Fig 7. Infectivity of AmB-resistant parasites and SMT gene expression in intracellular amastigotes.

A & B) Infectivity and replication of parasites within primary bone marrow-derived murine macrophages. Stationary phase promastigotes were used to infect macrophages for variable lengths of time and infection burdens were quantified as both parasites per infected macrophage (A) and percentage of macrophages infected (B). Error bars denote standard deviation, n = 3. C) AmB sensitivity of intracellular amastigotes. Asterisks significant differences (P < 0.05) in IC₅₀ from wild-type, n = 3. D) Infectivity of wild-type and AmBRC/cl3 parasites in vivo. Parasites recovered from mouse lymph nodes were brought to stationary phase and injected into mouse footpads. Footpad size was measured over time. The mean of five mice per parasite line is shown, error bars represent standard deviation. E) Fold-change in expression of SMT genes in amastigotes compared to promastigotes in wild-type parasites. Asterisks significant differences (P < 0.05) in δCt between the two life cycle stages (statistically significant P values are 3.00 x 10⁻⁴ (total SMT) and 1.42 x 10⁻⁵ (LmxM.36.2380)), n = 3. F) Expression of SMT genes in intracellular amastigotes. RNA was derived from primary macrophages 72 hours after infection with stationary phase promastigotes. Asterisks denote statistically significant differences (P < 0.05) in δCt from wild-type, n = 3. Statistically significant P values are as follows: for total SMT, respective values for AmBRB/cl2, AmBRC/cl3 and AmBRD/cl2 are 0.0270, 0.00106 and 0.00164; for LmxM.36.2380, respective values for AmBRB/cl2, AmBRC/cl3 and AmBRD/cl2 are 0.00164, 6.31 x 10⁻⁶ and 1.95 x 10⁻⁴; for LmxM.36.2390, values for AmBRA/cl1, AmBRB/cl2, AmBRC/cl3 and AmBRD/cl2 are 0.0105, 0.0143, 0,0396 and 0,0121. See Methods for the statistical approach used in qRT-PCR assays.