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Department of Molecular Genetics and Microbiology, Box 3020, Duke University Medical Center, Durham, NC 27710, USA
Correspondence
John H. McCusker
mccus001{at}mc.duke.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A supplementary table of primers is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; reviewed by Jones & Fink 1982). In the yeast Saccharomyces cerevisiae, this pathway is regulated at the level of transcription by general control (Hinnebusch, 1992; Mountain et al., 1991), and of enzyme activity, particularly by threonine feedback inhibition of aspartate kinase (Hom3p) at the initial step of the pathway (Martin-Rendon et al., 1993; Ramos & Calderon, 1992). In addition to auxotrophy, a number of deleterious phenotypes have been attributed to threonine biosynthetic mutants (Arevalo-Rodriguez et al., 2004; Birrell et al., 2001, 2002; Care et al., 2004; Deutschbauer et al., 2002; Dunn et al., 2006; Enyenihi & Saunders, 2003; Giaever et al., 2002; Roberg et al., 1997), some defects of which, such as temperature sensitivity, salt sensitivity and being petite-negative, could also influence fungal survival in vivo. Moreover, the threonine-biosynthetic intermediate homoserine is also required for biosynthesis of methionine, itself a central metabolite, and threonine is required for isoleucine biosynthesis. Significantly, we and others have shown that various fungal methionine (Met2p, Met3p and Met6p) and isoleucine (Ilv2p), as well as threonine (Hom3p), biosynthetic enzymes are required for fungal survival in vivo and/or virulence (Kingsbury et al., 2004a, 2006; Nazi et al., 2007; Pascon et al., 2004; Yang et al., 2002).
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, b; Nazi et al., 2007; Pascon et al., 2004). In addition, auxotrophs show defects in known cryptococcal virulence traits such as the ability to proliferate at 37 °C, and melanin and capsule production (Kingsbury et al., 2004a, b; Pascon et al., 2004; Yang et al., 2002). We were therefore interested in evaluating the potential of threonine biosynthetic enzymes as anti-cryptococcal targets in C. neoformans. Several attempts to disrupt the homoserine and threonine biosynthetic gene HOM3 (encoding aspartate kinase, EC 2.7.2.4), and the threonine biosynthetic genes THR1 (encoding homoserine kinase, EC 2.7.1.39) and THR4 (encoding threonine synthase, EC 4.2.3.1) were unsuccessful. We demonstrate that this is because HOM3 and THR1, and likely other threonine biosynthetic enzymes, are essential for C. neoformans growth in most conditions. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) were isogenic with S288c and C. neoformans strains were isogenic with H99 serotype A Mat 
, (Perfect et al., 1993). One Shot Top10 Chemically Competent Escherichia coli (Invitrogen) was used for plasmid propagation. Standard yeast and bacterial media were used (Sherman et al., 1974; Sambrook et al., 1989). Where specified, media were supplemented with nourseothricin (Nat; 100 µg ml–1; Hans Knöll Institute für Naturstoff-Forschung, Jena, Germany), geneticin (200 µg ml–1; Life Technologies), proline (1 g l–1), sorbitol (1 M), bathocuproinedisulfonic acid (BCS; 200 µM), cupric sulfate (CuSO4; 25 µM), ascorbic acid (1 mM), threonine (2.5 mM), homoserine (2.5 mM), methionine (0.13 mM), Ala-Thr (2.5 mM) and Met-Leu (0.13 mM).
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). NCBI accession numbers for the predicted Hom3p, Thr1p and Thr4p included XP_572658, XP_572893 and XP_568789, respectively. Sequences were then BLASTed against the C. neoformans serotype A strain H99 sequence. Serotype A HOM3, THR1 and THR4 occurred in sequence with the NCBI accession numbers AACO02000077.1, AACO02000074.1 and AACO02000068.1, respectively.
We also attempted to isolate the C. neoformans HOM3, THR1 and THR4 cDNAs by complementation of the methionine and/or threonine auxotrophies of S. cerevisiae hom3
, thr1 and thr4 strains, using a C. neoformans cDNA library. Specifically, S. cerevisiae strains YJK2416 (hom3 ura3), YJK1358 (thr1 ura3) and S318 (thr4 ura3) were transformed by lithium acetate-mediated transformation (Gietz et al., 1995), with a library that contained C. neoformans H99 cDNAs under the control of the S. cerevisiae GAL1 promoter in the pYES2.0 vector (Invitrogen) (Suvarna et al., 2000). Ura+ transformants were screened for the acquisition of methionine and/or threonine prototrophy in the presence of galactose, but not glucose, as a carbon source. Plasmids that conferred prototrophy were isolated, propagated in E. coli DH10B, then analysed by restriction analysis, and sequenced by the Duke University Cancer Center Sequencing Facility. Plasmids included pJO373 (pYES2.0+C. neoformans THR1 cDNA, NCBI accession number EU623435), and pJO378 (pYES2.0+C. neoformans THR4 cDNA, NCBI accession number EU635873).
