Fig 1.
Analysis of the enzymatic activity of recombinant CpLDH protein.
(A) SDS-PAGE analysis of the nickel affinity column chromatography-purified His-tagged recombinant CpLDH protein stained with coomassie blue (Lane M: protein ladder; Lane LDH: CpLDH protein). (B and C) Enzyme kinetics of recombinant CpLDH protein for the reduction of pyruvate to lactate on pyruvate as substrate (B), and NADH as co-factor (C). (D and E) Enzyme kinetics of recombinant CpLDH protein for the oxidation of lactate to pyruvate on lactate as substrate (D), and NAD+ as co-factor (E). Insets are Lineweaver–Burk representations of the saturation curves. S, substrate; V, velocity of the reaction. The data shown represent means of three independent experiments.
Table 1.
CpLDH enzyme kinetics on substrates and co-factors.
Fig 2.
Effect of the diverse set compounds on the catalytic activity of recombinant CpLDH protein.
Individually reconstituted compounds were used at a final concentration of 20 μM in the reaction for the reduction of pyruvate to lactate with recombinant CpLDH protein as enzyme. The mean percent inhibition of CpLDH activity by each compound was derived by dividing the mean change in optical density (ΔOD340) of the reaction after 2 min in the presence of the compound by the mean ΔOD340 of the reaction without compound, and multiplying the product by 100. The baseline mean percent inhibition of 0 (buffer) was for the reaction without compound, but with an equivalent volume of solvent used to reconstitute compound. Compounds with mean percent inhibition values greater than 0 were designated as inhibitors of the activity of CpLDH, while those with mean percent inhibition values less than 0 were classified as augmenters. Each reaction was performed in triplicate, and the data shown represents means of three independent experiments.
Fig 3.
Effect of the Mechanistic Set IV compounds on the catalytic activity of recombinant CpLDH protein.
Individually reconstituted compounds were used at a final concentration of 20 μM in the reaction for the reduction of pyruvate to lactate with recombinant CpLDH protein as enzyme. The mean percent inhibition of CpLDH activity by each compound was derived by dividing the mean change in optical density (ΔOD340) of the reaction after 2 min in the presence of the compound by the mean ΔOD340 of the reaction without compound, and multiplying the product by 100. The baseline mean percent inhibition of 0 (buffer) was for the reaction without compound, but with an equivalent volume of solvent used to reconstitute compound. Compounds with mean percent inhibition values greater than 0 were designated as inhibitors of the activity of CpLDH, while those with mean percent inhibition values less than 0 were classified as augmenters (data points below 0 that are not shown in Fig 3 were 765 in total, and are listed in S4 Table together with their corresponding CpLDH percent inhibition values). Each reaction was performed in triplicate, and the data shown represents means of three independent experiments.
Table 2.
Affinity of the most favorable compound poses within lactate dehydrogenase active site.
Fig 4.
Analysis of the effect of varying concentrations of compound NSC158011 on the growth of Cryptosporidium parvum in HCT-8 cells.
Equal amounts of freshly excysted sporozoites of C. parvum were inoculated into HCT-8 cells in culture and varying concentrations of NSC158011 added at the time of infection (solid line) or added 2 h post-infection (p.i.) (dashed line). Control infected cells (dotted line) were treated immediately p.i. with volumes of DMSO equivalent to those used in the compound-treated cultures. The cells were analyzed for parasite infectivity and proliferation by an immunofluorescence assay after (A) 48 h, and (B) 72 h of culture. The fluorescence generated by intracellular C. parvum merozoites was quantified and is shown on the Y-axis representing the parasite load. The data shown represent means of three independent experiments with standard error bars and levels of statistical significance between groups indicated by asterisk (*, P < 0.05).
Fig 5.
Analysis of the effect of varying concentrations of compound NSC10447 on the growth of Cryptosporidium parvum in HCT-8 cells.
Equal amounts of freshly excysted sporozoites of C. parvum were inoculated into HCT-8 cells in culture and varying concentrations of NSC10447 added at the time of infection (solid line) or added 2 h post-infection (p.i.) (dashed line). Control infected cells (dotted line) were treated immediately p.i. with volumes of DMSO equivalent to those used in the compound-treated cultures. The cultures were analyzed for parasite infectivity and proliferation by an immunofluorescence assay after (A) 48 h, and (B) 72 h of culture. The fluorescence generated by intracellular C. parvum merozoites was quantified and is shown on the Y-axis representing the parasite load. The data shown represent means of three independent experiments with standard error bars and levels of statistical significance between groups indicated by asterisk (*, P < 0.05).
