Table 1.
List of target genes, their putative function and primer sequence used for amplification.
Table 2.
In vitro miltefosine susceptibility (IC50), MIL accumulation and thiol levels of L. donovani isolates from pretreatment group (LdPreTx), isolates obtained from visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) patients that relapsed after MIL treatment (LdRelapse) and experimental MIL resistant parasites (LdM30).
Fig 1.
In vitro MIL susceptibility, percent infectivity and MIL uptake in L. donovani parasites.
(A) MIL susceptibility at promastigotes stage of LdPreTx, LdRelapse and LdM30 with each individual value represented as mean IC50±SD from three separate assays. (B) MIL susceptibility at intracellular amastigote stage LdPreTx, LdRelapse and LdM30 with each individual value represented as mean IC50 ± SD from three separate assays (C) Mice peritoneal derived macrophages infected with LdPreTx or LdRelapse parasites at a 1:10 (cell/parasite) ratio. The percent infectivity was determined at 24h, 48h, and 72h post infection by counting number of infected cells out of 100 macrophages at 1000X magnification after staining with Diff-Quik. Data represents mean ± SD of three independent experiments each in duplicate. (D) Percent metacyclogenesis of promastigote population estimated based on negative selection of peanut agglutinin (%PNA- promastigote). Values represent mean ± SD of two independent experiments (E) MIL uptake, estimated using LC-MS in 1x108 promastigotes of LdPreTx, LdRelpase and LdM30. Data represents mean ± SD of two independent experiments, each in duplicate. Asterisks indicate significance (**P<0.01; and ***P<0.001).
Fig 2.
MIL induced oxidative stress (ROS level) and intracellular thiol content in L. donovani.
(A) Dose dependent accumulation of ROS in LdPreTx, LdRelapse and LdM30 at promastigote stage was assayed fluorometrically at 495 nm excitation and 535 nm emission wavelength using cell permeable probe H2DCF-DA (40nM). Data represents mean ± SD of three independent experiments, each performed in triplicate. Asterisks indicate significance (**P<0.01; and ***P<0.001). (B) Accumulation of ROS in macrophages infected with LdPreTx, LdRelapse, LdM30 parasites before and after MIL exposure (20μM), assayed fluorometrically at 495 nm excitation and 535 nm emission wavelength using cell permeable probe H2DCF-DA (30μM). Data represents mean ± SD of three independent experiments, each in triplicate. Asterisks indicate significance (*P<0.05; **P<0.01; and ***P<0.001). (C) Intracellular thiol content in LdPreTx, LdRelapse and LdM30 promastigotes, measured fluorometrically at 390 nm excitation and 520 nm emission wavelength. Data represents mean ± SD of two independent experiments each performed in triplicate. Asterisks indicate significance (**P<0.01; and ***P<0.001).
Fig 3.
Expression analysis of selected genes by real time PCR in clinical isolates of L. donovani at pretreatment (LdPreTx), relapse (LdRelapse), LdM30 parasites.
Fold change represents expression of target genes normalized to internal control (GAPDH and α-Tubulin) genes and relative to LdAG83. Data represents mean ± SD of two separate assays, each performed in triplicate. Asterisks indicate significance (*P<0.05; **P<0.01; and *** P<0.001).