Challenging bovine foot skin fibroblasts with digital dermatitis treponemes: effects on global gene expression

Purpose, aims and objectives: Bovine digital dermatitis (BDD) is a painful and ulcerative, inflammatory, infectious disease of digital skin in dairy and beef cattle and is one of the most economically important causes of severe cattle lameness worldwide. A polytreponemal aetiology has been consistently implicated in BDD pathogenesis with three key phylogroups implicated globally: Treponema medium, Treponema phagedenis and Treponema pedis. This study aimed to investigate the hypothesis that the three Treponema phylogroups implement distict mechanisms of pathogenesis within cells of the bovine foot skin tissue during infection. Using a next generation RNA sequencing (RNA-Seq) approach, the dysregulation of global mRNA expression was investigated in primary bovine foot skin fibroblasts following challenge with representative strains of the three predominant BDD Treponema phylogroups (T19, T320A and T3552B respectively). Transcriptome profiles were compared to that of a commensal treponeme, Treponema ruminis strain Ru1, in attempts to identify pathogenic signatures. Results and conclusions: Here, for the first time, we implicate fibroblasts as a likely source of interleukin-8 dysregulation in BDD lesions. Despite treponemes reportedly having atypical LPS, a strong pro-inflammatory response was elicited by the BDD treponemes in a similar manner to the Gram-negative LPS control, which appeared to be mediated through interleukin-17 signalling. Along with interleukin-17, several interesting gene targets were identified which may help to inform and drive future vaccine development and therapeutic approaches; particularly those of RGS16, GRO1, MAFF and ZC3H12A, which were only stimulated by BDD treponemes. Interestingly, the three BDD treponemes were found to elicit very distinct mechanisms of pathogenesis in fibroblasts. T. phagedenis and T. pedis spirochaetes were found to upregulate several genes associated with apoptosis. Both T. medium and T. pedis spirochaetes, but notably not T. phagedenis, stimulated genes associated with actin rearrangement and a loss of cell adhesion, which may promote their persistence and deeper invasion within host tissues. The upregulation of antimicrobial peptide precursor, DEFB123, by T. phagedenis spirochaetes may present a microbial ecological advantage to all treponemes within BDD infected tissue, explaining their dominance within lesions. Interestingly, T. medium phylogroup was the least stimulatory and appeared to induce immunosuppression via the dysregulation of genes such as TSC22D3. Together with findings that the commensal, T. ruminis, significantly dysregulated over three times the number of host genes compared to each BDD treponeme; this suggests that, alike the syphilis pathogen (Treponema pallidum), BDD treponemes have evolved to become “stealth pathogens” and avoid triggering a substantial host immune/inflammatory response to promote persistence and facilitate invasion of host tissues. Overall, this study has highlighted the complexities of BDD pathogenesis; three key Treponema phylogroups appear to elicit very distinct mechanisms of pathogenesis in bovine foot skin cells. Several interesting gene targets have been identified from this study which may help to inform and drive future vaccine development and therapeutic approaches. Overall design: RNA sequencing analysis was performed on RNA extracted from primary bovine foot skin fibroblast cell cultures following challenge for six hours with either control media (three experimental replicates), lipopolysaccharide from Salmonella enterica serotype Typhimurium (as an inflammatory stimulant control) (three experimental replicates), Treponema medium phylogroup strain T19 (three experimental replicates), Treponema phagedenis phylogroup strain T320A (three experimental replicates plus an extra technical replicate), Treponema pedis strain T3552B (three experimental replicates) or Treponema ruminis strain Ru1 (three experimental replicates plus an extra technical replicate). Following RNA extraction, these 20 samples underwent rRNA depletion and cDNA libraries were prepared using an Illumina NEBNext Ultra Directional RNA Library Prep Kit. Libraries undewent paired-end clustering and were subsequently sequenced on an Illumina HiSeq 4000 using version 1 chemistry, generating 2x 150 bp paired end reads. On average, we generated approximately 66 million sequenced reads per sample (range 44-102 million). FASTQ sequence files were trimmed to exclude adaptor sequences and poor quality reads using Cutadapt V1.2.1 and Sickle V1.200 respectively. Reference-based assembly was implemented using the Galaxy platform's main public server. Trimmed reads were mapped to the Bos taurus genome (UMD 3.1.1/bosTau8; Genbank assembly accession number: GCA_000003055.5) using TopHat2, with an overall average mapping rate of 86%. Mapped reads were assembled into transcripts using Cufflinks and a master transcriptome generated by Cuffmerge, using the Bos taurus genome as a reference. Differential gene expression profiles of fibroblasts (mean FPKM gene expression) were generated for each challenge group compared to the media control by implementing CuffDiff; using the master transcriptome assembly and mapped read (BAM) files for all corresponding replicates of the challenge and control group comparison. Differentially expressed genes with an adjusted p (q or FDR) value = 0.05 were considered statistically significant, with a further log2 fold change cut-off (= 1 and = -1) used to define biological significance; these criteria were used as a cut-off for SDE in all downstream analyses. Ingenuity pathway analysis was used to identify significantly enriched canonical pathways, diseases and biological functions. Technical replicates were obtained from cell monolayers seeded within different flasks from the same experimental culture.