Doi:10.1016/s0166-6851(03)00174-9

Molecular & Biochemical Parasitology 131 (2003) 77–81 The H region HTBF gene mediates terbinafine resistance Julio F.M. Marchini , Angela K. Cruz , Stephen M. Beverley , Luiz R.O. Tosi a Departamento de Biologia Celular e Molecular e Bioagentes Patogˆenicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ave. Bandeirantes, 3900, 14049-900 Ribeirão Preto—SP, Brazil b Department of Molecular Microbiology, Washington University Medical School, 660 South Euclid Ave., St. Louis, MO 63110, USA Received 15 March 2003; received in revised form 23 June 2003; accepted 30 June 2003 Keywords: Leishmania; Terbinafine resistance; H region; Gene amplification; In vitro transposition Leishmania spp. are the causative agent of leishmania- sistant to heavy metals (arsenite and antimonials) through sis, a disease that has an estimated global prevalence of 12 a still unclear mechanism The alternative pteridine million cases The disease has a broad spectrum of clin- reductase PTR1 confers resistance to methotrexate, and has ical presentations, from skin and subcutaneous lesions to been shown to limit pathogenesis within the mammalian visceral disease. The mainstream treatment for leishmania- host L. major subject to drug pressure may become sis consists of parenteral administration of pentavalent anti- resistant to terbinafine through H region amplification, as monials, which may result in dose-cumulative side effects.
observed in cell lines SF-R30 (selected in terbinafine) and Amphotericin B is an even more toxic and less effective al- PQ-R10 (selected in primaquine) erbinafine is an anti- ternative. For the treatment of complex cases, inhibitors of fungal drug that is capable of inhibiting squalene-epoxidase the sterol biosynthesis pathway, such as terbinafine, offer an a key enzyme in the biosynthesis of ergosterol, an attractive possibility as they target parasite-specific physio- essential component of the cell membrane. Terbinafine tox- icity thus arises from ergosterol deficiency and squalene The study of the ability of this organism to evade accumulation the genes mediating resistance chemotherapy is important not only to understand different to other agents that induce H region amplification have aspects of this protozoan’s biology but also to help the been identified, terbinafine resistance has not been mapped design of effective treatments. Gene amplification is a com- mon mechanism used for survival in Leishmania strains The effects of terbinafine on the parasite growth are com- selected for resistance to drugs such as methotrexate plex and were shown to elicit resistance. The identification Leishmania major selected in methotrexate, primaquine, of a terbinafine-resistance gene present within the H region or pentavalent antimonials derive resistance to these drugs took advantage of a strategy based upon gene inactivation through the amplification of a 48 kb locus in chromosome through insertional mutagenesis and its correlation with loss 23, the H region. P-glycoprotein A (PGPA) and Pteridine of phenotype. Considering the 48 kb span of the H locus, the reductase 1 (PTR1) are the two genes related to drug resis- resistance phenotype was constrained to a smaller fragment tance that have been identified within the H region. PGPA in order to reduce the number of genes to be inactivated by is an ATP-binding cassette which renders the parasite re- the insertional mutagenesis protocol. The H region had al-ready been divided into four fragments cloned into the shut-tle vector pSNAR Terbinafine resistance was assessed Abbreviations: PCR, polymease chain reaction; GFP, green fluores- cent protein; RTPCR, reverse transcriptase polymerase chain reaction; using not only L. major transfectants bearing each of the PFGE, pulse field gel electrophoresis; YIP, YPT interacting protein; PGPA, four clones, but also the SF-R30 strain and the wild-type cell P-glycoprotein A; PTR1, Pteridine reductase 1; ORF, open reading frame; line LT252 as positive and negative controls, respectively.
MOPS, 3-[N-morpholino] propane-sulfonic acid; HTBF, H region asso- The fold resistance at EC50 for these lines is presented in ciated terbinafine resistance; SNARE, soluble N-ethylmaleimide-sensitive Transfectants bearing the H region subfragments ∗ Corresponding author. Tel.: +55-16-602-3117; fax: +55-16-633-1786.
H1, H2, and H3 were not different from the wild-type cell E-mail address: luiztosi@fmrp.usp.br (L.R.O. Tosi).
line. However, the fold resistance exhibited by transfectant 0166-6851/$ – see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0166-6851(03)00174-9 J.F.M. Marchini et al. / Molecular & Biochemical Parasitology 131 (2003) 77–81 Fig. 1. Delimitation of the terbinafine-resistance gene within the H region. (A) SacI fragments of H4 (T1, T2, and T4) were subcloned into pELHYGand tested for terbinafine resistance; plus and minus signs represent resistance and sensitivity, respectively; black arrows represent inverted repeats (T3aand T3b); S = SacI site; sequencing of T1 revealed two complete open reading frames indicated by white arrows; the arrows on HTBF represent theplacement of mariner transposon insertion events; the insertion named 82 was used to originate the L82 transfectant. (B and C) Fold resistance of L.
