From the Department of Molecular Biology and
Pharmacology, Washington University School of Medicine,
St. Louis, Missouri 63110 and the ¶ Department of Medicinal and
Structural Chemistry, G.D. Searle and Company,
St. Louis, Missouri 63198
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cryptococcus neoformans is a fungal pathogen that causes chronic meningitis in 10% of patients with AIDS. Genetic and biochemical studies were conducted to determine whether myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a target for development of a new class of fungicidal drugs. A single copy of a conditional lethal C. neoformans NMT allele was introduced into the fungal genome by homologous recombination. The allele (nmt487D) produces temperature-sensitive myristic acid auxotrophy. This phenotype is due, in part, to under-myristoylation of a cellular ADP ribosylation factor (Arf) and can be rescued by forced expression of human Nmt. Two isogenic strains with identical growth kinetics at 35 °C were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 contained nmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 °C, survival of HMC1 depends upon its human Nmt. ALYASKLS-NH2, an inhibitor derived from an Arf, was fully depeptidized: p-[(2-methyl-1-imidazol-1-yl)butyl]phenyl-acetyl was used to represent the GLYA tetrapeptide, whereas SKLS was replaced with a chiral tyrosinol scaffold. Kinetic studies revealed Ki (app) values of 1.8 ± 1 and 9 ± 2.4 µM for purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was 2-fold lower for CPA8 compared with HMC1. A single dose of 100 µM produced a 5-fold greater inhibition of protein synthesis in CPA8 versus HMC1. The strain specificity of these responses indicates that the fungicidal effect was Nmt-dependent. These two strains may be useful for screening chemical libraries for Nmt-based fungicidal compounds with relatively little activity against the human enzyme.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cryptococcus neoformans is a haploid yeast that causes systemic infection in immunocompromised humans. The organism has tropism for the central nervous system where it produces chronic meningitis. The incidence of infection in patients with acquired immune deficiency syndrome is ~10% (1). New ways of treating cryptococcal meningitis are needed given the limitations of currently available fungicidal and fungistatic agents (e.g. Refs. 2 and 3). An ideal drug target would be a fungal gene product that is expressed under the conditions of infection and that is essential for the viability of the organism. The metabolic pathway and/or substrate specificities of the target protein should be distinguishable from those of the human host. Finally, it would be desirable if the protein is common to many fungal pathogens and is required for their survival.
Studies of C. neoformans pathogenesis have revealed several
virulence genes. They encode products involved in capsule formation (CAP59 and CAP64), synthesis of melanin
(CNLAC1), and mating (MF). GPA1
functions as a regulator of each of these functions (4). None of these
genes is essential for viability (4-8).
Genetic studies have shown that ADE2 (phosphoribosylaminoimidazole carboxylase) is necessary for the growth of C. neoformans in cerebrospinal fluid (9) and that calcineurin is required for survival at 37 °C (10). A number of other C. neoformans genes have been isolated, but their necessity for growth and/or infection has either not been evaluated by direct genetic tests or the results of such tests have been ambiguous (e.g. Refs. 11-18). One reason for the paucity of such tests is that targeted gene disruption is limited by several factors in C. neoformans. Homologous recombination appears to be inefficient (4-7, 19). There are only a few selectable markers available for conducting such tests (12, 20, 21). Moreover, in the absence of a conditional lethal allele, disruption of an essential gene in this haploid organism will produce death, precluding further analysis of the function of the gene.
The C. neoformans gene encoding myristoyl-CoA:protein N-myristoyltransferase (EC 2.1.3.97) (NMT)1 fulfills many of the criteria for an anti-fungal target. Nmt is a monomeric enzyme that catalyzes the co-translational transfer of myristate, a 14-carbon saturated fatty acid, from CoA to the N-terminal Gly residue of nascent proteins. Cellular N-myristoylproteins have diverse biological functions (22). The enzyme appears to be ubiquitously expressed in eukaryotes, including the two organisms that are the principal causes of systemic fungal infections in immunosuppressed humans, C. neoformans and Candida albicans (23, 24). In vitro studies of purified orthologous Nmts have shown that their acyl-CoA substrate specificities are highly conserved, whereas their peptide substrate specificities are divergent. These differences in peptide recognition have been exploited to develop species-selective peptidomimetic inhibitors (Refs. 25-29; reviewed in Ref. 30). In addition, genetic tests have established that NMT is essential for the growth and viability of both C. neoformans and C. albicans (19, 31).
