Department of Animal Health, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA1
Author for correspondence: Michael C. Justice. Tel: +1 732 594 3941. Fax: +1 732 594 6708. e-mail: michael_justice{at}merck.com
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ABSTRACT |
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Keywords: translation, elongation, sordarin specificity, protein synthesis
Abbreviations: eEF2, eukaryotic elongation factor 2; EF-G, prokaryotic elongation factor G
The GenBank accession numbers for the sequences reported in this manuscript are AF107286AF107291, AF292693 and AF248644.
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INTRODUCTION |
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The sordarins display varying levels of antifungal activity when tested against different species of pathogenic fungi (Dominguez et al., 1998 ; Herreros et al., 1998
). For example, sordarin and its glycone-substituted derivatives inhibit growth of species such as Ca. albicans, Candida glabrata, Candida tropicalis and Cryptococcus neoformans, while other species including Candida parapsilosis and Candida krusei are insensitive to high levels of these compounds. The activity of the sordarins has been demonstrated in whole-cell assays and in cell-free translation systems, consistent with the role of these compounds as protein synthesis inhibitors (Dominguez et al., 1998
).
In an effort to understand the molecular basis for the fungal specificity of sordarin and its selectivity amongst different species of fungi, we have cloned and characterized the eEF2s from various sordarin-sensitive and -insensitive species. Our results show that amino acid residues at three specific positions in fungal eEF2 are primarily responsible for the observed differences in sordarins spectrum. Moreover, we demonstrate that the corresponding residues in mammalian eEF2 are sufficient to confer a sordarin-insensitive phenotype to S. cerevisiae eEF2 and therefore account for sordarins fungal specificity.
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METHODS |
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Cloning and expression of fungal EFT2 genes.
The EFT2 genes encoding eEF2 from Candida species were cloned by PCR using synthetic oligonucleotides that were designed based on the published ORF sequence of Ca. albicans EFT2 (GenBank entry Y09664). The 5' primer contains a DraI restriction enzyme recognition sequence and the 3' primer has XhoI and MluI sites for subcloning purposes (oligonucleotide sequences for cloning and modification of the genes encoding the eEF2 proteins described in this manuscript are available on request). The Ca. glabrata EFT2 gene was cloned using primers based on the ORF of the S. cerevisiae EFT2 gene.
Genomic DNA from Ca. glabrata, Candida guilliermondii, Ca. krusei, Candida lusitaniae, Ca. parapsilosis and Ca. tropicalis species was prepared by standard procedures and purified with a QIAamp Tissue Kit (Qiagen) according to the manufacturers specifications. PCR products were amplified from genomic DNA with the synthetic oligonucleotides described above using Klentaq polymerase (Clontech) according to the manufacturers recommendations.
At least three independent PCR reaction products were obtained from genomic DNA from each of the Candida species. Following initial sequence identification of three identical EFT2 PCR products from each fungal species, the products were cloned into the vector pGEM-T Easy (Promega), followed by transformation into the Escherichia coli strain DH5 (Life Technologies). Plasmid DNA was prepared with Qiaspin columns (Qiagen) according to the manufacturers instructions and digested with appropriate restriction enzymes to verify the identity of the constructs. At least four clones of each of the Candida eEF2 genes in pGEM-T Easy were sequenced with an ABI Prism 377 DNA Sequencer according to the manufacturers recommendations (PE-Applied Biosystems). Sequences were analysed using Sequencher DNA analysis software (Gene Codes). These sequence data were compared with the original sequence obtained from the PCR products to verify the identity of each Candida species EFT2 obtained. The Candida EFT2 genes were excised as 5'-DraI to 3'-XhoI fragments from the pGEM-T Easy plasmids, gel-purified using QIAEX (Qiagen) and ligated into the expression vector YCpEFT2 (Justice et al., 1998
) digested with HpaI and XhoI. The final constructs contained the Candida species EFT2 genes expressed from the S. cerevisiae EFT2 promoter. All of the EFT2 clones described above contain about 25 nucleotides of PCR primer sequence at the 5' and 3' ends of the Candida species eEF2s and include the start and stop codons that were derived from either Ca. albicans EFT2 (Ca. lusitaniae, Ca. parapsilosis and Ca. tropicalis) or S. cerevisiae EFT2 (Ca. glabrata). The fungal EFT2 constructs were transformed into the S. cerevisiae
eft1
eft2 deletion strain YEFD12h. Transformants were selected for leucine prototrophy and subsequently plated on 5-fluoroorotic acid agar medium to select strains that have lost pURA3-EFT1 that encodes S. cerevisiae eEF2 while retaining the plasmid encoding the heterologous eEF2. For Ca. guilliermondii and Ca. krusei, primers were designed based on the consensus sequence of a Candida species EFT2 nucleotide alignment. These were used to amplify a region corresponding to nucleotides 14001800 in the gene encoding eEF2 with the corresponding genomic DNA as a template. These PCR products were sequenced, cloned and verified as described above. The Candida EFT2 sequences described in this paper were submitted to GenBank (AF107286AF107291 and AF292693). The deduced full-length amino acid sequences of the eEF2s were aligned using the CLUSTAL W program (Thompson et al., 1994
).
