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Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

McCall LI, El Aroussi A, Choi JY, Vieira DF, De Muylder G, Johnston JB, Chen S, Kellar D, Siqueira-Neto JL, Roush WR, Podust LM, McKerrow JH - PLoS Negl Trop Dis (2015)

Bottom Line: While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available.In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51.While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani.

View Article: PubMed Central - PubMed

Affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.

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Essentiality of CYP51 in L. donovani.A, Persistence of CYP51 gene even after correct targeting of two knockout cassettes into the CYP51 locus. Correct targeting of the knockout cassettes was verified by PCR using one primer within the knockout cassette and one upstream of CYP51 (primers 7 and 8 (hygromycin) or 7 and 9 (puromycin)). CYP51 was amplified with primers within the coding region (primers 10 and 11). B, Targeting strategy. Primers 7 and 8 or 7 and 9 and expected PCR product sizes to confirm correct targeting of chromosomal CYP51 alleles are indicated by arrows (step 4). C, Loss of chromosomal CYP51 in the presence of episomal CYP51 complementation but not in strains complemented with the empty vector. PCR was performed using primers 12+13. D, pXNG4 loss monitored by qPCR during GCV selection or in the absence of either positive or negative selection (untreated samples). pXNG4 Ct values were normalized to SAT levels and to samples under positive selection (treated with nourseothricn (NTC) in the absence of GCV). E, GFP positive, propidium iodide (PI) negative levels were monitored by flow cytometry. Percentage of GFP+ PI- cells were normalized to the levels for week 1. *, p<0.05 compared to HKO+CYP+PAC.
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pntd.0003588.g002: Essentiality of CYP51 in L. donovani.A, Persistence of CYP51 gene even after correct targeting of two knockout cassettes into the CYP51 locus. Correct targeting of the knockout cassettes was verified by PCR using one primer within the knockout cassette and one upstream of CYP51 (primers 7 and 8 (hygromycin) or 7 and 9 (puromycin)). CYP51 was amplified with primers within the coding region (primers 10 and 11). B, Targeting strategy. Primers 7 and 8 or 7 and 9 and expected PCR product sizes to confirm correct targeting of chromosomal CYP51 alleles are indicated by arrows (step 4). C, Loss of chromosomal CYP51 in the presence of episomal CYP51 complementation but not in strains complemented with the empty vector. PCR was performed using primers 12+13. D, pXNG4 loss monitored by qPCR during GCV selection or in the absence of either positive or negative selection (untreated samples). pXNG4 Ct values were normalized to SAT levels and to samples under positive selection (treated with nourseothricn (NTC) in the absence of GCV). E, GFP positive, propidium iodide (PI) negative levels were monitored by flow cytometry. Percentage of GFP+ PI- cells were normalized to the levels for week 1. *, p<0.05 compared to HKO+CYP+PAC.

Mentions: We generated half knockout L. donovani parasites (HKO strains) in which a single CYP51 allele was replaced with either a puromycin or hygromycin resistance marker (Fig. 1A; see Fig. 2B for the knockout approach). CYP51 is located on chromosome 11, which is disomic in reference L. donovani genomes [37], but trisomic in some clinical L. donovani isolates [31]. In addition, the Leishmania genome contains many direct and indirect repeats that can promote extrachromosomal element formation under drug pressure or for essential genes [31,38]. Prior to targeting another CYP51 allele, we therefore verified CYP51 copy number in parasites transfected with the first knockout cassette, resistant to either hygromycin (HygR HKO strains) or puromycin (PAC HKO strains). HKO strains contained half of the CYP51 DNA content found in wild-type, indicating loss of one out of two alleles (Fig. 1B). Furthermore, CYP51 protein levels were decreased two to five fold in half knockout strains. Complementation with an episomal CYP51 gene restored protein expression to levels comparable to wild-type L. donovani (Fig. 1C).


Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

McCall LI, El Aroussi A, Choi JY, Vieira DF, De Muylder G, Johnston JB, Chen S, Kellar D, Siqueira-Neto JL, Roush WR, Podust LM, McKerrow JH - PLoS Negl Trop Dis (2015)

Essentiality of CYP51 in L. donovani.A, Persistence of CYP51 gene even after correct targeting of two knockout cassettes into the CYP51 locus. Correct targeting of the knockout cassettes was verified by PCR using one primer within the knockout cassette and one upstream of CYP51 (primers 7 and 8 (hygromycin) or 7 and 9 (puromycin)). CYP51 was amplified with primers within the coding region (primers 10 and 11). B, Targeting strategy. Primers 7 and 8 or 7 and 9 and expected PCR product sizes to confirm correct targeting of chromosomal CYP51 alleles are indicated by arrows (step 4). C, Loss of chromosomal CYP51 in the presence of episomal CYP51 complementation but not in strains complemented with the empty vector. PCR was performed using primers 12+13. D, pXNG4 loss monitored by qPCR during GCV selection or in the absence of either positive or negative selection (untreated samples). pXNG4 Ct values were normalized to SAT levels and to samples under positive selection (treated with nourseothricn (NTC) in the absence of GCV). E, GFP positive, propidium iodide (PI) negative levels were monitored by flow cytometry. Percentage of GFP+ PI- cells were normalized to the levels for week 1. *, p<0.05 compared to HKO+CYP+PAC.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359151&req=5

pntd.0003588.g002: Essentiality of CYP51 in L. donovani.A, Persistence of CYP51 gene even after correct targeting of two knockout cassettes into the CYP51 locus. Correct targeting of the knockout cassettes was verified by PCR using one primer within the knockout cassette and one upstream of CYP51 (primers 7 and 8 (hygromycin) or 7 and 9 (puromycin)). CYP51 was amplified with primers within the coding region (primers 10 and 11). B, Targeting strategy. Primers 7 and 8 or 7 and 9 and expected PCR product sizes to confirm correct targeting of chromosomal CYP51 alleles are indicated by arrows (step 4). C, Loss of chromosomal CYP51 in the presence of episomal CYP51 complementation but not in strains complemented with the empty vector. PCR was performed using primers 12+13. D, pXNG4 loss monitored by qPCR during GCV selection or in the absence of either positive or negative selection (untreated samples). pXNG4 Ct values were normalized to SAT levels and to samples under positive selection (treated with nourseothricn (NTC) in the absence of GCV). E, GFP positive, propidium iodide (PI) negative levels were monitored by flow cytometry. Percentage of GFP+ PI- cells were normalized to the levels for week 1. *, p<0.05 compared to HKO+CYP+PAC.
Mentions: We generated half knockout L. donovani parasites (HKO strains) in which a single CYP51 allele was replaced with either a puromycin or hygromycin resistance marker (Fig. 1A; see Fig. 2B for the knockout approach). CYP51 is located on chromosome 11, which is disomic in reference L. donovani genomes [37], but trisomic in some clinical L. donovani isolates [31]. In addition, the Leishmania genome contains many direct and indirect repeats that can promote extrachromosomal element formation under drug pressure or for essential genes [31,38]. Prior to targeting another CYP51 allele, we therefore verified CYP51 copy number in parasites transfected with the first knockout cassette, resistant to either hygromycin (HygR HKO strains) or puromycin (PAC HKO strains). HKO strains contained half of the CYP51 DNA content found in wild-type, indicating loss of one out of two alleles (Fig. 1B). Furthermore, CYP51 protein levels were decreased two to five fold in half knockout strains. Complementation with an episomal CYP51 gene restored protein expression to levels comparable to wild-type L. donovani (Fig. 1C).

Bottom Line: While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available.In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51.While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani.

View Article: PubMed Central - PubMed

Affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.

Show MeSH
Related in: MedlinePlus