Plasmid and strain construction.
In vitro C. neoformans thr4 : : NAT1, thr4 : : NEO, thr1 : : NAT1 and hom3 : : NAT1 targeting cassettes were constructed using a modified PCR fusion technique (Davidson et al., 2002). To construct the thr4 : : NAT1 and thr4 : : NEO targeting cassettes, the first rounds of PCR amplified 5' and 3' THR4 sequence from H99 genomic DNA (primer pairs ZY125+JO257 and ZY126+JO255, respectively; see Supplementary Table S1 for sequences of all primers used in this study), and the NAT1 cassette from pGMC200 (McDade & Cox, 2001) or the NEO cassette from pJAF1 (Fraser et al., 2003) (primers JO254+JO256). The gel-purified products were used as a template in the final fusion PCR with primers ZY125+ZY126. To construct the thr1 : : NAT1 targeting construct, the first round of PCR consisted of amplification of 5' and 3' THR1 sequence from H99 genomic DNA (primer pairs JO298+JO303 and JO302+JO300, respectively), and the NAT1 cassette from pGMC200 (primers JO301+JO304), then products were combined for the fusion PCR using primers JO298+JO300. Construction of the hom3 : : NAT1 cassette consisted of amplification of 5' and 3' HOM3 sequence from H99 genomic DNA (primer pairs JO318+JO316 and JO320+JO315, respectively), and NAT1 from pGMC200 (primers JO314+JO317), followed by fusion of products in the final PCR with primers JO318+JO320. All gel-purified constructs were cloned into pCR2.1-TOPO (Invitrogen) according to the manufacturer's instructions, resulting in pJO193 (thr4 : : NAT1), pJO221 (thr4 : : NEO), pJO249 (thr1 : : NAT1), and pJO260 (hom3 : : NAT1).
The NAT1-PCTR4-1 cassette-containing plasmid pJO306 was also created by fusion PCR. The first round of PCR consisted of amplification of NAT1 from pGMC200 (primers JO357+JO412) and PCTR4-1 from template pCTR4.2 (Ory et al., 2004) (primers JO408+JO409). The purified products were used as a template in the final fusion PCR using primers JO357+JO409, and the gel-purified final product was cloned into pCR2.1-TOPO.
In vitro targeting cassettes were constructed to place HOM3 and THR1 under control of the CTR4-1 promoter. To create the NAT1-PCTR4-1-HOM3 cassette, HOM3 upstream and the 5' gene sequence were PCR-amplified from H99 genomic DNA (primer pairs JO414+JO415 and JO416+JO413), and NAT1-PCTR4-1 was amplified from pJO306 (primers JO357+JO409). The products were combined in the fusion PCR (primers JO413+JO414), and the resulting product was gel-purified and cloned into pCR2.1-TOPO (pJO310). The first round of PCR for construction of the NAT1-PCTR4-1-THR1 cassette consisted of amplification of THR1 upstream and the 5' gene sequence (primer pairs JO359+JO360 and JO410+JO362), and NAT1-PCTR4-1 from pJO306 (primers JO357+JO409). The products were used as a template for the final fusion PCR using primers JO359+JO362, and the resulting product was cloned into pCR2.1-TOPO (pJO308). All plasmid constructions were confirmed by restriction digestion and PCR analyses.
