Mitochondrion 18 (2014) 7–11

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Mutation of the mitochondrial large ribosomal RNA can provide pentamidine resistance to Saccharomyces cerevisiae Ş. Tomris Örs, Emel Akdoğan, Cory D. Dunn ⁎ Department of Molecular Biology and Genetics, Koç University, Sarıyer, İstanbul 34450, Turkey

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Article history: Received 10 May 2014 Received in revised form 24 July 2014 Accepted 13 August 2014 Available online 21 August 2014 Keywords: Trypanosome Pneumocystis Mitochondrial translation Anti-microbial Antibiotic

a b s t r a c t Pentamidine is used to treat several trypanosomal diseases, as well as opportunistic infection by pathogenic fungi. However, the relevant targets of this drug are unknown. We isolated dominant mutations providing pentamidine resistance to Saccharomyces cerevisiae, one of which was localized to mitochondrial DNA. Nextgeneration sequencing revealed alteration of a widely conserved base at the peptidyl transferase center of the mitochondrial 21S ribosomal RNA. Our results provide a potential rationale for the toxicity of this drug to patients, and we discuss whether blockade of mitochondrial translation is the mechanism by which pathogenic fungi or protists are killed by pentamidine. © 2014 The Authors. Elsevier B.V. and Mitochondria Research Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction Pentamidine is used to treat infection by trypanosomatids or by the fungus Pneumocystis jirovecii (Soeiro et al., 2013). However, the precise molecular target(s) of this drug are unknown (Alsford et al., 2013; Baker et al., 2013; Werbovetz, 2006). Much evidence suggests that pentamidine can affect mitochondrial function in organisms susceptible to killing by pentamidine. For example, pentamidine can rapidly reduce the mitochondrial electrochemical potential (ΔΨmito) across the inner membrane of trypanosomid mitochondria (Lanteri et al., 2008; Vercesi and Docampo, 1992). Pentamidine also prompts damage to or loss of the kinetoplast DNA (Hentzer and Kobayasi, 1977; Shapiro and Englund, 1990), which encodes the machinery used for parasite oxidative phosphorylation. In Saccharomyces cerevisiae, pentamidine treatment leads to an inhibition of mitochondrial translation (Zhang et al., 2000), and overexpression of Pnt1p, a polypeptide involved in export of mtDNA-encoded protein domains from the mitochondrial matrix (He and Fox, 1999) can provide a measure of pentamidine resistance (Ludewig and Staben, 1994). Importantly, pentamidine causes significant patient toxicity (Soeiro et al., 2013), and a large-scale comparison of several mitochondrial parameters upon treatment of a myoblast cell line with hundreds of different drugs suggested that myopathy

Abbreviations: mtDNA, mitochondrial DNA; EtBr, ethidium bromide; BAM, binary sequence alignment/map format; rRNA, ribosomal ribonucleic acid. ⁎ Corresponding author at: Department of Molecular Biology and Genetics, Koç University, Rumelifeneri Yolu, Sarıyer, İstanbul 34450, Turkey. Tel.: +90 212 338 1449; fax: +90 212 338 1559. E-mail address: [email protected] (C.D. Dunn).

associated with pentamidine treatment might be linked to mitochondrial dysfunction (Wagner et al., 2008). By taking a genetic approach to identify potential mitochondrial targets of pentamidine, we discovered that mutation of the mitochondrial ribosome can provide pentamidine resistance. 2. Materials and methods 2.1. Yeast strains and culture conditions Yeast media and genetic techniques are as described in Adams et al. (1997). YEPGE medium contains 3% glycerol and 3% ethanol. Gene disruptions were performed as detailed in Sikorski and Hieter (1989). Strains were cultured at 30 °C. Pentamidine isothionate (Sigma, St. Louis, MO) was added at the indicated concentrations. Pentamidine in aqueous solution is unstable (Martindale et al., 1972), leading to potential variation in the effective drug concentration during plate assays. We also noted that pentamidine is sensitive to inactivation in molten agar at high temperature, and so one should quickly cool media to which pentamidine has been added to minimize variability in pentamidine efficacy between plate batches. Plates were also protected from exposure to light before use. Pentamidine-resistant mutants were isolated by culture in liquid YEPGE containing pentamidine isothionate for ~9 h before plating at high density to solid medium also containing drug. Where indicated, ethidium bromide (EtBr; Thermo-Fisher Scientific, Waltham, MA) was used at 25 μg/ml to destroy mtDNA. Serial dilution assays were performed as in Garipler et al. (2014). Cytoduction was accomplished by patching kar1 cyh2 ρ− strain CDD723 to either WT strain CDD725 or PNT3-1 strain CDD726, followed by spreading the