Full-length cDNA clones encoding the Cr. neoformans EFT2 homologue were isolated from a lambda ZAP II cDNA library (NIH AIDS Research and Reference Reagent Program catalogue number 2077, contributed by Dr Jeffrey Edman). Approximately 106 p.f.u. from the amplified library were screened at reduced stringency (40% formamide/5xSSPE/35 °C) using a gel-purified 1·9 kb HindIII fragment from the protein coding region of S. cerevisiae EFT2 as hybridization probe. Twenty hybridization-positive clones were plaque-purified, subcloned by in vivo excision to pBluescript SK (Stratagene) and characterized. Seventeen clones had inserts approximately 2·7 kb in length; several of these were sequenced and the sequence submitted to GenBank (AF248644). The ORF of the Cr. neoformans EFT2 gene was modified for expression in S. cerevisiae by PCR. The 5' primer introduced a HpaI site for cloning and appended a portion of the S. cerevisiae EFT2 5'-untranslated region to the Cr. neoformans protein coding region and destroyed a HpaI site at the 5' end of the Cr. neoformans gene. The 3' primer introduced an MluI site for subcloning. The product of the PCR reaction was cloned, sequenced to confirm PCR accuracy and ligated to YCpEFT2 digested with HpaI and MluI. As described above, this construct was then transformed and assayed for function in the S. cerevisiae strain YEFD12h.
Construction of substituted S. cerevisiae eEF2s.
Site-directed mutagenesis of the Ca. parapsilosis gene encoding eEF2 to create S521Y and N523S single substitution mutants and the S521Y N523S doubly substituted mutant was accomplished by the PCR overlap extension technique (Ho et al., 1989 ). Modified S. cerevisiae eEF2 genes encoding amino acid residues from Ca. glabrata, Ca. guilliermondii, Ca. krusei, Ca. lusitaniae, Ca. parapsilosis, Cr. neoformans or human eEF2 were constructed by cassette mutagenesis as described by Prodromou & Pearl (1992)
.
Plasmid DNA from each fungal EFT2 construct was used to transform the S. cerevisiae eft1
eft2 deletion strain YEFD12h to leucine prototrophy and strains harbouring only the fungal EFT2 constructs were selected as described above. S. cerevisiae strains containing the fungal EFT2 constructs were also assayed for the predicted sordarin-resistance phenotype by plating cells on medium containing sordarin at 5 or 10 µg ml-1. Plates were incubated at 29 °C for approximately 3 d until colonies appeared. Growth inhibition assays to generate dose response curves were performed as previously described with individual assays performed at least three times (Justice et al., 1998
).
Biochemical assays.
S. cerevisiae post-ribosomal extracts were prepared from cultures grown to mid-exponential phase in YPAD medium as described previously (Justice et al., 1999 ). The potency of sordarin was assessed in in vitro translation systems as described previously (Justice et al., 1998
, 1999
), using 0·05A260 of wild-type ribosomes and 3 µg S. cerevisiae post-ribosomal extract for each 12·5 µl assay. Binding studies with a labelled sordarin derivative and unlabelled sordarin (Justice et al., 1998
) were employed to establish the lower limit of sordarin sensitivity using approximately 0·25 pmol eEF2 per assay.