NMT represents a unique example of an essential C. neoformans gene where a conditional lethal allele has been
generated by homologous recombination. This allele was based on a
mutant Saccharomyces cerevisiae NMT1 allele
(nmt1-451D). nmt1-451D contains a single nucleotide substitution that results in replacement of an absolutely conserved Gly, located 5 residues from the C terminus of the enzyme, with an Asp (32). This substitution reduces the affinity of the enzyme
for myristoyl-CoA (33). The analogous mutation in C. neoformans
NMT is Gly487 Asp (nmt487D). A strain
containing several copies of nmt487D, including one at the
endogenous locus, was produced (19). It is a myristic acid auxotroph.
Withdrawal of myristate produces rapid growth arrest and death within
4 h (19). Virulence studies using isogenic NMT and
nmt487D strains and an immunosuppressed rabbit model of
cryptococcal meningitis established that genetic attenuation of Nmt
activity allows the host to rid itself of an otherwise fatal infection
(19).
In the present study, we generated a strain of C. neoformans with a single copy of nmt487D at the endogenous gene locus. This strain was used to correlate cellular protein N-myristoylation with growth and viability. Moreover, strains expressing wild type C. neoformans or human Nmt were employed to show that a species-selective, fully depeptidized inhibitor of the acyltransferase produces fungicidal effects through an Nmt-dependent mechanism.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains and Media-- Strains generated during the course of these studies are described in Table I. Two C. neoformans strains, MO49 (ade2, NMT) and CMD2 (ADE2, nmt487D), have been described previously (19, 20). A strain derived from MO49 with an unmapped insertion of ADE2 was named CAP8 in an earlier report (19) but is now renamed CPA8 (Cryptococcus with prototropy for adenine) to avoid confusion with capsule-deficient mutants (34). The following media were used: YPD (1% yeast extract, 2% peptone, 2% glucose); YPD supplemented with 1% Brij 58 (Sigma) and 500 µM myristate or palmitate (NuChek Prep); YNB (yeast nitrogen base; Bio 101); and RPMI 1640 (with glutamine and without bicarbonate; Life Technologies, Inc.).
Expression Vectors-- pCN36-23 was employed for forced expression of a C. neoformans ADP-ribosylation factor (Arf) in C. neoformans. The plasmid contains three components: (i) the C. neoformans GAL7 promoter from pGUST11 (35) containing an NcoI site in place of the NdeI site at the initiator Met codon (engineered by PCR using 5'-TGTTCCGCCGGTGGAAAGAAGCAGG-3' and 5'-GGGCCATGGTCAAGAGGGGATTGAGC-3' as primers and pGUST11 as the template DNA); (ii) the open reading frame of the C. neoformans ARF gene (24), obtained by PCR of genomic DNA from strain MO49 (the PCR reaction contained 5'-GGGGCCATGGGCCTTTCTGTCTCC-3' which incorporates an NcoI site at the initiator ATG, and 5'-CCTTGCGGCCGCGTGCGAAGGTATAAATGGAC-3' which incorporates an NotI site at the 3' end of the coding sequence); and (iii) an NotI fragment from pTelHyg (21) that includes sequences for maintenance in Escherichia coli and a hygromycin resistance cassette. The ARF insert in pCN36-23 was sequenced in its entirety to verify that no unanticipated mutations had been introduced by PCR.
pHS20-57 was used to express human Nmt in C. neoformans. This plasmid consists of five components: (i) a 1.4-kb fragment from C. neoformans NMT that includes transcriptional regulatory elements from its 5'-nontranscribed domain. This fragment spans the region from the HindIII site of pCN25-28 (19) to the ATG of the coding sequence where an NdeI site was engineered by PCR; (ii) a 1.7-kb NdeI/EcoRI fragment from pBB218 which includes the coding sequence of a human Nmt cDNA (36); (iii) an EcoRI/BglII fragment from the polylinker of pSP70 (Promega); (iv) a 2.4-kb BamHI/EcoRI fragment from pCN25-28 containing sequences 3' to the stop codon of C. neoformans NMT (19); and (v) pGEM4Z (Promega) linearized by digestion with HindIII and EcoRI.Transformation of C. neoformans-- Strains M049 and CMD19 (Table I) were transformed using a Bio-Rad Biolistic PDS-1000/HE particle delivery system and published protocols (19, 20). The diameter of the gold particles was 0.6 µm.