http://dx.doi.org/10.1016/j.mito.2014.08.004 1567-7249/© 2014 The Authors. Elsevier B.V. and Mitochondria Research Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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Fig. 1. An allele providing pentamidine resistance is inherited in a non-Mendelian fashion. (A) PNT3-1 provides dominant pentamidine resistance on non-fermentable medium. Diploid strains BMA64 (WT) and CDD693 (PNT3-1) were cultured in YEPD medium, then subjected to serial dilution and either spotted upon YEPGE medium and incubated for 2 d or, rather, spotted upon YEPGE to which pentamidine was added at a concentration of 10 μg/ml, then incubated for 7 d. (B) All four spores produced from a PNT3-1 diploid are pentamidine-resistant. Diploid strains BMA64 (WT) and CDD693 (PNT3-1) were sporulated. Resultant haploid progeny proliferating on YEPD medium were struck to YEPGE and incubated for 2 d or struck to YEPGE containing pentamidine added to 10 μg/ml and incubated for 3 d.

mated cells on YEPD at a density at which single colonies were apparent, then replica-plating to YEPGE medium containing 3 μg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO) to select for cytoductants. Cytoduction was further confirmed by examining nuclear-background-specific markers. Genotypes of all strains used in the course of this study, along with details of their construction and oligonucleotide sequences, are provided in Supplementary Table 1.

25 bp) were sequenced on the Illumina MiSeq platform to a read length of 150 bp. Sequencing reads have been deposited into the Sequence Read Archive of the National Center for Biotechnology Information under accession number PRJNA246119. Bioinformatic analysis of genomic sequence to identify single nucleotide polymorphisms was performed as in Mutlu et al. (2014), and VarScan2 output is provided as Supplementary Table 2.

2.2. Genomic DNA isolation and next-generation sequencing

3. Results and discussion

Genomic DNAs from strain BMA64, strain CDD693, and a pool of wild-type haploids derived from strain CDD682 were isolated from saturated cultures essentially as in Looke et al. (2011). Paired-end library preparation was performed at the European Molecular Biology Laboratory Genomics Core Facility (Heidelberg, Germany) using the Illumina protocol for preparation of genomic DNA sequencing libraries. Paired-end library fragments averaging 300 bp (standard deviation of

The recovery of dominant mutations providing drug resistance to S. cerevisiae is one avenue that may successfully reveal either the protein or the pathway targeted by that drug (Heitman et al., 1991). Among numerous scenarios for how alteration of a gene product might provide dominant resistance to a chemical agent, a mutation might block drug access to the catalytic site of an enzyme, thereby preserving functionality. Under these circumstances, localizing the mutation would directly

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Fig. 2. PNT3-1 is linked to the mitochondrial genome. (A) Destruction of mtDNA by EtBr prevents transmission of PNT3-1 to a diploid cell. Haploid strains were permitted to mate on YEPD medium, followed by culture on SD medium lacking histidine, then selection on SC medium lacking uracil (SC-Ura) to obtain diploids. From the SC-Ura plate, diploids were struck to YEPGE and incubated for 2 d or struck to YEPGE containing pentamidine added at 10 μg/ml and incubated for 4 d. Genotypes and treatment conditions are as indicated, and the identities of mated strains are as follows. Top wedge, CDD706 mated to CDD708. Left wedge, CDD707 mated to CDD708. Right wedge, CDD707 mated to CDD709. Bottom wedge, CDD706 mated to CDD709. (B) Pentamidine resistance is maintained following cytoduction of the PNT3-1 allele. Strains CDD727 and CDD728 were treated as in Fig. 1A and incubated for 4 d (YEPGE) or 7 d (YEPGE + pentamidine).

