Cotrim

SUMMARY

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets or partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

KEYWORDS: Leishmania major; Pentamidine; Drug resistance; Gene transfection; Overexpression; Gene mapping; Transposable element.

INTRODUCTION

Leishmania is a flagellated protozoan and the causative agent of lcishmaniasis, an human infection that can develop as cutaneous, mucocutaneous, or visceral lesions according to the mononuclear phagocyte system infected, parasite species or host susceptibility2.12.

Despite recent developments in the chemotherapy for lcishmaniasis6, effective drug treatment remains difficult because of the many combinations of syndromes, the resistance of Leishmania species and the lack of adequate clinical trials. Antimony, the most effective agent to treat the disease is difficult to administer, present potential toxic effects and are considered expensive for large scale OcId regiments.

Few other antileishmanial drugs have been as extensively used as antimonials for treatment of lcishmaniasis. Amphotcricin B and Pcntamidine (PKN) have generally been chosen as parcntcral alternatives for human cases of poor response to the antimonial regimen. In spite of their toxic renal effects and the little knowledge of their mode of action in the parasite, these two agents are now being used with the benefit of new formulations or dosage regimens12,18

A better understanding of the mode of action of these agents would bring new insights into the antilcishmanial chemotherapy. From this perspective, the identification of parasite gcnomic loci involved in the development of resistance to these compounds might be of great value. Such approach is possible through the use of molecular techniques such as gene lransfcction, which employs specific shuttle vectors capable of replication within the parasite, as well as in bacteria. For instance, the vector cLHYG accepts up to 40 kb inserts of Leishmania genomic DNA and allows autonomous replication of molecules at a high copy number9,15,20. In previous work we demonstrated that resistance-related genes carried by cLHYG can be overcxpressed under drug pressure, yielding specific drug resistance phenotypes in L. major Friedlin Al strain (LmFAl)5.

Here we describe the use of this overcxpression/sclection approach in the presence of PEN, and the mapping of isolated inserts aiming at functional gene identification.

MATERIAL AND METHODS

Parasite strains, cultures and drugs: Leishmania major strain Friedlin Al (LmFAl) is a clonal avirulent line derived from the Friedlin Vl line (MHOM/1L/80/ Friedlin) after multiple passages in vitro1. Cells were grown in M199 media11, transfectcd by electroporation and plated on M199 semisolid media5,14, containing Pentamidinc (PEN), obtained from Sigma Chemical Company (St. Louis, MO). For translations of cosmids into Leishmania, plates contained 40 µg/ml of hygromycin B (HYG) were used for selection of transfcctcd lines. To identify cosmid-bearing lines exhibiting PKN resistance, 106 wild type or cosmid-library transfcctcd cells were plated on 100 mm M199 plates, containing increasing concentrations of the selective drug of interest. Macroscopic colonies were counted after 10-15 days of incubation, and recovered into M199 medium holding appropriate concentrations of selective drug.

Cosmid libraries and transfection: A library containing 30-40 kb inserts of Fricdlin Vl gcnomic DNA were constructed into the E. coli-Leishmania cLHYG shuttle vector as described20. Maxi preparation of cosmid library DNA was prepared by SDS/alkali lyscs plus PRG precipitation and sterile DNAs were transfected into I-riedlin Al lines by clcctroporation, using 14-40 Mg DNA per independent transfection and plating on M199 semi-solid media containing 40 Mg/ml HYG5,9,14,20.

Analysis of PEN resistance: PKN resistance of cosmid transfcctcd cells were performed after DNA ovcrexpression by successive passages in increasing concentrations of HYG up to 500 µg/ml5. Parasite numbers were determined using a Coulter Counter (model ZBI) after 2-3 days of incubation at 26 °C. The inhibition concentration for 50% inhibition (IC50) was dclined as that drug concentration which resulted in a 50% decrease in cell number, measured at the time when control cultures lacking drug had reached late log phase (typically less than 10^sup 7^/mI)11. Bxpcrimenl variations occurring during the tests were controlled by statistical tests for drug resistance utilized the parameter of fold resistance, defined as the average ratio of the experimental cell line IC^sub 50^ divided by the wild-type IC^sub 50^ measured in the same experiment, over (n) independent experiments.

Molecular techniques: Cosmid DNA was recovered from a liquid culture with 10^sup 7^ Leishmania colls by an alkalinc-SDS lyses plus PEG precipitation, as described9,10,20. DNA was then transformed in E. coli (DH5-a strain) and isolated from these cells by SDS/alkali-phcnol extraction and ethanol precipitation. Cosmid DNAs were first mapped by total digestion with restriction enzymes that have no sites within cLHYG vector DNA, like HindIII or A'coRV. Deletions were obtained by partial digestion with 0.1U of HindIII per pg of cosmid DNA, followed by sclf-ligation also at partial conditions5.