major cell lines and transfectants at EC50 for terbinafine relative to that of the wild-type LT252, CC1 clone (MHOM/IR/84/LT 252); log-phase cellswere seeded at a cell density of 105 cells ml−1 into drug-free medium as well as in media containing terbinafine ranging from 2 to 8 ␮g ml−1; thedrug-free culture growth was monitored daily until it reached late-log phase (0.8 to 1.0 × 107 cells ml−1), when the terbinafine EC50 was determinedas the concentration that decreased the cell growth rate by 50%; mean and S.D. are shown; ∗Values significantly higher than the wild-type value byStudent’s t test (P < 0.05); ∗∗Values significantly higher than all other lines and transfectants by Student’s t test (P < 0.05). Terbinafine resistantcell lines were SF-R30 and PQ-R10 stable transfectants were initially selected on 32 ␮g ml−1 of hygromycin B and increased to 500 ␮g ml−1 inindicated transfectants by doubling steps; transfectants were named according to Clayton et al The fold-resistance is the ratio of terbinafine EC50sfor experimental and LT252 cells, measured in the same experimenta.
H4 (LT[pSNAR8H4]) was comparable to that of the resis- ent mechanisms of terbinafine resistance in addition to the tant cell line PQ-R10 and significantly higher than that ob- served for wild-type cells (P < 0.05). Although fold re- In order to limit the locus that has imparted terbinafine re- sistance is typically low (about two-fold), it is consistently sistance to Leishmania, the H4 fragment was restricted with found in terbinafine resistant lines regardless of the selec- SacI, which generated five fragments (Two of the tive force leading to H region amplification. The cell lines resulting fragments encompass the inverted repeats present selected in terbinafine are an exception in which the fold in the right end of the H locus. The remaining fragments resistance is significantly higher Different mechanisms were subcloned into shuttle vectors pELHYG or pELHYGII might play a role in bringing about terbinafine resistance. In and transfected into the parasite. L. major transfectants T1 fact, Leishmania resistance to sterol biosynthesis inhibitors (LT[pELHYG32T1]) and T4 (LT[pELHYGII32T4]) were iso- can be achieved by the expression of many loci in- lated and kept under 32 ␮g ml−1 hygromycin B. The concen- cluding the H locus, as shown in SF-R30 and PQ-R10 lines.
tration of hygromycin B in culture was raised to 500 ␮g ml−1 Therefore, we cannot exclude the possibility that terbinafine in doubling steps so as to increase the plasmid copy num- selected cell lines, such as SF-R30, have developed differ- ber. The transfectants T1500 (LT[pELHYG500T1]) and T4500 J.F.M. Marchini et al. / Molecular & Biochemical Parasitology 131 (2003) 77–81 (LT[pELHYGII500T4]) allowed the analysis of the effect of prenylated to a geranylgeranyl group that mediates attach- this phenomenon on the terbinafine-resistance phenotype.
ment to membranes pool of YPT exists in the cytosol, Control cell lines, as well as the different transfectants noted chaperoned by GDP dissociation inhibitor (GDI). Interac- above, were used in a terbinafine resistance-assay ( tion with YIP1 promotes the dissociation of the YPT/GDI The fold resistance of transfectants T4500 and T132 was not heterodimer, allowing YPT to reinsert into membranes different from that observed for the wild-type cells. Nev- A YPT homologue gene has already been identified in L. ma- ertheless, the transfectant T1500 exhibited a fold resistance jor and its product has been localized in the parasite Golgi that was significantly higher than that observed for wild-type cells and comparable to that exhibited by the transfectant The predicted HTBF is a 21 kDa protein containing four H4. These results not only consistently linked the resistance membrane-spanning domains, with both C and N terminus phenotype to the H locus, but also narrowed the search for oriented toward the cytoplasmic face of the membrane.
the resistance gene to only 2.8 kb contained in T1 fragment.
Kyte-Doolittle hydrophobicity plot analysis also indicated Primer-island sequencing of the segment cloned into pEL- that HTBF is potentially an integral membrane protein.
HYG revealed two complete open reading frames (ORFs) Southern blot analysis of pulse field gel electrophoresis within the T1 locus (accession number AY227807). ORF2 (PFGE)-separated chromosomes, as well as SacI digested and ORF3 had typical upstream and downstream putative genomic DNA, suggested that HTBF is present in one copy AG trans-splicing acceptor sites and pyrimidine-rich tracks in the chromosome 23 of L. major (data not shown).
