Identification of point mutations previously identified in MIL-resistant <i>L</i>. <i>donovani</i> (T421N, L856P, W210*, M1) and <i>L</i>. <i>major</i> (G582D, M547del).

<p>Previously identified mutations were sequenced and are indicated with an asterisk (*) and highlighted in bold font, using <i>L</i>. <i>major</i> FVI wild-type as the reference strain. No mutations were detected in any of the resistant lines.</p><p>Identification of point mutations previously identified in MIL-resistant <i>L</i>. <i>donovani</i> (T421N, L856P, W210*, M1) and <i>L</i>. <i>major</i> (G582D, M547del).</p

Susceptibility of MIL-resistant <i>L</i>. <i>major</i> FVI populations generated by step-wise selection and determined by EC₅₀ analysis.

<p>1×10⁶ Log-phase parasites were incubated in the presence of a range of drug concentrations for 48 hours at 27°C, and the surviving cells were quantified with Cell Titer Blue proliferation assay using a Typhoon FLA-9500 laser scanner. Populations of parasites were grown in increasing concentrations of MIL ranging from 10 μM (R10) to 40 μM (R40), showing increased resistance to MIL. Horizontal dashed line represents WT threshold for MIL resistance. “Rno” are resistant lines grown in the absence of MIL for at least 75 passages. Results are the average of triplicate experiments ± SD.</p

Growth curves of <i>L</i>. <i>major</i> WT and MIL-resistant promastigotes growing in the presence of 30μM MIL or absence of MIL selection.

<p>Log-phase promastigotes cultures were counted daily until they reached stationary phase. Concentration was determined microscopically by counting in a Neubauer chamber. Results are the average of triplicate experiments ± SD.</p

Virulence of WT and MIL-resistant <i>L</i>. <i>major</i>.

<p>R40 demonstrate attenuated virulence <i>in vivo</i> compared with WT promastigotes. 1×10⁶ WT (n = 5) and R40 (n = 6) metacyclic promastigotes were injected into the footpads of female BALB/c mice. Lesion size was recorded weekly by taking measurements of footpad thickness with a Vernier caliper, results are averages ± SD.</p

Metacyclogenesis in WT and MIL-resistant <i>L</i>. <i>major</i>.

<p><i>L</i>. <i>major</i> promastigotes resistant to MIL exhibit increased metacyclogenesis as determined by qRT-PCR of SHERP expression relative to housekeeping gene GAPDH and normalized to WT expression levels (left). 5-day stationary parasites were subjected to peanut agglutination and Ficoll-400 gradients and percentage of metacyclics is shown (right). Results are the average of triplicate experiments ± SD. Statistical differences determined with a Student’s <i>t</i> test relative to control values (* <i>p</i><0.05)</p

Host cell infection assay.

<p>Early stages of macrophage invasion are similar between <i>L</i>. <i>major</i> WT and R40, as determined by infection of RAW264.7 murine macrophages. Metacyclic parasites were incubated in the presence of macrophages at a MOI of 10 metacyclic parasites per macrophage and cells were collected at 6h, 12h, 24h, and 48h. Samples were stained and infection was determined through light microscopy. <b>(A)</b> The percentage of infected macrophages, and <b>(B)</b> the number of parasites/100 cells were recorded. Results are the average of triplicate experiments ± SD. Statistical differences determined with a Students <i>t</i> test relative to control values (* <i>p</i><0.05; ** <i>p</i><0.01)</p