Nucleotide sequencing strategy and reactions: To determine and facilitate the nuclcotide sequence of the gene related with PEN resistance, random transposon insertion pools using the mosK mariner transposable clement21. For sequencing reactions, a PCR-bascd reaction kit was done with Thermo Sequenasc fluorescent labeled primer cycle sequencing kit with 7-dcaza-dGTP (Amersham Pharmacia). DNA sequencing was done on an ALF Express System (Amersham Pharmacia) automated sequencing. Nucleotide sequence of DNA inserts containing the mariner mosK were done using the 5' and 3' ends of the transposable element as primers island with primers directed to these extremities: mosK F 5'-CCGAGAGAGATGGGAAAAATG-3' and mosK R 5'-GGTTGACACTTCACA AGGTC-3'.

Analysis of the sequence was performed using the DNASTAR software (Madison, Wl) and Clone Manager 5(TM). We also used for confirmation and final deduction of the nuclcotide sequence of the coding region; comparison with (he data from the Leishmania major Genome Project at Sanger Center Web Server (www.sangcr.ac.uk).

RESULTS

Selection of cosmids containing loci capable of render Leishmania resistant to PEN was carried out after platting a LmPAl transfcctant cosmid library with increasing concentrations of PRN (8 to 27 µM). The number of colonies obtained was compared to that of a control consisting of parental or cLHYG transfcctcd LmHAl cells as previously described (Table I). Control experiments yielded parasite colonies up to 17 µM PRN but not on higher concentrations. In contrast, LmRA 1 cosmid library transfcctants yielded 20 colonies on 23 µM PEN. No colonies were observed at 27 µM PRN. The cosmid DNAs from 7 colonies showing differential PRN survival were recovered and analyzed by restriction digestion. Wc were able to isolate two cosmid populations (cosPRNl-A and cosPRN1-B) presenting inserts related to the same locus, which was named PENI.

Both cosmid DNAs were transfected back into LmFAl cells to confirm their role in PBN resistance, and we could observe that transtcctcd cells present statistical significant levels of PEN resistance when compared to LmFAl wild type cells4.

The PENI locus was mapped by restriction analysis. The DNAs of cosPENl-A (with an insert of approximately 25 kb), and cosPENl-B (with approximately 32 kb) were digested with five restriction enzymes. The representation of Hind III prototype cosmid maps is shown in Fig. 1 A. It is noteworthy that cosPENl-A is a "natural" deletion of cosPENl-B, being entirely contained within the latter. In order to limit the resistance phcnotypc locus (Fig. IA), the smaller cosPENl-A was used to produce deletions by partial digestion with Hindlll, followed by self-ligation. The deletion cosPENl-A [Delta]HindIII, with an insert of approximately 15 kb, was the only construct that remained able to render PEN resistance after transfcction (Fig. 1 A). A second set of deletions using EcoKV partial digestion was generated from cosPENl-A AHincllll, and none confers PEN resistance after transfection, suggesting that the gene contains an internal EcoRV site. A 5 kb Sail fragment from cosPENl-A [Delta]Hind III DNA was subcloncd into the shuttle vector pSNBR (Fig. IA)3. The resulting construct was named pSNBR/5 kb Sail and did not confer PEN resistance after transfection into parental LmFAl cells4.

Primer island sequencing was used to characterize the construct pSNBR/5 kb Sail. The mariner inosK in vitro transposition system was used to generate random transposon insertion into the target DNA21. The transposable element used (inosK) carries the Tn903 kanamycin resistance gene and allows the selection of insertion events following transformation into E. colt. Initially, mosK insertions were mapped by HindiU restriction analysis in order to determine the position of the transposon insertions along the 5 kb plasmid DNA. The presence of two Hinclll] sites within the target DNA and one Hindlll site within the transposable clement allowed the discrimination between insertion events that occurred into the cloned fragment and those in the vector backbone DNA (Pig. IB).

Specific primers located at the 5'- and 3'-ends of the transposon were used in the sequencing of insertion events (Pig. 1 B). The sequence generated from mosk insertions was assembled into a fragment of 4,787 bp corresponding to the total DNA insert of the construct pSNBR/5 kbSa/l (GcnBank (TM) accession number AY251609). The S'-cnd of the insert contains an open reading frame (OKH) of 3,696 nucleotidcs, which codes for a 1,232 amino acids (Pig. IB), and was named Pentamidine Resistance Protien-1 (PRPl)4.