An incomplete open reading frame, ORF1, lacking a The transcription of HTBF was studied using Northern 5 -end was identified upstream of ORF2. Comparison with blot analysis of total RNA extracted from Leishmania cell the public sequence databases revealed the homology of lines LT252 and SF-R30, as well as from transfectants the 570 bp ORF2 to genes annotated in the human, worm, T4500, T132, T1500, and L82. The 570 bp HTBF probe rec- and yeast genomes. No significant sequence homology was ognized predominantly a 1.9 kb transcript in T132, T1500, found for 333 bp ORF1 and 360 bp ORF3.
and SF-R30, which was absent in LT252 line or transfec- Since transfection of segment T1 clearly rendered L. ma- tant T4500 (Although the basal level of the tran- jor resistant to terbinafine, this fragment was selected as a script was not detected, its expression is evident in the H target for an insertional mutagenesis protocol. Inactivation region-amplified cell line and in the transfectants carrying of genes within T1 was carried out in vitro using the mariner the intact recombinant HTBF. Increased drug pressure led transposition system Specific pairs of primers for each to higher transcript levels in T1500 when compared to T132.
gene within T1 were used in a colony polymease chain re- A 5 kb transcript, which could correspond to read-through action (PCR) protocol in order to identify useful insertion transcription across the circular episome was also de- events. Primers LT007 and LT008 were selected to screen tected in transfectants carrying pELHYG-T1, but not in the insertion events into ORF2, revealing 5 out of 96 events that SF-R30 line. As a consequence of the insertion into HTBF, had the transposable element inserted within the 570 bp ORF.
the transcripts detected in transfectant L82 were 2 kb larger One of the insertions disrupting ORF2, named 82 ( than those found in T1500. HTBF transcripts were also was transfected into L. major. The transfectant obtained, L82 detected in T1500 and SF-R30 through a RT-PCR method (LT[pELHYG500T1::/GFP∗K82]), was used in a terbinafine (Being a more sensitive method, the RT-PCR resistance-assay where T1500, H4, and LT252 were the pos- also detected the basal level of transcript in the LT252 line itive and negative controls, respectively. The transfectant and transfectant T4500. As expected, a 2 kb larger band L82, in which the insertion occurred 141 bp downstream of was also amplified from L82 total RNA. Reactions that did ORF2 ATG, presented a fold resistance that was not differ- not include the reverse transcriptase confirmed the absence ent from the wild-type, indicating the loss of the terbinafine of contaminating DNA in the template samples (data not resistance-phenotype Therefore, the integration of the mariner element/GFP∗K at one fourth of the putative HTBF could mediate resistance to terbinafine through gene length was enough to inactivate the terbinafine resis- a general mechanism involving the increased formation tance associated to the 2.8 kb fragment T1. ORF2 was thus and/or the redirection of vesicles. Studies on the mecha- named H region associated terbinafine resistance (HTBF).
nism of metal resistance in Leishmania mediated by the The BLAST analysis of HTBF revealed that its predicted ABC transporter PGPA suggested that this protein acts on amino acid sequence had significant homology to the YIP1 metal-thiol conjugates PGPA is associated to intra- protein of Saccharomyces cerevisiae. The predicted HTBF cellular membranes in structures proposed to be part of and the yeast protein shared 23.9% identity at the amino acid exocytic and endocytic pathways in Leishmania level and there was 38.4% identity between this HTBF and findings indicated that PGPA confers resistance to arsenite the Caenorhabditis elegans homologue. Overall, the Leish- and antimonials by sequestration of metals into vesicles mania HTBF shared 27.0% identity with the domain consen- that could be exocytosed through the flagellar pocket. In sus of YIP1 protein from different organisms (COG5080).
this scenario, HTBF would improve the process of vesicle In the yeast, YIP1 interacts with YPT, a protein related to trafficking and/or docking. It is noteworthy that L. tarento- vesicle docking and trafficking YPT is a Rab/GTPase lae H region-derived amplicons containing PGPA are likely J.F.M. Marchini et al. / Molecular & Biochemical Parasitology 131 (2003) 77–81 Fig. 2. Analyses of HTBF transcripts. (A) Northern analysis of total RNA in the cell lines LT252 (lane 1); SF-R30 (lane 6) and transfectantsT4500 (lane 2); T132 (lane 3); T1500 (lane 4) and L82 (lane 5); the black arrowhead indicates the ∼3.9 kb transcript in the transfectant bearingthe transposon insertion into HTBF. Total RNA from promastigote forms was extracted using TRIzol® reagent (Gibco BRL) and manipulated asdescribed elsewhere The HTBF probe was amplified by PCR with primers LT007 (5 -GCGCCCGGGCATATGCTCAACGAGGTGC) and LT008(5 -CGCGGATCCTAAATACCAACCAGA). (B) RT-PCR amplification products using HTBF primers (LT007/LT008) and total RNA from LT252 (lane1); T4500 (lane 2); T1500 (lane 4); L82 (lane 5); and SF-R30 (lane 6); the white arrowhead indicates the amplification product of 2.5 kb which representsthe insertion of element/GFP∗K into HTBF; reactions that did not include the reverse transcriptase confirmed the absence of contaminating DNA inthe template samples (data not shown); reverse transcriptase-PCR was performed with Ready-To-GoTM RT-PCR Beads (Amersham Pharmacia Biotech)according to the manufacturer’s specifications.
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