Fig 1.
A, The canonical pathway of sterol biosynthesis in fungi, Leishmania, vertebrates and land plants. B, Structure of ergosterol, cholesterol and stigmasterol molecule with atom numbering. C, Reaction showing the enzymatic conversion of β- sitosterol into stigmasterol in the presence of CYP710C1 (C-22 desaturase).
Table 1.
Primers used for generation of the NEO specific linear replacement cassette fragments.
Table 2.
Primers used for molecular characterization of genetically manipulated parasites.
Fig 2.
Multiple sequence alignment of CYP710 proteins using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/).
For analysis, we used the following sequences: Leishmania donovani (LdBPK_303610.1), Trypanosoma cruzi (TcCLB.510101.50), Selaginella moellendorffii (Uniprot ID: D8QPW3) and Arabidopsis thaliana CYP710A1 (Uniprot ID: O64697), Arabidopsis thaliana CYP710A2 (Uniprot ID: O64698), Arabidopsis thaliana CYP710A3 (Uniprot ID:A0A178VR86), Arabidopsis thaliana CYP710A4 (A0A178VVZ9). Residues conserved in all five proteins are indicated by an asterisk. Residues having similar, or very similar physiochemical character is indicated by a dot or colon, respectively. The four conserved motifs are highlighted by a rectangular box.
Fig 3.
Sequence logos of the conserved CYP motifs of Leishmania and their comparison with Trypanosoma cruzi, Arabidopsis thaliana and Selaginella moellendorffii.
The most characteristic motif FXXGXRXCXG (d) corresponds to the heme-binding domain. The second conserved motif EXLR (b) and the third consensus PER (c) form E–R–R triad and are essential for stabilization of the core structure. AGXDTT (a) is known to contribute to oxygen binding and activation.
Fig 4.
Expression and characterization of recombinant CYP710C1.
A. The SDS-PAGE analysis of whole cell lysate of uninduced and induced E.coli BL21 (DE3) cells transformed with pET-30a-CYP710C1. M, molecular weight marker; Lane 1 uninduced bacterial cell lysate; Lane 2 induced bacterial cell lysate; Lane 3, Flow through; Lane 4 Purified eluted fraction of recombinant CYP710C1 on Ni2+ -nitrilotriacetic acid affinity resin with 100 mM imidazole. B, Western blot analysis of recombinant protein using anti-CYP710C1 antibody. Lane 1, negative control, Lane 2, 50 ng rCYP710C1 protein, Lane 3, 100 ng rCYP710C1 protein C, Western blot analysis of cell lysates (~ 40 μg) prepared from L. donovani wild-type promastigotes and amastigotes probed with an anti-CYP710C1 antibody (1:3000). Loading was normalized with β- Actin (42 kDa). D, Histogram representing normalized means from densitometric analysis of the immunoblots shown in panel (D) was quantified using ImageJ software. E, RT-PCR analysis of CYP710C1 mRNA levels in the cell lysates of amastigotes and promastigotes. The results represent mean ± SD with n = 3. * p < 0.05; **, p < 0.01, *** p < 0.005 and ns indicates not significant (p > 0.05).
Table 3.
Comparison between sterols in promastigotes and axenic amastigotes of L. donovani.
Identification is based on matches with the NIST library after fragmentation. Abundance relative to the total sterol content is indicated in each cell line and the fold change of that [32]. Results are the mean % of total sterols ± S.D. of three independent experiments.
Fig 5.
Reaction products from the recombinant CYP710C1 assays were analysed in GC-MS total ion chromatograms.
A Ion chromatogram of standard sterols at specific retention times (RTs): a, stigmasterol-trimethylsilyl (TMS) (RT = 25.09 min; molecular ion [m/z] = 484) b, β-sitosterol-TMS (RT = 26.2; m/z = 486). B, Ion chromatogram showing peak ‘a’ of stigmasterol as Stigmasta-5, 22-dien-3-ol, (Stigmasta-5,22-dien-3-ol is the IUPAC name of stigmasterol, structure is shown in Fig 1) at 0 μM (negative control, where no stigmasterol peak was obtained), 75 μM, 100 μM, 125 μM and 150 μM concentrations of the substrate (β-sitosterol) showing a higher peak at 125 μM concentration. The coupled enzyme assay was done to confirm the desaturase activity of rCYP710C1 (100 μg/ml) with 50 mM potassium phosphate, pH 7.25, 100 mM NADPH, and sterol substrate (β-sitosterol) supplemented with 0.1 unit/mL of a purified recombinant NADPH-P450 reductase (Cytochrome P450 reductase). The pattern of fragment ions with m/z values of 484 and 412 was attributed to stigmasterol whereas 486 and 414 were used to identify β-sitosterol according to the NIST 14 mass spectral library. Three replicates of each reaction show high reproducibility concerning the peak area.
Table 4.
CYP710C1 desaturase activity at different concentrations of substrate (β- sitosterol).
Identification is based on matches with the NIST library after fragmentation. Mean values of three replicates are shown.
Fig 6.
Characterization of CYP710C1 OE.