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RESULTS |
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The results described above underscore the importance of residues 521 and 523 in determining sordarin specificity in Ca. parapsilosis. Other species of Candida which are not sensitive to sordarin do have Tyr and Ser at positions 521 and 523 in their eEF2s, respectively (Fig. 2), but differ at positions 519 (Ca. guilliermondii) or 524 (Ca. krusei). Cr. neoformans eEF2, which is partially insensitive to sordarin, differs from S. cerevisiae eEF2 at positions corresponding to residues 519, 521, 523 and 524. To further evaluate the role of these residues in conferring sordarin specificity in the context of S. cerevisiae eEF2, we created a set of substituted S. cerevisiae eEF2s by replacing amino acids between 517 and 524 with residues from the corresponding region in either Ca. glabrata, Ca. guilliermondii, Ca. krusei, Ca. lusitaniae, Ca. parapsilosis or Cr. neoformans eEF2 (Table 2
). In addition, to assess the contribution of individual amino acids in conferring sordarins selectivity, we generated a set of substituted S. cerevisiae eEF2 constructs that differed from each other by a single amino acid in this region (Table 3
). All eEF2 constructs were assayed for sordarin sensitivity. As seen in Table 3
, S. cerevisiae eEF2s substituted with residues found in either Ca. parapsilosis, Ca. lusitaniae or Ca. krusei are fully insensitive to sordarin whereas the mutants with Ca. glabrata, Ca. guilliermondii or Cr. neoformans residues are partially sensitive (IC50 0·635 µg ml-1). The data also suggest that individual residues may play a synergistic role in sordarins selectivity; e.g. single amino acid substitutions between 517 and 524 from Cr. neoformans eEF2 result in little resistance to sordarin whereas multiple substitutions result in an S. cerevisiae eEF2 that is quite insensitive (up to 800-fold) to sordarin (see Tables 2
and 3
). Moreover, the identity of the amino acid substitution at a given position determines the extent to which the eEF2 construct becomes insensitive to sordarin. For example, the E524P substituted S. cerevisiae eEF2 (as seen in Ca. krusei eEF2) is completely insensitive to sordarin, while E524D (characteristic of Cr. neoformans eEF2) has no significant effect on sordarin sensitivity in whole-cell assays (Table 3
).
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Human eEF2 and sordarin insensitivity
Earlier studies revealed that sordarin had no effect on protein synthesis in mammals and plants despite the fact that the soluble translation factors are highly conserved within eukaryotes (Justice et al., 1998 ). To investigate the role of mammalian eEF2 in sordarin insensitivity, we aligned the deduced protein sequence of human eEF2 with that of the fungal eEF2s. Comparison of human and fungal eEF2 proteins showed that although there are highly conserved regions, there are also conspicuous differences, including some in the sordarin-specificity region corresponding to S. cerevisiae residues 517524 (Fig. 2
). To examine whether this region contributes to the lack of sordarin sensitivity of mammalian eEF2, a substituted S. cerevisiae eEF2 with amino acids 517524 replaced by the corresponding residues from human eEF2 was generated and assayed for growth inhibition in sordarin-containing medium. As seen in Table 2
, the wild-type S. cerevisiae eEF2 is sensitive to sordarin, whereas the substituted S. cerevisiae eEF2 is fully insensitive to the compound. This result, taken together with the results described above, demonstrates that the identity of eEF2 residues 517524 is sufficient to account for sordarins fungal specificity.
S. cerevisiae eEF2 differs from human eEF2 at six positions in the sordarin-specificity region. We examined the role of these individual residues in conferring sordarin insensitivity with mutant S. cerevisiae eEF2 constructs that had single amino acid replacements from the human eEF2 between residues 517 and 524. The results from growth inhibition studies showed that a single substitution at position 523 to the corresponding amino acid from the human eEF2 rendered the S. cerevisiae construct insensitive to sordarin while the change at 521 from Tyr to Ile conferred partial insensitivity (Table 3). Amino acid substitutions at the other positions in this region did not significantly change the efficacy of sordarin. The results clearly suggest that the eight-amino-acid region of human eEF2 is sufficient to confer sordarin insensitivity on S. cerevisiae that is equal to that observed with mammalian eEF2 (see Fig. 1
). Similar to the fungal eEF2s, the amino acid corresponding to S. cerevisiae residue 523 in human eEF2 is the major determinant of sordarin efficacy.
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DISCUSSION |
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In this paper, we demonstrate that the fungal selectivity of sordarin can be mapped to an eight-amino-acid region within eEF2. To show this, heterologous fungal eEF2s and S. cerevisiae substituted eEF2s were expressed in S. cerevisiae. The observation that eEF2s from several Candida species were able to functionally complement S. cerevisiae eEF2 was not unexpected since this had been previously shown with eEF2 from Ca. albicans (Mendoza et al., 1999 ). However, we were surprised to find that the highly related fungal eEF2s from Ca. tropicalis and Cr. neoformans could not functionally substitute for S. cerevisiae eEF2. Interestingly, we also found that Ca. tropicalis eEF2 exhibited a dominant negative lethal phenotype when expressed in S. cerevisiae (M. Shastry and others, unpublished results). These findings suggest that there are critical determinants for species-specific function of eEF2. The vast majority of data describing the interaction of elongation factors with the ribosome come from studies done in prokaryotes. For example, it has been shown that the eukaryotic translocase eEF2 does not function with the prokaryotic 70S ribosome and conversely the prokaryotic EF-G is not functional in the context of the 80S ribosome (Grasmuk et al., 1977
; Taira et al., 1972
). In a recent study, Uchiumi et al. (1999)
swapped the L8 complex consisting of L7/L12 and L10 proteins in E. coli with its counterpart from rat, comprising P1, P2 and P0 proteins, and showed that the hybrid ribosome displayed eEF2-dependent but not EF-G-dependent GTPase activity. This hybrid system demonstrates that the stalk protein complex determines the specificity of the ribosometranslocase interaction. These results correlate with our unpublished data (M. C. Justice and others) showing that some combinations of sordarin-resistant alleles of EFT2 and P0 are synthetically lethal while others result in a synergistic effect on sordarin resistance. Uchiumi et al. (1999)
also showed that the 23S rRNA plays a vital role in this interaction. Further, cryoelectron microscopy studies of the eukaryotic ribosomeeEF2 complex stabilized by sordarin suggest that the interaction between the eEF2 and stalk region of the large subunit is more extensive than in prokaryotes (Gomez-Lorenzo et al., 2000
). These results underscore the close interplay between the ribosomal stalk complex, elongation factor 2 and the rRNA during translocation.