The targeting cassettes were PCR-amplified from their respective plasmids, and introduced into strain H99 by biolistic transformation (Toffaletti et al., 1993). For transformation with the thr4 : : NAT1, thr4 : : NEO, thr1 : : NAT1, and hom3 : : NAT1 constructs, transformation was performed on YPD+sorbitol, and after a 4 h incubation, cells were scraped off plates and spread on YPD+NAT or G418 plates to select for transformants. Transformants were purified and plated on SD to screen for acquisition of auxotrophy. For transformation with the NAT1-PCTR4-1-HOM3 and NAT1-PCTR4-1-THR1 constructs, cells were plated on YPD+sorbitol+BCS and incubated for 2–3 h prior to transformation to allow for expression from PCTR4. Following transformation, plates were incubated for 4 h and cells were replated on YPD+BCS+NAT. Purified transformants were screened for acquisition of auxotrophy on SD+CuSO4+ascorbic acid plates (PCTR4-1-repressing conditions). The NAT1-PCTR4-1-THR1 genotype in strain H99-73 and NAT1-PCTR4-1-HOM3 genotype in strain H99-76 was confirmed by PCR (primer pairs JO281+JO300 and JO506+JO280 for H99-73, and JO281+JO320 and JO505+JO280 for H99-76) and Southern hybridization analysis (Fig. 2).
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; Wach et al., 1994). Gene deletions were confirmed by PCR, and by acquisition of methionine and/or threonine auxotrophy.
Manipulation of nucleic acids.
Plasmid DNA from E. coli was extracted using the QIAprep Spin Miniprep kit (Qiagen), according to the manufacturer's instructions. Extraction of plasmid DNA from S. cerevisiae, and genomic DNA from C. neoformans for PCR analysis, was performed as described previously (Hoffman & Winston, 1987). Genomic DNA from C. neoformans for Southern hybridization analysis was isolated as described previously (Yang et al., 2002), 2 µg of which was digested with various restriction enzymes, separated by electrophoresis on a 0.75 % (w/v) agarose gel, denatured and transferred to a nylon membrane (Roche), as described previously (Sambrook et al., 1989).
RNA for Northern analyses was prepared from cells that had first been grown to a density of approximately 2x108 cells ml–1, in 50 ml YPD+BCS. Cells were harvested, washed twice in sterile water, then split four ways and incubated with shaking in 50 ml YPD+BCS or YPD+CuSO4+ascorbic acid, at 25 °C or 37 °C. Following incubation for 5 h, RNA was isolated as described previously (Yang et al., 2002). Each sample was prepared in duplicate, and 10 µg duplicates of each preparation were separated in a 1 % (w/v) agarose-formaldehyde gel, and transferred to a nylon membrane.
Probes for Southern and Northern hybridizations were prepared from gel-purified PCR products. Specifically, probes for Southern hybridizations were amplified using primer pairs JO413+JO414 (HOM3) and JO362+JO506 (THR1). Primer pairs for amplification of Northern hybridization probes included JO770+JO772 (HOM3), JO298+JO362 (THR1), JO223+JO225 (GPD) and JO765+JO766 (CTR4) (see Table S1 for primer sequences). Probes were labelled with [-32P]dCTP (Perkin-Elmer) using the RediprimeII Random Prime Labelling System (Amersham Biosciences), according to the manufacturer's instructions. Blots were prehybridized and hybridized in ULTRAhyb buffer (Ambion), and washed according to the manufacturer's instructions. Membrane signal was visualized using a Typhoon 9200 Variable Mode Imager (Molecular Dynamics), and band signal intensity was quantified using ImageQuaNT 5.2 software (Molecular Dynamics).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and the avirulence or inability to survive in vivo of various amino acid auxotrophs, we were interested in assessing the potential of threonine biosynthetic enzymes as antifungal drug targets in the human-pathogenic fungus C. neoformans. In particular, we focused on, first, the aspartate kinase (encoded by HOM3), the initial and key feedback regulatory enzyme of the pathway. The avirulence of other cryptococcal methionine auxotrophs (Nazi et al., 2007; Pascon et al., 2004; Yang et al., 2002) indicates that C. neoformans is unable to supplement this auxotrophy in the in vivo environment, thus we reasoned that the combined threonine and methionine auxotrophies of hom3 mutants should be even more detrimental to survival in vivo and/or virulence. Moreover, HOM3 is required for the in vivo survival of S. cerevisiae (Kingsbury et al., 2006). We were also interested in the final two steps of threonine biosynthesis, catalysed by homoserine kinase (encoded by THR1) and threonine synthase (encoded by THR4). Mutation of these genes in S. cerevisiae results in a plethora of deleterious phenotypes in addition to auxotrophy (Birrell et al., 2001, 2002; Care et al., 2004; Deutschbauer et al., 2002; Dunn et al., 2006; Enyenihi & Saunders, 2003; Giaever et al., 2002; Roberg et al., 1997), which may also influence in vivo survival and/or virulence.