lead to identification of the drug target. However, identification of dominant mutations in S. cerevisiae has typically required arduous mapping or genomic library construction (Forsburg, 2001). Excitingly, with the advent of next-generation sequencing, discovery of dominant alleles need not be labor intensive. Based on a previous report suggesting that pentamidine resistance can be mediated by dominant alleles (Hatfield et al., 1991), we isolated spontaneously arising mutations in diploid cells that would provide resistance to pentamidine added at 10 µg/ml to non-fermentable YEPGE medium, a concentration previously demonstrated to severely inhibit the proliferation of cells cultured under conditions where oxidative phosphorylation was required (Lanteri et al., 2004; Ludewig et al., 1994). Among the candidates isolated, the PNT3-1 allele provided clear pentamidine resistance (Fig. 1A) and is further described in this work. Another mutation conferring pentamidine resistance yet not localized to PNT3 will be described elsewhere. With the initial intention of performing bulk segregant analysis, we sporulated mutant diploids and tested haploid progeny for pentamidine resistance. We were surprised to find that all four progeny spores produced by meiosis of diploids carrying the PNT3-1 mutation were equally resistant to pentamidine (Fig. 1B), which is inconsistent with Mendelian inheritance of the PNT3-1 allele. Due to the previous reports linking pentamidine's toxic effects to mitochondrial function, we suspected that the mitochondrial genome (mtDNA) might harbor the PNT3-1 allele. To examine this possibility, we asked whether destruction of mtDNA in PNT3-1 haploid cells using

ethidium bromide (EtBr) (Goldring et al., 1970) would prevent transmission of pentamidine resistance to a diploid strain following mating. Indeed, diploids generated by mating EtBr-treated PNT3-1 cells to wild-type (WT) cells were not pentamidine-resistant, in contrast to diploids which were instead generated by mating untreated PNT3-1 cells to EtBr-treated WT cells (Fig. 2A). To further test linkage of the PNT3-1 allele to the mitochondrial genome, we used cytoduction to transfer mtDNA from a haploid PNT3-1 strain to a new nuclear background. Although the recipient nuclear background used during cytoduction is innately more resistant to pentamidine than the W303 background donor (Supplementary Fig. 1), those yeast cytoduced with mtDNA carrying PNT3-1 exhibited a two to three fold increase in pentamidine resistance over yeast cytoduced with WT mtDNA (Fig. 2B and Supplementary Fig. 1). To find the mtDNA alteration providing pentamidine resistance, we sequenced the entire genomic DNA of our starting strain, the derivative PNT3-1 strain, and a control pool of WT haploids. Indeed, a single point mutation, converting a conserved cytosine at position 1955 to a thymine, was found within the gene encoding the mitochondrial 21S ribosomal RNA (rRNA) of the PNT3-1 strain. No other mtDNA sequence variations not found in the parental strain were uncovered in the PNT3-1 isolate. Sanger sequencing confirmed that this change existed in the pentamidine-resistant strain resulting from cytoduction of the PNT3-1 allele (Fig. 3A). C1955 is localized to “domain V” within the secondary structure map (Amunts et al., 2014) of the 21S rRNA (Fig. 3B) at

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Fig. 3. The PNT3-1 allele alters a conserved base found within many mitochondrial and prokaryotic large rRNAs. (A) C1955 of the S. cerevisiae mitochondrial 21S rRNA gene is changed to a T in PNT3-1 mtDNA. A portion of 21S rRNA from strains CDD727 (WT) and CDD728 (PNT3-1) was amplified by primers 566 and 567, then examined by Sanger sequencing. Chromatograms demonstrating the relevant sequence change are provided. (B) PNT3-1 is localized to the portion of the 21S rRNA that provides peptidyl transferase activity. 21S rRNA secondary structure at domain V (Amunts et al., 2014) is illustrated, and the location of PNT3-1 is indicated (red arrowhead). Red coloration denotes the portion of the 21S addressed in (C). (C) C1955 of the S. cerevisiae mitochondrial large rRNA is conserved within several prokaryotic and prokaryote-derived ribosomes, but not among kinetoplast-encoded large rRNAs. The position of C1955 within S. cerevisiae mitochondrial 21S rRNA and orthologous sequences is marked with a black arrowhead. Sequences displayed are recovered from the following Genbank entries: S. cerevisiae mitochondrial 21S rRNA, NC_001224.1; E. coli 23S rRNA, NC_000913.2 (sequence shown is identical in all seven 23S rRNA operons); S. aureus 23S rRNA, NC_002952.2 (sequence shown is identical in all five 23S rRNA operons); P. jirovecii large rRNA encoded by rn1 (Svedberg value of large ribosomal subunit unknown), NC_020331.1; Homo sapiens mitochondrial 16S rRNA, NC_012920.1; T. brucei mitochondrial 12S rRNA, X02547.1; L. major mitochondrial 12S rRNA, FJ349263.1.