A, PCR analysis showing insert fall out confirmation after restriction digestion from the cloned pSP-α-blast-α vector. Lane 1–4, size of CYP710C1 gene and the vector is 1.5 kb and 4.5 kb respectively B, Western blot analysis of protein cell lysates prepared from L. donovani wild-type Bob promastigotes and CYP710C1 OE probed with the anti-CYP710C1 antibody. Loading was normalized with β- actin (42 kDa). C, Histograms representing normalized means from densitometric analysis of the immunoblots shown in panel (B), as quantified using ImageJ software D, RT-PCR analysis of CYP710C1 mRNA levels in the cell lysates of CYP710C1 OE and WT promastigotes. The results represent mean ± SD with n = 3. * p < 0.05; **, p < 0.01, *** p < 0.005 and ns indicates not significant (p > 0.05). E, Ion chromatograms showing peak ‘a’ of stigmasterol after GC-MS analysis of reaction products of CYP710C1 activity by using an equal amount of cell lysate of WT and CYP710C1 OE and with 125 μM of β-sitosterol. Three replicates of each reaction of extraction show high reproducibility about the peak area.
Table 5.
CYP710C1 desaturase activity in WT and CYP710C1 OE by using 125 μM concentration of substrate (β- sitosterol).
Representative stigmasterol level as detected by GC-MS analysis of reaction products of CYP710C1 activity in CYP710C1 OE and WT. Identification is based on matches with the NIST library after fragmentation. Mean values of three replicates are shown.
Table 6.
Comparison between sterols in promastigotes of L. donovani (Bob) wild-type (WT), CYP710C1 OE and heterozygous mutants (CYP710C1/NEO).
Identification is based on matches with the NIST library after fragmentation. Abundance relative to the total sterol content is indicated in each cell line and the fold change of that. Results are the mean % of total sterols ± S.D. of three independent experiments.
Table 7.
Comparison between sterols in axenic amastigotes of L. donovani (Bob) wild-type (WT), CYP710C1 OE and heterozygous mutants (CYP710C1/NEO).
Fig 7.
Characterization of CYP710C1 heterozygous mutants.
A, Restriction map of the LdCYP710C1 genomic locus and location of the primers used for confirmation by PCR-based analysis along with the expected band sizes. B, PCR analysis of heterozygous mutants (CYP710C1/NEO) to evaluate the specific integration of the replacement cassette by using NEO and LdCYP710C1 (WT) gene-specific primers. Genomic DNA from LdCYP710C1 heterozygous mutants (CYP710C1/NEO) and WT parasites was used as a template for PCR analysis. M indicates the molecular size markers in kilobases. Lane numbers in panel B indicate the primers (mentioned in panel A) used for each lane. C, Southern blot analysis of genomic DNA from wild-type (WT) (lane 1), heterozygous LdCYP710C1 (CYP710C1/NEO) (lane 2) mutant parasites. Genomic DNA was digested with XhoI, and BglII separated on a 0.6% agarose gel and probed with the 5’UTR of the LdCYP710C1 gene. In the lane of WT, digestion of the LdCYP710C1 gene locus with XhoI yielded a 1.2-kb band whereas in case CYP710C1/NEO there was an extra band of 4.3 kb in addition to 1.2 kb. Molecular sizes are shown to the right of the blot. D, Western blot analysis of cell lysates prepared from L. donovani wild-type promastigotes and CYP710C1 heterozygous mutants (CYP710C1/NEO) probed with the anti-CYP710C1 antibody. Loading was normalized with β- actin (42 kDa) E, Histograms representing normalized means from densitometric analysis of the immunoblots shown in panel (D) and in two other experiments, as quantified using ImageJ software F, RT-PCR analysis of CYP710C1 mRNA levels in the cell lysates of CYP710C1 heterozygous mutants (CYP710C1/NEO) and WT promastigotes. G, Infectivity assay in which J774A.1 cells were infected with WT, CYP710C1/NEO and CYP710C1 OE parasites. H, Growth curve of WT, CYP710C1 OE and CYP710C1/NEO mutants. The results represent mean ± SD with n = 3. *** p < 0.005 the statistical difference from the wild-type control.
Table 8.
CYP710C1 desaturase activity in WT and CYP710C1/NEO by using 125 μM concentrations of substrate (β- sitosterol).
Representative stigmasterol level as detected by GC-MS analysis of reaction products of CYP710C1 activity in WT and CYP710C1/NEO. Identification is based on matches with the NIST library after fragmentation. Mean values of three replicates are shown.
Fig 8.
Effect of AmB on WT, CYP710C1 OE, and CYP710C1/NEO parasites.
In vitro drug sensitivity was measured by incubating promastigotes at a cell density of 2x 105 cells/ml in M199 medium supplemented with 10% FBS with a range of different concentrations of the AmB in 96 well plates. After 72 h, the viability of the cells was determined by the 50% inhibitory concentration (IC50) with the MTT assay. This value corresponds to the concentration of AmB which results in 50% inhibition of cell proliferation compared with untreated cells. CYP710C1 OE showed resistance to strains to anti-leishmanial drug AmB. CYP710C1 heterozygous L. donovani promastigotes display increased sensitivity to AmB. IC50 are given as mean ± S.D of at least two independent determinations with triplicates in each.
Table 9.
Drug sensitivity of wild, CYP710C1 OE and CYP710C1/NEO promastigotes.
Values were determined in promastigotes of wild-type, CYP710C1 OE and CYP710C1/NEO of L. donovani after 72 h of drug addition. IC50 are given as mean ± S.D of at least two independent determinations with triplicates in each. *nd- not determined.