Taken together, these observations suggest that there are critical interactions between ribosomal proteins and eEF2 that are important for species-specific expression of eEF2. Despite a wealth of biochemical data describing interactions between EF-G and the ribosome, there is relatively little known about the specific interactions of eEF2 and the ribosome. The reagents produced in this study should prove useful for identification of critical determinants of eEF2 function and interactions with other macromolecules in the translational apparatus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Dominguez, J. M. & Martin, J. J.(1998). Identification of elongation factor 2 as the essential protein targeted by sordarins in Candida albicans. Antimicrob Agents Chemother 42, 2279-2283.
Dominguez, J. M., Kelly, V. A., Kinsman, O. S., Marriott, M. S., Gomez de las Heras, F. & Martin, J. J.(1998). Sordarins: a new class of antifungals with selective inhibition of the protein synthesis elongation cycle in yeasts. Antimicrob Agents Chemother 42, 2274-2278.
Dominguez, J. M., Gomez-Lorenzo, M. G. & Martin, J. J.(1999). Sordarin inhibits fungal protein synthesis by blocking translocation differently to fusidic acid. J Biol Chem 274, 22423-22427.
Gomez-Lorenzo, M. G. & Garcia-Bustos, J. F.(1998). Ribosomal P-protein stalk function is targeted by sordarin antifungals. J Biol Chem 273, 25041-25044.
Gomez-Lorenzo, M. G., Spahn, C. M. T., Agrawal, R. K. & 7 other authors (2000). Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17·5 resolution. EMBO J 19, 27102718.
Grasmuk, H., Nolan, R. D. & Drews, J.(1977). Interchangeability of elongation factor-Tu and elongation factor-1 in aminoacyl-tRNA binding to 70 S and 80 S ribosomes. FEBS Lett 82, 237-242.[Medline]
Hauser, D. & Sigg, H. P.(1971). Isolation and decomposition of sordarin. Helv Chim Acta 54, 1178-1190.[Medline]
Herreros, E., Martinez, C. M., Almela, M. J., Marriott, M. S., De Las Heras, F. G. & Gargallo-Viola, D.(1998). Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carini and filamentous fungi. Antimicrob Agents Chemother 42, 2863-2869.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R.(1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 51-59.[Medline]
Justice, M. C., Hsu, M.-J., Tse, B., Ku, T., Balkovec, J., Schmatz, D. & Nielsen, J.(1998). Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis. J Biol Chem 273, 3148-3151.
Justice, M. C., Ku, T., Hsu, M.-J., Carniol, K., Schmatz, D. & Nielsen, J.(1999). Mutations in ribosomal protein L10e confer resistance to the fungal-specific eukaryotic elongation factor 2 inhibitor sordarin. J Biol Chem 274, 4869-4875.
Mendoza, A., Serramia, M. J., Capa, L. & Garcia-Bustos, J. F.(1999). Translation elongation factor 2 is encoded by a single essential gene in Candida albicans. Gene 229, 183-191.[Medline]
Phan, L. D., Perentesis, J. P. & Bodley, J. W.(1993). Saccharomyces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin. J Biol Chem 268, 8665-8668.
Prodromou, C. & Pearl, L. H.(1992). Recursive PCR: a novel technique for total gene synthesis. Protein Eng 5, 826-829.
Taira, H., Ejiri, S. & Shimura, K.(1972). The interaction of elongation factor 2 with ribosomes from silk gland. Formation of an EF-2-ribosome-GDP complex. J Biochem (Tokyo) 72, 1527-1535.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Uchiumi, T., Hori, K., Nomura, T. & Hachimori, A.(1999). Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding. J Biol Chem 274, 27578-27582.
Received 20 July 2000;
revised 4 October 2000;
accepted 9 October 2000.