a stretch of bases that are highly conserved at the peptidyl transferase site of many prokaryotic and mitochondrial large rRNAs (Fig. 3C). The orthologous base, C2055 (under the standard naming convention), is found within the Escherichia coli 23S rRNA at the peptidyl transferase domain (Dunkle et al., 2010), and similarly to S. cerevisiae, E. coli translation is inhibited by pentamidine under certain experimental conditions (Amos and Vollmayer, 1957; Prouty and Goldberg, 1972). Furthermore, this base is conserved in the gram-positive bacteria Staphylococcus aureus, in which protein synthesis is also inhibited by pentamidine (Gale and Folkes, 1967). Interestingly, the presence of a cytosine at this position, rather than the adenine found at the analogous

position of eukaryotic cytosolic ribosomes, has been suggested to dictate the efficacy of several antibiotics that directly bind to bacterial and to mitochondrial ribosomes (Dunkle et al., 2010). Importantly, C1955 of the S. cerevisiae 21S rRNA and nearby bases are also conserved in the large mitochondrial ribosomal subunit of P. jirovecii, suggesting that this organism's demise during pentamidine treatment might result from a blockade of mitochondrial translation. Finally, conservation of C1955 is apparent in human mitochondrial ribosomes, raising the possibility that the documented toxicity of pentamidine to patients may result, at least in part, from an inhibition of mitochondrial protein synthesis.

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Might pentamidine, as in S. cerevisiae, block mitochondrial protein synthesis in parasitic protozoa, thereby making the mitochondrial ribosome a relevant target of this drug among trypanosomids? Trypanosomid mitochondrial ribosomes are highly divergent from other eukaryotes, both at the overall structural level (Zikova et al., 2008) and at the rRNA sequence level (de la Cruz et al., 1985a, 1985b; Sloof et al., 1985). In fact, S. cerevisiae C1955 is not conserved within the 12S large mitochondrial rRNA of Trypanosoma brucei, nor within the Leishmania major mitochondrial 12S rRNA (Fig. 3C), yet both organisms can be killed by pentamidine. Moreover, dyskinetoplasic T. brucei, lacking the mitochondrial genome and its encoded ribosomes, remains quite sensitive to pentamidine (Damper and Patton, 1976; Gould and Schnaufer, 2014). Finally, since a single base change near the peptidyl transferase site of the mitochondrial ribosome can reduce the pentamidine sensitivity of S. cerevisiae, we might expect that such a mutation might also be commonly achievable within a large population of trypanosomes under high selection for pentamidine resistance across many millions of patients. Yet, pentamidine resistance apparently does not easily evolve in the wild (Alsford et al., 2013; Bray et al., 2003), and such a 12S rRNA mutation providing pentamidine resistance has not been reported for Trypanosoma or Leishmania. Taken together, if the mitochondrial ribosome is a target of pentamidine in trypanosomids, it is certainly not the only relevant target; the immediate effects of pentamidine on ΔΨmito (Lanteri et al., 2008; Vercesi and Docampo, 1992) may be of equal or greater importance. Our identification of a pentamidine-resistance allele within the mitochondrial 21S rRNA of S. cerevisiae suggests a potentially direct, physical interaction of this drug with the mitochondrial translational apparatus, as previously confirmed for other drugs that directly bind prokaryotic or prokaryote-derived ribosomes and whose efficacy can be altered by base changes to the large rRNA. However, directed biochemical assays are clearly required to demonstrate pentamidine binding to specific regions of the mitochondrial or bacterial ribosome. Moreover, saturated mutagenesis of the S. cerevisiae mitochondrial ribosome, accomplished by manganese treatment (Putrament et al., 1973) or by the use of error-prone DNA polymerase (Foury and Vanderstraeten, 1992) might identify additional alleles within the 21S rRNA whose location might shed further light on the role of the mitochondrial ribosome in pentamidine sensitivity. Considering the similarity of the human mitochondrial 16S rRNA and the yeast 21S rRNA at the location of the PNT3-1 mutation, further investigation focused upon the possible effect of pentamidine on human mitochondrial translation seems imperative. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2014.08.004. Acknowledgments We thank Geneviève Dujardin for providing the CK01 strain, and we thank Gülayşe İnce Dunn, Görkem Garipler, and Alena Zíková for critical review of the manuscript. Support for this work was provided by a European Molecular Biology Organization Installation Grant to C.D.D. and by Koç University's College of Sciences. References Adams, A., Gottschling, D., Kaiser, C., Stearns, T., 1997. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Plainview, NY. Alsford, S., Kelly, J.M., Baker, N., Horn, D., 2013. Genetic dissection of drug resistance in trypanosomes. Parasitology 140, 1478–1491. Amos, H., Vollmayer, E., 1957. Effect of pentamidine on the growth of Escherichia coli. J. Bacteriol. 73, 172–177. Amunts, A., Brown, A., Bai, X.C., Llacer, J.L., Hussain, T., Emsley, P., Long, F., Murshudov, G., Scheres, S.H., Ramakrishnan, V., 2014. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489. Baker, N., de Koning, H.P., Maser, P., Horn, D., 2013. Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol. 29, 110–118. Bray, P.G., Barrett, M.P., Ward, S.A., de Koning, H.P., 2003. Pentamidine uptake and resistance in pathogenic protozoa: past, present and future. Trends Parasitol. 19, 232–239. Damper, D., Patton, C.L., 1976. Pentamidine transport and sensitivity in brucei-group trypanosomes. J. protozool. 23, 349–356.

