Whole Genome Analysis and Biomarker Identification of Leptospira spp.

Abstract

Leptospirosis is the most common zoonotic disease worldwide. In this work, a pathogenomics approach was used to associates distinctive phenotypic features with genomic and transcriptomic characteristics in clinical strains of Leptospira interrogans (L. interrogans). In addition, ORFeome phage display was used to identify seroreactive peptides. Six L. interrogans isolated from Malaysia were studied and compared clinically and tested for virulence in the guinea pig model. SMRT sequencing was carried out on the PacBio RSII and complemented using an Illumina MiSeq. Two L. interrogans strains from both extremes of the virulence spectrum were subjected in triplicates to RNA-seq using Illumina HiSeq2500. All six L. interrogans strains were typed with multilocus sequence typing (MLST), core genome MLST and single nucleotide polymorphism (SNP) genotyping. Two ORFeome phage display libraries of Leptospira spp. genomes from Malaysian strains and WHO reference strains were constructed. Subsequently, five seroreactive synthetic peptides were constructed and validated for immunogenicity with 16 control and 16 patients sera by titration ELISA. Clinically, the patient infected with strain Langkawi presented with the most severe clinical course, while strain 1530d had mild leptospirosis. These findings correlated well with those of the animal study. Both cgMLST and SNP genotyping are comparable to ST clustering in MLST, however, SNP genotyping resulted in a higher coverage of the core genome i.e. 77.5% compared to 57.7% in cgMLST. There is remarkable genome plasticity in nearly all genomes, which is mainly driven by plasmids and insertion sequences elements. At logarithmic phase culture, genes associated with chemotaxis were expressed significantly more highly in strain Langkawi compared to strain 1530d, most likely explains the greater virulence of strain Langkawi compared to 1530d. In the phage display approach, two peptides (SIR16-D1 and SIR16-H1) were identified to have good accuracy for acute leptospirosis detection (area under the ROC curve: 0.86 and 0.78 respectively). Genome plasticity were gained through horizontal gene transfer and homologous recombination as an adaptive strategy in a variety of ecological niches. Differences in pathogenicity of all the strains appeared to be largely determined by genomic make-up. ORFeome phage display proved to be a powerful technique for the identification of leptospiral immunogenic peptides.