We identified the putative C. neoformans H99 HOM3, THR1 and THR4 genes from the C. neoformans serotype A strain H99 through sequence similarity with the respective predicted ORFs in serotype D. Furthermore, cDNAs matching the predicted THR1 and THR4 genes were isolated from a C. neoformans H99 cDNA library based on the ability to confer threonine prototrophy to S. cerevisiae thr1 and thr4 strains, respectively (Fig. 3), thus verifying that the identified genes encoded the predicted enzyme activities. We were unable to isolate the HOM3 cDNA by complementation of a S. cerevisiae hom3 strain, however, likely due to under-representation of the HOM3 cDNA in the library. Consistent with this, we were unable to PCR-amplify the HOM3 cDNA from the library DNA. The C. neoformans H99 HOM3, THR1 and THR4 genes were predicted to contain six, two and five introns, respectively. The predicted C. neoformans Hom3p, Thr1p and Thr4p sequences were highly similar to the corresponding proteins in S. cerevisiae, with approximately 50, 56 and 50 % amino acid identity, respectively.
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C. neoformans HOM3 and THR1 are essential
To determine whether threonine biosynthetic genes are essential in C. neoformans, we replaced the endogenous promoters of HOM3 and THR1 with the PCTR4-1 promoter (Ory et al., 2004), thereby placing the genes under copper-repressible control (Fig. 2
). Growth of the wild-type (H99), PCTR4-1-HOM3 (H99-76) and PCTR4-1-THR1 (H99-73) strains was compared by plating 10-fold spot dilutions of strains, pre-grown in YPD+BCS, onto YPD+BCS (promoter-inducing conditions) and YPD+CuSO4+ascorbic acid (promoter-repressing conditions) (Fig. 4). After incubation at 30 °C for 3 days in promoter-inducing conditions, PCTR4-1-HOM3 and PCTR4-1-THR1 strains grew considerably, although less well than the wild-type as judged by colony size, indicating that HOM3 and THR1 were expressed at different from normal level in these strains. However, no colony formation was observed for the PCTR4-1-HOM3 and PCTR4-1-THR1 strains in promoter-repressing conditions. Since the yeast extract and Bacto peptone in YPD contains significant levels of threonine and methionine for supplementation of Thr and Met auxotrophies (Difco Manual, 11th edition), these results indicate that THR1 and HOM3 are essential in C. neoformans, even in the presence of abundant threonine and methionine.
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; Pascon et al., 2004; Yang et al., 2002). Since both PCTR4-1-HOM3 and PCTR4-1-THR1 strains were unable to grow under promoter-repressing conditions, the growth deficiency on YPD may instead be due to C. neoformans being unable to satisfy the threonine auxotrophy in this medium.
Growth of the PCTR4-1-HOM3 and PCTR4-1-THR1 strains was compared by spot dilutions on minimal medium containing ammonium (SD) or proline [SD(Pro)] as the nitrogen source, supplemented with various combinations of homoserine, methionine and threonine amino acids or dipeptides, in PCTR4-1-repression conditions (Fig. 5). While no colony formation of strains was observed in PCTR4-1-repression conditions in the absence of amino acid supplements, residual growth similar to that present on YPD in repressing conditions was observed, which we attribute to a basal level of gene expression still occurring, and/or utilization of cell reserves accumulated during the pre-growth in gene-expressing conditions. Strains grew no better when the amino acids methionine and threonine were added to either SD or SD(Pro) medium than on media lacking amino acids. Strains were also unable to grow when SD medium was supplemented with threonine and methionine dipeptides, but growth was enhanced above background levels when the threonine and methionine dipeptides were added to SD(Pro) medium. Furthermore, growth of the PCTR4-1-THR1 strain required only threonine dipeptides, while growth of the PCTR4-1-HOM3 strain required both methionine and threonine dipeptides, or homoserine, providing further evidence that these genes indeed confer homoserine kinase and aspartate kinase activities, respectively.
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, b). Combined, these results indicate that C. neoformans possesses fewer amino acid permeases, permeases that have a lower transport velocity, or more permeases that are subject to ammonium repression. Although YPD medium contains significant levels of peptides, our results indicate that the lack of growth in this medium is likely due to nitrogen repression, and/or transport of other peptides outcompeting transport of threonine-containing peptides.