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de la Cruz, V.F., Lake, J.A., Simpson, A.M., Simpson, L., 1985a. A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae. Proc. Natl. Acad. Sci. U. S. A. 82, 1401–1405. de la Cruz, V.F., Simpson, A.M., Lake, J.A., Simpson, L., 1985b. Primary sequence and partial secondary structure of the 12S kinetoplast (mitochondrial) ribosomal RNA from Leishmania tarentolae: conservation of peptidyl-transferase structural elements. Nucleic Acids Res. 13, 2337–2356. Dunkle, J.A., Xiong, L., Mankin, A.S., Cate, J.H., 2010. Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc. Natl. Acad. Sci. U. S. A. 107, 17152–17157. Forsburg, S.L., 2001. The art and design of genetic screens: yeast. Nature reviews. Genetics 2, 659–668. Foury, F., Vanderstraeten, S., 1992. Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity. EMBO J. 11, 2717–2726. Gale, E.F., Folkes, J.P., 1967. Actions of pentamidine on the metabolism of Staphylococcus aureus. Biochim. Biophys. Acta 144, 467–470. Garipler, G., Mutlu, N., Lack, N.A., Dunn, C.D., 2014. Deletion of conserved protein phosphatases reverses defects associated with mitochondrial DNA damage in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 111, 1473–1478. Goldring, E.S., Grossman, L.I., Krupnick, D., Cryer, D.R., Marmur, J., 1970. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J. Mol. Biol. 52, 323–335. Gould, M.K., Schnaufer, A., 2014. Independence from kinetoplast DNA maintenance and expression is associated with multidrug resistance in Trypanosoma brucei in vitro. Antimicrob. Agents Chemother. 58, 2925–2928. Hatfield, C., Kasarskis, A., Staben, C., 1991. Pentamidine sensitivity and resistance in Saccharomyces cerevisiae as a model for pentamidine effects on Pneumocystis carinii. J. protozool. 38, 70S–71S. He, S., Fox, T.D., 1999. Mutations affecting a yeast mitochondrial inner membrane protein, Pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix. Mol. Cell. Biol. 19, 6598–6607. Heitman, J., Movva, N.R., Hall, M.N., 1991. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909. Hentzer, B., Kobayasi, T., 1977. The ultrastructural changes of Leishmania tropica after treatment with pentamidine. Ann. Trop. Med. Parasitol. 71, 157–166. Lanteri, C.A., Trumpower, B.L., Tidwell, R.R., Meshnick, S.R., 2004. DB75, a novel trypanocidal agent, disrupts mitochondrial function in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 48, 3968–3974. Lanteri, C.A., Tidwell, R.R., Meshnick, S.R., 2008. The mitochondrion is a site of trypanocidal action of the aromatic diamidine DB75 in bloodstream forms of Trypanosoma brucei. Antimicrob. Agents Chemother. 52, 875–882. Looke, M., Kristjuhan, K., Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCR-based applications. BioTech. 