contains a residue of 217 Da not found in all other Campylobacter species tested [29]. Given this, our phylogeny and SNP analysis was extended to the 13 genes characterised within the N-glycosylation pathway and present in the C. concisus strains (pglABCDEFGHIJK, wbpO and galE). While the phylogenetic analysis did not group the strains according to a specific phenotype (Additional file 1: Figure S1), a further 4 non-synonymous SNPs were identified, all of which were present within known functional domains (Table 3). As N-linked glycosylation of surface proteins appears to enhance C. jejuni fitness by protecting bacterial proteins from cleavage by gut proteases [30], differences within this pathway may influence the role C. concisus strains play within the gut.
Comparative genomic analyses of Campylobacter concisus and Campylobacter jejuni
Comparative genomic analyses of Campylobacter concisus and Campylobacter jejuni . A: The core genomes of C. concisus and C. jejuni were identified. B: Enrichment of genes specific to C. concisus core genome. C: Enrichment of genes specific to C. jejuni core genome. Enrichment tests of all Gene Ontology terms were analysed using Fisher’s Exact Test with Multiple Testing Correction of FDR (Benjamini and Hochberg).
This analysis identified a possible competitive advantage of C. jejuni in terms of iron acquisition, as the genomes of this bacterium contained both the ferritin FtnA involved in iron storage, and the Fe3+ transport system FbpABC, which were all absent in the C. concisus strains. Specificity towards iron was also observed by the presence of the Fe/Mn superoxide dismutase in C. jejuni, whereas both the Cu/Zn and Fe/Mn superoxide dismutases were present in C. concisus. One significant difference was identified within the peptidoglycan biosynthesis pathway, whereby only C. concisus was found to contain the flagellar P-ring protein PbpC. This finding is of particular interest given our previous observation that two SNPs within pbpC were associated with motility and adherence (Table 3). Moreover, the fact that the peptidoglycan biosynthesis pathway clusters the highly invasive C. concisus strains together when compared to the other C. concisus strains (Figure 5), would suggest that differences in this pathway may significantly influence the pathogenic potential of C. concisus.
The C4-Dicarboxyrate transport system DctPQM was present within the genomes of all C. concisus strains and absent in the C. jejuni genomes (Additional file 1: Table S6). This system has been reported to be involved in sensing and differentiating between aerobic and anaerobic respiration [31], which may relate to the fact that C. concisus can grow anaerobically whereas C. jejuni cannot. This finding is in line with the identification of major differences within the respiration pathways of these two organisms, specifically those related to nitrogen and sulfur respiration. For example, genomes of the C. concisus strains all contained the nitric oxide reductase NorB and the nitrous oxide reductase NosZ, which were absent in the genomes of the C. jejuni strains. Significantly, C. jejuni strains contained the two associated proteins NrfA and NrfH involved in nitrite respiration, whereby NrfH functions to anchor NrfA into the membrane [32]. In contrast, C. concisus contains NrfC and NrfD which have been linked to both nitrite and sulfite respiration [33, 34]. Moreover, while both organisms contain NapG and NapH, C. concisus also contains a NapC/NirT cytochrome c family protein, which is absent in C. jejuni. In addition to these findings, we found that in C. jejuni 2-oxoglutarate is converted to L-glutamate through the glutamate synthase GltD, which is then converted to L-1-Pyrroline-5-carboxylate through the activity of the proline dehydrogenase PutA. In contrast, in C. concisus the glutamate dehydrogenase GdhA produces ammonia through its conversion of L-glutamate to 2-oxoglutarate. The hydroxylamine reductase Hcp (present in C. concisus; absent in C. jejuni) can employ ammonia, water and an acceptor to produce hydroxylamine, which can then be converted to nitrite through the activity of hydroxylamine oxidase Hao (present in C. concisus, absent in C. jejuni). This nitrite may feed into the nitrite respiration pathway, thereby giving C. concisus a means to respire through the amino acid L-glutamate.
