All leptospiral strains were aligned to reference sequences for the six genes in the NCBI GenBank, if adequate sequences were available. Accession numbers for L. interrogans serovar Copenhageni strain Fiocruz L1-130 are AE016823.1 and for L. borgpetersenii serovar Hardjo-bovis strain L550: CP000348.1. Accession numbers

for the Treponema outgroup are AE017226.1, see more CP001843.1 and CP000805.1. For DNA extraction, each strain was cultured for seven days. Six millilitres of the cultured organisms were centrifuged at 14.000 rpm, 4°C for 10 min, the pellet was then washed once with PBS and either stored at −30°C or used directly for DNA extraction. Extraction was performed using the QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. PCR for each target gene was performed using 25 mM MgCl2 (included in the 10x standard reaction buffer, NEB, Frankfurt am Main, Germany), 0.2 mM dNTP`s (NEB), 1 U Taq DNA Polymerase (NEB) and 1 μl template DNA. Amplification Ferroptosis phosphorylation parameters were set according to Ahmed et al. [33],

using the Master Cycler® pro system (Eppendorf AG, Hamburg, Germany). PCR products were visualized in 1.6% agarose gels. Products were then purified using the peqGOLD Gel Extraction Kit (Peqlab, Erlangen, Germany) following the manufacturer’s instruction. Five nanograms per μl of the purified product were sequenced by Eurofins MWG Operon (Ebersberg, Germany). All

strains were sequenced twice. Sequence analysis was performed by using the MEGA4 Software and Neighbor Joining trees were constructed for each gene and for each leptospiral strain according to Ahmed et al. [33]. 16S rRNA gene sequencing 16S rRNA gene sequencing was performed with the bacterial universal primers 27f (agagtttgatcmtggctcag) and 1392r (acgggcggtgtgtgtrc) (see GATC Biotech AG, Konstanz, Germany; http://​www.​gatc-biotech.​com, free universal primers). PCR was performed using HotStarTaq® Master Mix (Qiagen, Hilden, Germany) with the following profile: 15 min at 95°C for initial denaturation, 35 cycles of 30 sec at 95°C, 30 sec at 56°C and 1.5 min at 72°C, followed by a final extension step of 72°C for 5 min. Endonuclease PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequence analyses were performed using the Cycle Sequencing Kit (Applied Biosystems, Carlsbad, California, USA) following the manufacturer’s instructions. Sequencing was carried out on Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Carlsbad, California, USA) and the sequences were analyzed using the 16S rRNA gene database of SmartGene (Lausanne, Switzerland). A Maximum Likelihood phylogenetic tree of all 28 leptospiral 16S rRNA gene sequences was computed with PHYLIP dnaml (SmartGene).