50, 325–328. Ludewig, G., Staben, C., 1994. Characterization of the PNT1 pentamidine resistance gene of Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 38, 2850–2856. Ludewig, G., Williams, J.M., Li, Y., Staben, C., 1994. Effects of pentamidine isethionate on Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 38, 1123–1128. Martindale, W., Blacow, N.W., Wade, A., Pharmaceutical Society of Great Britain, Department of Pharmaceutical Sciences, 1972. The Extra Pharmacopoeia: Incorporating Squire's “Companion”, 26th ed. Pharmaceutical Press, London. Mutlu, N., Garipler, G., Akdogan, E., Dunn, C.D., 2014. Activation of the pleiotropic drug resistance pathway can promote mitochondrial DNA retention by fusion-defective mitochondria in Saccharomyces cerevisiae. G3 4, 1247–1258. Prouty, W.F., Goldberg, A.L., 1972. Effects of protease inhibitors on protein breakdown in Escherichia coli. J. Biol. Chem. 247, 3341–3352. Putrament, A., Baranowska, H., Prazmo, W., 1973. Induction by manganese of mitochondrial antibiotic resistance mutations in yeast. Mol. Gen. Genet. 126, 357–366. Shapiro, T.A., Englund, P.T., 1990. Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. Proc. Natl. Acad. Sci. U. S. A. 87, 950–954. Sikorski, R.S., Hieter, P., 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27. Sloof, P., Van den Burg, J., Voogd, A., Benne, R., Agostinelli, M., Borst, P., Gutell, R., Noller, H., 1985. Further characterization of the extremely small mitochondrial ribosomal RNAs from trypanosomes: a detailed comparison of the 9S and 12S RNAs from Crithidia fasciculata and Trypanosoma brucei with rRNAs from other organisms. Nucleic Acids Res. 13, 4171–4190. Soeiro, M.N., Werbovetz, K., Boykin, D.W., Wilson, W.D., Wang, M.Z., Hemphill, A., 2013. Novel amidines and analogues as promising agents against intracellular parasites: a systematic review. Parasitology 140, 929–951. Vercesi, A.E., Docampo, R., 1992. Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca 2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ. Biochem. J. 284 (Pt 2), 463–467. Wagner, B.K., Kitami, T., Gilbert, T.J., Peck, D., Ramanathan, A., Schreiber, S.L., Golub, T.R., Mootha, V.K., 2008. Large-scale chemical dissection of mitochondrial function. Nat. Biotechnol. 26, 343–351. Werbovetz, K., 2006. Diamidines as antitrypanosomal, antileishmanial and antimalarial agents. Curr. Opin. Investig. Drugs 7, 147–157. Zhang, Y., Bell, A., Perlman, P.S., Leibowitz, M.J., 2000. Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae. RNA 6, 937–951. Zikova, A., Panigrahi, A.K., Dalley, R.A., Acestor, N., Anupama, A., Ogata, Y., Myler, P.J., Stuart, K., 2008. Trypanosoma brucei mitochondrial ribosomes: affinity purification and component identification by mass spectrometry. Mol. Cell. Proteomics 7, 1286–1296.

Mutation of the mitochondrial large ribosomal RNA can provide pentamidine resistance to Saccharomyces cerevisiae.

Pentamidine is used to treat several trypanosomal diseases, as well as opportunistic infection by pathogenic fungi. However, the relevant targets of t...
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