Genomic investigation into strain heterogeneity and pathogenic potential of the emerging gastrointestinal pathogen Campylobacter ureolyticus

Abstract

The recent detection and isolation of C. ureolyticus from patients with diarrhoeal illness and inflammatory bowel diseases warrants further investigation into its role as an emerging pathogen of the human gastrointestinal tract. Regarding the pathogenic mechanisms employed by this species we provide the first whole genome analysis of two C. ureolyticus isolates including the type strain. Comparative analysis, subtractive hybridisation and gene ontology searches against other Campylobacter species identifies the high degree of heterogenicity between C. ureolyticus isolates, in addition to the identification of 106 putative virulence associated factors, 52 of which are predicted to be secreted. Such factors encompass each of the known virulence tactics of pathogenic Campylobacter spp. including adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (ciaB and 16 virB-virD4 genes) and toxin production (S-layer RTX and ZOT). Herein, we provide the first virulence catalogue for C. ureolyticus, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology.

Figure 1. DSM 20703 V's ACS-301-V-Sch3b Synteny Map.

Syntenic affinities (conserved blocks) across the whole genome sequences of ACS-301-V-Sch3b and DSM 20703 were determined using BLAST+ with DSM 20703 used to build the database and using BLASTN to find synteny within the ACS-301-V-Sch3b genome. The ideogram was built using Circoletto where ribbons represent the local alignments and the colours blue, green, orange and red, respectively representing the 25% blocks up to the maximum score of 100% and histogram on top of the ideograms, counting how many times each band has hit the specific part of the sequence.

Figure 2. SDS-PAGE of C. ureolyticus Soluble Proteins.

(A) Protein profiles of 7 Campylobacter ureolyticus strains. Phoretix 1D pro was employed to cluster the lanes based on banding patterns. The Dendrogram is an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) Dice coefficient distance tree. (B) Original SDS-PAGE gel, with variable regions highlighted. Lane 1: Page Ruler Plus Ladder, lane 2: CIT001, lane 3: CIT002, lane 4: CIT004, lane 5: CIT 005, lane 6: CIT007, lane 7: CIT009, lane 8: DSM 20703.

Figure 3. Bioinformatics based predictions of the C. ureolyticus secretome.

(A) 288 proteins were predicted to be secreted by C. ureolyticus DSMZ 20703 using the SignalP 4.1 server for classically secreted proteins and the SecretomeP 2.0 server for non-classically secreted proteins. Of the 288 proteins, 187 proteins were predicted to be secreted by the SecretomeP 2.0 sever alone, 57 proteins by the SignalP 4.1 alone and 44 proteins were identified by both servers. Additionally, 117 proteins of 28 were hypothetical, the remaining 171 proteins were associated with a diverse range of functions relating to membrane transport, protein metabolism, respiration, stress response and at least 25 proteins with putative virulence roles. (B) SignalP4.1 and SecretomeP2.0 servers predicted 269 proteins are secreted by C. ureolyticus ACS-301-V-Sch3b. 165 proteins were predicted to be secreted by the SecretomeP 2.0 server alone, a further 65 proteins were identified by the Signal 4.1 server and 39 proteins were identified by both servers. Of the 269 secreted proteins, a total of 94 were hypothetical proteins, the remaining 175 proteins were predicted to have a wide range of functions including DNA metabolism, cell wall synthesis, sulphur and nitrogen metabolism and at least 28 proteins with putative virulence roles.

Figure 4. Evolutionary relationships of taxa Zona Occluden Toxin (ZOT).

The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. DSMZ 1 and DSMZ 2 correspond to the ZOT paralogs in C. ureolyticus DSM20703. ACS-301-V-Sch-3b:C. ureolyticus ACS-301-V-Sch-3b.