Table 3.
NSC158011 and NSC10447 IC50 values on CpLDH enzyme and C. parvum.
Fig 6.
Analysis of the effect of varying concentrations of paromomycin on the growth of Cryptosporidium parvum in HCT-8 cells.
Equal amounts of freshly excysted sporozoites of C. parvum were inoculated into HCT-8 cells in culture and varying concentrations of paromomycin dissolved in sterile distilled water was added at infection (solid line) or 2 h post-infection (p.i.) (dashed line). Control infected cells (dotted line) were treated immediately p.i. with volumes of sterile distilled water equivalent to those used in the paromomycin-treated cultures. The cultures were analyzed for parasite infectivity and proliferation by an immunofluorescence assay after (A) 48 h, and (B) 72 h of culture. The fluorescence generated by intracellular C. parvum merozoites was quantified and is shown on the Y-axis representing the parasite load. The data shown represent means of three independent experiments with standard error bars and levels of statistical significance between groups indicated by asterisk (*, P < 0.05).
Table 4.
Real-time PCR quantification of C. parvum DNA in fecal samples of treated or untreated infected mice.
Table 5.
Real-time PCR quantification of C. parvum DNA in fecal samples of treated or untreated infected mice.
Table 6.
Real-time PCR quantification of C. parvum DNA in fecal samples of treated or untreated infected mice.
Fig 7.
Histopathological analysis of the effect of Cryptosporidium parvum infection in the lower small intestines of mice with or without NSC158011 treatment.
Mice infected with C. parvum were maintained untreated (Infected no treatment), treated with paromocycin (Infected+Paromomycin (100 mg/kg)) or treated with compound NSC158011 at 400 mg/kg (Infected+NSC158011 (400 mg/kg)) for 8 days. Uninfected mice were maintained as control (Uninfected Control). After 8 days, mice were sacrificed and the lower intestinal tissue processed for histology and stained with hematoxylin and eosin. (A) Uninfected control mice samples depicted intact intestinal epithelium with prominent villi. (B) In contrast, infected mice without treatment depicted denuded villi. Both (C) paromomycin and (D) NSC158011 treated infected mice depicted intact intestinal epithelium with prominent villi that were comparable to the uninfected control. The images are representative of samples analyzed from 3 mice per treatment group. (E) The mean percentage of denuded intestinal villi in 4 randomly chosen microscopic fields per sample from the uninfected (Uninf), infected untreated (Inf), Infected treated with paromomycin (Inf+P), and infected treated with 400 mg/kg NSC150811 (Inf+150811) mice. The data shown represent means for samples from three mice per group with standard error bars and levels of statistical significance depicted (*P < 0.05).
Fig 8.
Histopathological analysis of the effect of Cryptosporidium parvum infection in the lower small intestines of mice with or without NSC10447 treatment.
Mice infected with C. parvum were maintained untreated (Infected no treatment), treated with paromocycin at 100 mg/kg (Infected+Paromomycin (100 mg/kg)) or treated with compound NSC10447 (Infected+NSC10447) at varying doses (250, 500 or 1000 mg/kg) for 8 days. Uninfected mice were maintained as control (Uninfected Control). After 8 days, mice were sacrificed and the lower intestinal tissue processed for histology, and stained with hematoxylin and eosin. (A) Uninfected control mice samples depicted intact intestinal epithelium with prominent villi. (B) In contrast, infected mice without treatment depicted denuded villi. Infected mice treated with (C) 100 mg/kg Paromomycin, (D) 250 mg/kg NSC10447, (E) 500 mg/kg NSC10447, and (F) 1000 mg/kg NSC10447 depicted intact intestinal epithelium with prominent villi that were comparable to the uninfected control. The images are representative of samples analyzed from 3 mice per treatment group. (G) The mean percentage of denuded villi in 4 randomly chosen microscopic fields per sample from the uninfected (Uninf), infected untreated (Inf), infected treated with 100 mg/kg paromomycin (Inf+P), infected treated with 250 mg/kg NSC10447 (Inf+250), infected treated with 500 mg/kg NSC10447 (Inf+500), and infected treated with 1000 mg/kg NSC10447 (Inf+1000) mice. The data shown represent means for samples from 3 mice per group with standard error bars and levels of statistical significance depicted (*P < 0.05).