Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major

Coelho, Adriano C.; Beverley, Stephen M.; Cotrim, Paulo C.

doi:10.1016/s0166-6851(03)00162-2

Cited by 123 publications

(90 citation statements)

References 38 publications

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“…The Cos-Seq technique could be used with these new molecules to pinpoint their putative targets, which could be helpful for further drug improvements. Some of the genes identified by Cos-Seq are clearly drug-resistance genes, however, such as the ABC transporters MRPA and PRP1, whose implications in SbIII and PTD resistance, respectively, are well established (18,40).…”

Section: Discussionmentioning

confidence: 99%

“…S9 and Table S3). The gene encoding the ABC transporter pentamidine resistance protein 1 (PRP1), which is known to confer PTD resistance (18), was enriched as part of cosmid LinJ.31b, and its role in resistance was confirmed by cosmid recombineering (Fig. 5C).…”

Section: High-throughput Screening Of the Five Major Antileishmanial mentioning

confidence: 99%

“…Gain-of-function screening using genomic cosmid libraries is also a proven approach to studying drug resistance in Leishmania (12). Cosmid-based functional cloning was first implemented for studying lipophosphoglycan biosynthesis (13) and later successfully applied to study nucleoside transport (14,15) and drug resistance (16)(17)(18)(19)(20)(21). The technique has proven powerful, albeit with limitations; for instance, it is not easily amenable to high-throughput screening, because clones require individual characterization, and is biased toward the selection of cosmids conferring dominant phenotypes, leaving out less conspicuous candidates.…”

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confidence: 99%

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Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

Gazanion

Fernández-Prada

Papadopoulou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.

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Section: Discussionmentioning

confidence: 99%

Section: High-throughput Screening Of the Five Major Antileishmanial mentioning

confidence: 99%

mentioning

confidence: 99%

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Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

Gazanion

Fernández-Prada

Papadopoulou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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“…Se han identificado otros fenotipos asociados con la resistencia a metales y la resistencia cruzada (50,53,(58)(59)(60)68,70). La localización de los genes responsables de la resistencia a drogas también ha sido posible con mutaciones puntuales que llevan a pérdida (69) o ganancia de la función (57,58).…”

Section: Biosíntesis De Glicoconjugados Los Mutantes Deunclassified

Manipulación genética y el estudio del parásito protozoario Leishmania.

Cortázar

Walker

2004

biomedica

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Durante los últimos 15 años se ha dado paso al entendimiento de muchos aspectos de la genómica funcional de Leishmania gracias a los avances en la metodología de transfección de ADN dentro de la célula de este protozoario, la eliminación y la complementación de genes por medio de recombinación homóloga y las estrategias para la selección de células transfectadas. Estos acercamientos tienen el potencial de brindar información sobre la expresión génica y la función de las proteínas en el contexto del parásito intacto. Dado que el genoma de Leishmania muestra una carencia acentuada de los factores conocidos de iniciación de la transcripción y que la expresión génica está regulada casi completamente a nivel postranscripcional (a través del empalme de los ARNm y los mecanismos que involucran el procesamiento diferencial de la región no traducida 3' del ARNm (3'UTR), la transfección génica representa una herramienta útil para la identificación y el análisis funcional de los genes de interés así como de los mecanismos que dirigen su regulación. El desarrollo de los sistemas de manipulación genética también ha abierto nuevos horizontes para la identificación de genes esenciales involucrados en la virulencia, la supervivencia intracelular y la resistencia a drogas de Leishmania, así como para la validación de proteínas específicas del parásito como nuevos blancos quimio e inmunoterapéuticos. En esta revisión presentamos los avances más recientes en el campo de la manipulación genética en Leishmania, los cuales permiten análisis estructurales, funcionales y de fenotipo, por medio de la eliminación y complementación génica a través de la transfección transitoria o permanente de genes en este parásito.Palabras clave: Leishmania, transfección de ADN, expresión génica, eliminación y complementación génica, genómica funcional. Genetic manipulation and the study of the protozoan parasite LeishmaniaDuring the last 15 years, many aspects of the functional genomics of Leishmania have been revealed due to advances in DNA transfection, gene disruption and complementation through homologous recombination, and efficient strategies for the selection of transfected cells. These strategies have provided information about gene expression and protein function in the context of the intact parasite. The genome of Leishmania shows a marked deficiency of known transcription initiation factors, and gene expression is regulated almost entirely at the posttranscriptional level through trans-splicing of mRNAs and novel control mechanisms involving differential processing of 3' -untranslated regions (3'-UTRs) of mRNAs. Therefore, gene transfection represents a useful tool for the identification and functional analysis of genes of interest as well as the mechanisms that direct their regulation. The development of genetic manipulation systems has provided opportunities for the study of genes involved in virulence, intracellular survival and drug resistance of Leishmania, as well as for the functional validation of specific parasite proteins as new ch...

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“…Este gene codifica o transportador de membrana PRP1 (Pentamidine Resistance Protein), da superfamília de transportadores ABC (ATP Binding Cassette), e é capaz de conferir resistência a pentamidina às células selvagem do parasita após trasnfecção e super expressão gênica (Coelho et al, 2003;Coelho et al, 2006;Coelho et al, 2007).…”

Section: 2unclassified

Identificação e caracterização cromossomal de 9 loci de Leishmania (L.) major relacionados com resistência a inibidores da via de biossíntese do ergosterol

Camizotti¹

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Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major

Cited by 123 publications

References 38 publications

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

Manipulación genética y el estudio del parásito protozoario Leishmania.

Identificação e caracterização cromossomal de 9 loci de Leishmania (L.) major relacionados com resistência a inibidores da via de biossíntese do ergosterol

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