DISCUSSION

Southern blotting analysis revealed that the PENl locus is clearly distinct from those loci previously identified (data not shown), which are involved in the LmFAl resistance to antifolates, nucleosidcs and sterol biosynthesis inhibitors5. Comparison of amino acid sequences in GenBank(TM) through BLAST analysis' revealed a significant identity with proteins belonging to the ABC (ATP-Bjnding Cassette) transporters superfamily. In many organisms these proteins are involved in the transport of a variety of compounds through biological membranes13. The ABC transporters superfamily includes the P-glycoprotcin (PGP) described in L. major and in other Leishmania species such as L. tarentolae and L. tropica, as well as in other trypanosomatids such as Trypanosoma cruzi3,8,16,19. The nuclcotide identity between PGPs of these organisms and the PRPl described here varies from 30 to 40%4. The comparative analysis also suggested that the predicted gene is not entirely contained within pSNBR/5 kb Sail plasmid insert due to a missing 5' portion. all PGPs from other trypanosomatids are coded by ORHs that are larger than the 3,696 bp found for PRPl. This confirms our observation that the pSNBR/5 kb Sail construct was not able to confer PFlN resistance to LmFAI cells after transfection4.

The ABC transporter PGPA renders Leishmania resistant to heavy metals (arsenite and aniimonials). Subcellular localization of PGPA in Leishmania revealed that the protein is present in intracellular membranes, suggesting that PGPA confers resistance to arsenite and antimonials by sequestration of metals into vesicles that could be exocytosed17. The elucidation of the role of the PRPl in PRN sensitivity and/or resistance in Leishmania will not only contribute to the study of the ability of this organism to evade chemotherapy, but also to the design of effective treatments.

With the conclusion of the Leishmania genome sequencing project in a near future, the use of this new methodology for mapping and interrupting Leishmania gene/loci, can contribute enormously for gene identification as a practical tool for this new functional genomic era.

RESUMO

Mapeaniento de um gene de Leishmania major que confere resistencia a pentamidina por delecao e inscrcao de elementos transposicionais

A Pentamidina (PEN) e um composto alternativo para o tratamento de pacienles com leishmaniose que apresentam resistencia ao antimonio, cujo alvo celular continua incerto. Uma abordagem para se identificar provaveis alvos seria a identificacao e super-cxpressao de genes capazes de mediar resistencia a PEN. A partir de uma genotcca construida com o DNA de Leishmania major cm um vctor - cosmidio que se dcsenvolve tanto em bacterias como nas celulas do parasita, isolamos um locus que apos transfeccao e capaz de produzir resistencia a PEN as celulas do parasita. Almejando o mapeamento desse locus de leishmania, o inserto clonado ncsse cosmidio foi dclctado alravcs de duas digestoes parciais succssivas com enzimas de restricao, seguida de transfeccao em celulas selvagens, super-expressao genica, inducao e testes funcionais na prescnca de PEN. Para determinai' o gene de Leishmania relacionado corn a resistencia a PEN, o sequenciamcnto de nucleotideos foi executado apos insercao de elementos transposicionais de Drosophila melanogaster no interior do inserlo dclctado para atuar como 'ilhas de iniciadorcs'. Descrevemos aqui o mapcamento desse locus, apos a inscrcao transpositional, que alcm de facililar o scqucnciamcnto dc nucleolidcos de grandes fragmentes de DNA, pcrmile umu rapida idcnliCicacao do gene rclacionado com esse fcnolipo. Hxpcrimentos posted ores revelaram ncste locus a prcscnca do gene de uma Glicoproteina-P de membrana, cujo papcl no metabolismo na Leishmania esta scndo analisado.

ACKNOWLEDGEMENTS

This work was supported by grants from The Pew Charitable Trust; TDFi-WHO; FAPESP (95/9305-9, 97/00541-7, 02-09562-7; CNPq (to PCC and ACC) and LIM-48-FMUSP.

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Received: I October 2003

Accepted: 10 March 2004

Adrian C. COELHO(1), Luiz R. O. TOSI(2) & Paulo C. COTRIM(1)

Abbreviations: ABC, ATP-binding cassette; kb, kilobase; LmFA1, Leishmania major Friedlin A1 strain; ORF, open rending frame; PEN, Pentamidine; PGP, P-glycoprotein.

(1) Institute de Meclicina Tropical, Universidade de Sao Paulo, Sao Paulo, SP, Brasil.

(2) Departamento de Biologia Celular e Molecular e Bioagentcs Patogenicos, Faculdacle de Mcdicina de Ribcirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brasil.

Correspondence to: Paulo C. Cotrim, lnsiitulo de Mcdicina Tropical, USP. Av. Dr. Eneas de Carvalho Aguiar 470; 4° andar, 05403-900 Sao Paulo, SP, Brasil. Tel. 5511-3066-7024; fax. 5511-3062-3622, E-mail: pccotrim@usp.br

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