In addition to nitrogen-associated respiration, the genomes of the C. concisus strains had enzymes within the sulfur metabolism pathway that were absent in C. jejuni, most importantly a sulfite reductase which has the ability to convert sulfite to hydrogen sulfide (Additional file 1: Table S6). This is of interest as high levels of H2S have been detected in patients with UC [35], and C. concisus has been associated with UC [12].
These above differences in the respiration pathways are supported by the finding that molybdenum ion binding is enriched in the C. concisus core genome when compared to the C. jejuni core genome (Figure 6B), as molybdenum enzymes catalyse the oxidoreduction of certain small molecules, as part of the regulation of nitrogen, sulfur and carbon cycles [36]. Indeed, the importance of these enzymes in the overall physiology of C. concisus is highlighted by the fact that the highly invasive strains shared a molybdopterin synthase that was absent in the other strains, and molybdopterin and molybdenum combine to form the molybdenum cofactor.
Conclusions
This study has confirmed the high genetic diversity observed among C. concisus isolates, but more importantly has identified several factors pertaining to the pathogenic potential of the emerging pathogen C. concisus. In particular, the peptidoglycan layer of this bacterium and conserved elements within the highly invasive strains such as the exotoxin 9 may play an important role in its virulence. Finally, novel differences within the respiration pathways of the well-known pathogen C. jejuni and the emerging pathogen C. concisus were identified, which may provide insights into their growth within the environment and the host.
Methods
Bacterial strains and DNA preparation
The six C. concisus strains sequenced in this study were isolated from patients with CD (UNSW2 and UNSW3), chronic gastroenteritis (UNSW1), acute gastroenteritis (UNSWCS and ATCC 51562) and a healthy subject (ATCC 51561). ATCC 51562 and ATCC 51561 were purchased from the American Type Culture Collection. UNSW1, UNSW2, UNSW3 and UNSWCS were isolated as part of previous study [13] which was approved by the Research Ethics Committees of the University of New South Wales and the South East Sydney Area Health Service-Eastern Section, Sydney (Ethics No.: 06/164). Written consent was obtained from all subjects, or their guardians, participating in that study. Information on the strains is listed in Table 1. C. concisus strains were grown on Horse Blood Agar (HBA) plates [Blood Agar Base No. 2 supplemented with 6% defibrinated horse blood (Oxoid)], and incubated at 37°C under microaerobic conditions with H2 (generated using Campylobacter Gas Generating Kits (Cat. #. BR0056A, Oxoid)) for 48 h. Bacterial DNA was extracted using the Puregene Core Kit (Qiagen) according to the manufacturer’s instructions.
Sequencing, assembly and annotation
Genome sequencing of the six C. concisus strains was performed using a single multiplexed lane in a Hi-Seq Illumina sequencer. The libraries were prepared using the TruSeq DNA sample preparation kit (Illumina) following the manufacturer’s instructions. Briefly, 1 μg of DNA was sheared using the Covaris, followed by end-repair, A-tailing and ligation of adapters. Size selection was performed with the Pippin prep (Sage Science) selecting for 300–400 bp inserts, which were amplified using 10 cycles of PCR. The libraries were multiplexed in two lanes of the HiSeq 2000 (Illumina) and sequenced as 100 bp paired-end reads. Customised automated python scripts were developed to run two assembly programs, Velvet v1.2.08 and SOAPdenovo v1.05, and the overlap parameter k-mer varied between 51–91 to get an optimised assembly. The scaffolding tool SSPACE-BASIC-2.0_linux-x86_64 [37] was employed by providing the average fragment size of 320 bp with a tolerance of ± 50 bp, and the forward-reverse orientation of the paired-end reads to generate compact scaffolded assemblies for the individual genomes. The Rapid Annotation using Subsystem Technology (RAST) [38], a service for annotating bacterial and archaeal genomes, was used for gene definition and annotation for the individual assemblies. Genome scale alignments were performed using the Mauve alignment tool [17] and CGView [39]. Genomic data for already sequenced C. concisus strains BAA-1457 and UNSWCD were obtained from the GenBank repository (Table 1). The UNSWCD strain has previously been sequenced by our group [15].
