Genomanalyse und Identifikation von Biomarkern von Leptospira spp.

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.Leptospirose ist die weltweit häufigste Zoonose-Erkrankung. In dieser Arbeit wurde ein pathogenomischer Ansatz verwendet, um phänotypische Merkmale mit Genom und Transkriptom Daten von klinischen Stämmen von Leptospira interrogans (L. interrogans) zu verknüpfen. Zusätzlich wurde ORFeome-Phagendisplay verwendet, um seroreaktive Peptide zu identifizieren. Sechs aus Malaysia isolierte L. interrogans Stämme wurden untersucht,klinisch verglichen und auf Virulenz im Meerschweinchenmodell getestet. Die SMRT-Sequenzierung wurde am PacBio RSII durchgeführt und mit einem Illumina MiSeq ergänzt. Zwei L. interrogans-Stämme aus beiden Extremen des Virulenzspektrums wurden mittels RNA-seq untersucht. Alle sechs L. interrogans-Stämme wurden mittels MLST Core-Genom-MLST und (SNP) typisiert. Zwei ORFeome-Phagendisplaybibliotheken von Leptospira spp. Genomen vMalaysischer und WHO Referenzstämmen wurden konstruiert. Anschließend wurden fünf seroreaktive synthetische Peptide konstruiert und mit 16 Kontrollseren und 16 Patientenseren durch Titrations-ELISA auf Immunogenität validiert. Klinisch zeigte sich der mit dem Stamm Langkawi infizierte Patient dieschwerwiegendsten Symptome, während der Stamm 1530d eine leichte Leptospirose aufwies. Diese Befunde korrelierten gut mit denen der Tierstudie. Sowohl cgMLST als auch SNP-Genotypisierung sind vergleichbar mit ST-Clustering in MLST, jedoch führte die SNP-Genotypisierung zu einer höheren Abdeckung des Kerngenoms, d. H. 77,5%, verglichen mit 57,7% in cgMLST. In fast allen Genomen gibt es eine bemerkenswerte Genom-Plastizität, die hauptsächlich von Plasmiden und Insertionssequenzelementen bestimmt wird. In der Kultivierung wurden Gene, die mit Chemotaxis in Verbindung stehen, im Stamm Langkawi im Vergleich zum Stamm 1530d signifikant höher exprimiert. Dies erklärt höchstwahrscheinlich die höhere Virulenz des Stammes Langkawi im Vergleich zu 1530d. Beim Phagendisplay-Ansatz wurden zwei Peptide (SIR16-D1 und SIR16-H1) mit hoher Genauigkeit für den Nachweis akuter Leptospirosen identifiziert (AUC: 0.86 bzw. 0.78). Die Genomplastizität wurde durch horizontalen Gentransfer und homologe Rekombination als adaptive Strategie in verschiedenen ökologischen Nischen erlangt. Die Unterschiede in der Pathogenität aller Stämme schienen weitgehend durch die Genomzusammensetzung bestimmt zu sein. Das ORFeome-Phagendisplay erwies sich als leistungsfähige Methode zur Identifizierung von immunogenen Leptospiral-Peptiden

Proteomic Analysis of Urine Exosomes Reveals Renal Tubule Response to Leptospiral Colonization in Experimentally Infected Rats

BACKGROUND: Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. CONCLUSIONS: We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance