MLP analysis using capillary electrophoresis was modified from Botterel et al.[14]. Alleles were amplified in a multiplex PCR in a 50 μl final volume containing 20 ng DNA, 1X PCR-Buffer II (Applied Biosystems, CP673451 molecular weight Madrid, Spain), 0.2 mM of each deoxynucleotide triphosphate, 5 mM of MgCl2, and 0.15 μM of each primer and 1U of AmpliTaq Polymerase (Applied Biosystems). Sense CDC3 primer was labelled with 4, 7, 2′, 4′, 5′, 7′-hexachloro-6-carboxyfluorescein (HEX), EF3 antisense primer with 6-carboxyfluorescein (FAM)

and HIS 3 sense primer was labelled with 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1.4-dichloro-6-carboxyfluorescein SGC-CBP30 chemical structure (NED). Primers were synthesized by Sigma-Aldrich (Sigma-Aldrich, Madrid, Spain). PCR reactions were performed in a GeneAmp PCR system 9700 (Applied Biosystems). The cycling conditions included a first step for preincubation (activation of the enzyme) and denaturation of the DNA template at 95°C during 5 minutes. Next steps consisted in an amplification program of 30 cycles as follow: denaturation at 95°C for 30 s, annealing at 55°C for 30 ON-01910 in vivo s and extension at 72°C for 1 min with a final extension step of 7 min at 72°C. To assess the size of the fragments, 1 μl of the

PCR products was added to 9 μl of Formamide Hi-Di (Applied Biosystems, Madrid, Spain) and 1 μl of the internal size standard ROX 500 (Applied Biosystems, Madrid, Spain). Capillary electrophoresis was run using the ABI 3730 XL (Applied Biosystems, Madrid, Spain) sequencer. Fragment size for the different alleles was calculated with GeneMapper version 3.0

(Applied Biosystems, Madrid, Spain). In addition, a HRM-based analysis was performed using singleplex PCRs with each pair of primers without any modification of the reaction conditions. Control population was selected based on MLP results. Strains included as control were: CL 7484, CL 7498, CL 7504, CL 7513, CL 7694, ATCC 64548 and ATCC 64550 (Figure 1). Seven different genotypes for the three markers were chosen (Figure 1). Figure 1 Difference plots for the normalized and temperature shifted melting curves for microsatellite from control population and patient strains. A) Tolmetin CDC3 marker; B) EF3 marker and C) HIS3 marker. After PCR, HRM analysis was performed in a LightCycler 480 system (Roche, Madrid, Spain). To obtain the HRM curves, 1 μl of LightCycler® 480 ResoLight Dye (Roche, Madrid, Spain) was added to PCR products and the reactions were incubated at 95°C 1 min, followed by a renaturation step of 40°C for 1 min. Melting curves were generated by ramping from 65° to 95° at 0.02°C/s, 25 acquisitions/°C. HRM curves were plotted using the automated grouping option provided by the software and by manual editing for each microsatellite marker. Normalization conditions for each microsatellite marker are shown in Table 4.

However, in contrast, the pathogenic strain L. santarosai was not found to synthesize identifiable nonulosonic acid species at detectable levels (Figure 2). We also performed analyses on L. biflexa serovar Patoc. In this case, we observed the presence of Kdo by HPLC and mass spectrometry, but identifiable NulO molecules selleck inhibitor were not present at detectable levels (not shown). Figure 2  Leptospira  express mainly di-  N  -acetylated nonulosonic acids. Nonulosonic acids were released from Leptospira isolates and fluorescently derivatized with DMB followed by HPLC as described in Materials

and Methods. Selected peaks were subjected to electrospray ionization mass spectrometry. Pse and Leg refer to the di-N-acetylated nonulosonic acids pseudaminic and legionaminic acids, closely related isomers with an identical DMB-derivatized mass of 451. Kdo is a related eight-carbon backbone monosaccharide common to the core region of lipopolysaccharide. All MS data are shown from 400–500 m/z, except for representative MS data shown for peak b (Kdo), shown from 300–400 m/z. Each of these strains was analyzed in 2–3 independent experiments with similar results. Interestingly, HPLC analysis of the two different genome strains of L. interrogans (serovar Copenhageni strain L1-130 and

serovar Lai strain 56601) gave distinct results. While L. interrogans serovar Lai YAP-TEAD Inhibitor 1 mouse expresses di-N-acetylated nonulosonic acid (Figure 2, m/z Immune system 433), strain L1-130 (serovar Copenhagenii) exhibited a peak with mass and retention time Src inhibitor consistent with Neu5Ac (m/z 408, hydrated 426, and hydrated sodium salt 448) (Figure 3A-B). Additional MS2 analysis consistently reduced this trio of masses almost exclusively to the parent mass of 408 (Figure 3B), as expected based on the behavior of standard Neu5Ac derivatized in parallel (Figure 3C). Since the common animal sialic acids Neu5Ac and Neu5Gc were

found in the standard culture media used for Leptospira (EMJH, Figure 4A), experiments were designed to exclude the possibility that L. interrogans strain L1-130 may incorporate exogenous sialic acid from the culture media. Unfortunately, the lack of a readily available genetic system for Leptospira rules out gene deletion as an approach to demonstrate endogenous synthesis. However, leptospires grown in defined serum-free media without sialic acids (as confirmed by HPLC) still produced a Neu5Ac peak, confirming that L. interrogans strain L1-130 synthesizes Neu5Ac and this sugar is not acquired from growth media (Figure 4B). Figure 3  Leptospira interrogans  genome strain expresses sialic acid (Neu5Ac). HPLC analysis demonstrates peaks consistent with Kdo and Neu5Ac in Leptospira interrogans str. L1-130. Confirmation of the L1-130 Neu5Ac peak assignment was performed by parallel derivatization and LCMS analysis of Neu5Ac (Sigma). The structure of DMB-derivatized Neu5Ac has a protonated exact mass (m+H) of 426.1.