Investigation of plasmids in the assembled genomes
BLAST searches were performed using the genes from the previously identified plasmids within C. concisus strains UNSWCD and BAA-1457 to check for conservation in the sequenced C. concisus genomes. Detailed annotations and mapping of the gene synteny of the conserved genes on individual C. concisus genomes was done using custom scripts. Moreover, a set of all possible genes from plasmids identified in epsilon-Proteobacteria was downloaded from NCBI. The presence of these genes in the six C. concisus strains was investigated using BLAST searches.
Defining the core and pan genomes
The core genome for the eight C. concisus strains was defined using an iterative BLAST method. The UNSWCD proteome from NCBI was used as the starting reference genome. These sets of proteins were put through blast (tblastn, 40% homology and 40% length hit) in a sequential order against the rest of the C. concisus genomes. Customised scripts parsed out the intersecting genomes during each of the iterations, and thus, the genes found to be conserved in all the C. concisus genomes formed the core genome. The pan genome (that is the set of genes found in at least one C. concisus strain) was defined using a similar iterative BLAST strategy. All the genes not in the core were combined and a non-redundant set of genes was defined from the above group by performing an all versus all BLAST search. This gene set was then added to the core gene set to get the final pan genome for C. concisus.
Enrichment analysis using blast2GO
Functional enrichment analyses of the core and pan genomes were performed using the annotation and analysis tool Blast2GO [40]. Following annotation, the statistical analysis package in Blast2GO which uses Fisher’s exact test with multiple testing correction of false discovery rate [41] was applied and enrichment graphs were generated. A similar method was employed for the analysis of gene ontology enrichment across C. concisus and C. jejuni.
Multi-species, multiple pathway comparisons
A multi-species, multi-pathway comparison utility was developed for filtering KEGG pathways displaying similar as well as varying conservation patterns across sequences bacterial species of interest. The aim was to develop an unbiased method to analyse all KEGG pathways across a given set of species without having any prior knowledge about their possible biological importance in the group of species under study. Similar to KAAS [42] (which gives functional annotation of genes by BLAST comparisons against the manually curated KEGG GENES database), the genes defined for individual C. concisus strains were subjected to BLAST alignments against the KEGG GENES database to obtain KO identifiers. The KO identifiers were then mapped onto KEGG pathways. Customised scripts were written to allow comparison for individual KEGG pathways at gene component levels across selected species, to mark their conservation patterns. Pathways with complete or partial conservation for the given set of C. concisus and C. jejuni strains included in this analysis were separated out for further analysis.
Phylogeny and SNP analysis
Phylogenetic trees were generated using the MEGA 5.05 tool [43] by applying the neighbour-joining method with 1000 bootstrap replications. For phylogenies based on pathways, pairwise orthologs for each of the gene components of individual pathways were filtered out in all the C. concisus strains, and genes were then concatenated and aligned to the remaining strains using ClustalW [44].
For the SNP analysis, orthologs of individual gene components of selected KEGG pathways found conserved across all C. concisus genomes were aligned using local ClustalW alignment software. Non-synonymous SNP changes (resulting in amino acid changes) which displayed grouping by known phenotype categorisations were filtered out using customised python scripts. Pfam domain analysis of the proteins containing these SNPs was performed using Interproscan [45] integrated in Blast2GO.
Availability of supporting data
Draft genomes of the Campylobacter concisus strains sequenced in this study have been deposited in GenBank available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), and accession numbers of these genomes are provided in Table 1. All other supporting data are included as additional files.
Notes
Declarations
Acknowledgements
NOK is supported by an Early Career fellowship from the National Health and Medical Research, Australia. NOK and HMM acknowledge support from the University of New South Wales. NPD and MRW acknowledge support from the Australian federal government EIF Super Science Scheme, the New South Wales State Government Science Leveraging Fund (SLF) and the University of New South Wales.
Authors’ Affiliations
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