Temperature determines severity of phenotype
While the inability of a cryptococcal amino acid auxotroph to grow on YPD at 30 °C has not been previously demonstrated, it has been shown that SPE3-lys9 (lysine auxotrophic) and ilv2 (isoleucine and valine auxotrophic) mutants die in YPD at 37 °C (Kingsbury et al., 2004a, b). We therefore determined whether the incubation temperature affects growth of PCTR4-1-HOM3 and PCTR4-1-THR1 strains on YPD. Interestingly, reduction of the incubation temperature to 25 °C allowed the growth of the PCTR4-1-HOM3 strain in repressing conditions, while the PCTR4-1-THR1 strain was no better able to grow at this temperature than at 30 °C (Fig. 4).
Differences in the growth phenotype between the two strains could be attributable to either homoserine kinase having a role in addition to threonine biosynthesis in C. neoformans, or the block in the biosynthetic pathway caused by inhibition of homoserine kinase leading to the accumulation of a toxic intermediate. Consistent with this, elevated levels of the intermediate predicted to accumulate, homoserine, are toxic to mammalian (Rees et al., 1994) and bacterial cells (Kotre et al., 1973; O'Barr & Everett, 1971). Moreover, the accumulation of a toxic intermediate has been hypothesized to be responsible for toxic effects associated with other amino acid biosynthetic mutants (Arevalo-Rodriguez et al., 2004; Kingsbury et al., 2004a; Pascon et al., 2004; Suliman et al., 2007), and may explain deleterious phenotypes associated with THR1 and THR4 mutation in S. cerevisiae. In S. cerevisiae, the threonine pathway is regulated positively in response to threonine starvation, by upregulation of gene transcription and eliminating feedback inhibition (Hinnebusch, 1992; Martin-Rendon et al., 1993; Mountain et al., 1991; Ramos & Calderon, 1992). If the pathway is similarly regulated in C. neoformans, threonine starvation conditions, such as what Thr1p-inhibited C. neoformans would likely encounter in vivo, should result in increased flux through the threonine biosynthetic pathway, thus increased toxic intermediate accumulation, and hence intensified growth defects.
We also compared the growth of PCTR4-1-HOM3 and PCTR4-1-THR1 strains at 37 °C. While some background growth of both strains was still present at 30 °C in repressing conditions, growth was completely eliminated at 37 °C in repressing conditions. Surprisingly, growth was also eliminated in induction conditions, which may indicate that there is a greater difference between expression from the CTR4-1 promoter and endogenous THR1 and HOM3 expression at 37 °C, compared with 30 °C. To examine this, we compared HOM3, THR1 and CTR4 transcript levels following a 5 h incubation of the PCTR4-1-HOM3, PCTR4-1-THR1 and wild-type strains, in promoter-induction and repression conditions, at 25 and 37 °C (Fig. 6). Following normalization to the GPD housekeeping gene, we observed no obvious temperature-dependent changes in THR1, HOM3 or CTR4 transcript levels from the PCTR4-1-HOM3, PCTR4-1-THR1 and wild-type strains, respectively, grown in promoter-induction conditions; thus the CTR4 (and CTR4-1) promoter is not regulated by temperature. Furthermore, although HOM3 transcript levels were barely detectable at both temperatures, THR1 and HOM3 transcription levels in the wild-type did not appear to be enhanced at 37 °C compared with 25 °C. Results also show that HOM3 and THR1 transcripts expressed from PCTR4-1-HOM3 and PCTR4-1-THR1 strains in induction conditions were at higher than wild-type levels at both 30 and 37 °C. One possible explanation for the lack of growth in induction conditions is that higher expression of these genes might result in growth impairment by perturbing metabolic flux, interfering with general cell metabolism, which may be accentuated at higher temperatures. Given our SPE3-lys9 and ilv2 findings, the increased severity of growth defects at 37 °C in repressing conditions may be due to decreased threonine transport at this temperature.
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; Vallim et al., 2004). However, given the highly conserved nature of the threonine biosynthetic pathway between fungi, it is surprising to find that threonine biosynthetic genes are essential in C. neoformans, and to our knowledge, this is the first documented case of threonine biosynthetic gene necessity in fungi. The essential nature of these genes, particularly at 37 °C, makes aspartate kinase and homoserine kinase excellent candidates for anti-cryptococcal drug targets.
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ACKNOWLEDGEMENTS |
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Edited by: J. F. Ernst
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Received 16 April 2008;
revised 20 May 2008;
accepted 27 May 2008.
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