When strain MO49 was transformed with pCN28-14 (nmt487D; 19), cells were selected based on their ability to grow at 24 °C on YNB supplemented with 0.8 g/liter complete synthetic medium without adenine (CSM-ade: Bio 101), 500 µM myristate, 1% Brij 58, and 1 M sorbitol. When strain CMD19 was transformed with pCN36-23 (ARF), cells were selected at 24 °C on YPD containing 200 units/ml hygromycin (Calbiochem). When CMD19 was transformed with either pCN25-28 (C. neoformans NMT) or pHS20-57 (human NMT), cells were selected for their capacity to grow at 35 °C on YPD without supplements. NMT copy number was quantitated by scanning Southern blots of genomic DNA.Growth Curves-- Fresh colonies were picked from plates into YPD, and the liquid cultures were adjusted to an A595 = 0.1. Aliquots (250 µl) were inoculated into 5 ml of YPD or YPD containing 500 µM myristate and 1% Brij 58. Cultures were then incubated with shaking at 24 or 37 °C. Aliquots (10 µl) were withdrawn at various time points and diluted 1:10 with phosphate-buffered saline (PBS), and the A595 of the sample was determined using a Molecular Devices Thermomax microplate reader. All strains were grown at each condition in triplicate, and each A595 measurement was done in triplicate.
Western Blots of C. neoformans Nmt-- Five-milliliter cultures of C. neoformans strains were grown overnight at 24 °C in YPD supplemented 500 µM myristate and 1% Brij 58. The cells were then pelleted by centrifugation at 900 × g for 10 min at room temperature, washed once in PBS, pelleted once more, and then were resuspended in 0.25 ml of a solution containing 2% SDS, 80 mM Tris, pH 6.8, and 2 mM Pefabloc (Boehringer Mannheim). Following addition of 0.5 ml of zirconia/silica beads (0.5 mm diameter, Biospec Products), the cells were vortexed (3 pulses of 1 min, alternating with 1 min of incubation on ice). The resulting lysates were boiled for 5 min and clarified by centrifugation at 12,000 × g for 5 min. Glycerol (final concentration = 5% v/v), 2-mercaptoethanol (2%), and bromphenol blue (0.002%) were added. Equal amounts of lysate were fractionated by electrophoresis through 10% polyacrylamide gels containing 0.1% SDS (37). The separated proteins were then transferred to Immobilon-P (Millipore) transfer membranes (38). Protein blots were probed with a previously characterized rabbit anti-C. albicans Nmt sera (final dilution = 1:1000-fold in blocking buffer; Refs. 24 and 38). Antigen-antibody complexes were detected using reagents and protocols supplied in the Tropix Western-Light kit.
Arf Protein Mobility Gel Shift Assay--
Fifty-milliliter
cultures of C. neoformans strains were grown overnight in
YPD, 500 µM myristate, 1% Brij 58 at 24 °C to an A595 2.0-3.0. The cultures were then
centrifuged as above, washed twice in 25 ml of PBS, and resuspended in
4-6 ml of YPD. A 1-ml aliquot was added to 10 ml of YPD or YPD, 500 µM myristate, 1% Brij 58. Following a 2-h incubation at
24 or 37 °C, cells were pelleted, washed once in 5 ml of PBS, and
resuspended in 0.5 ml of a solution containing 4% SDS and 0.125 M Tris-HCl, pH 6.8. Two volumes of 0.5 mm zirconia/silica
beads were added, and cells were disrupted by vortexing as described
above. Cleared lysates were prepared and denatured, and the cellular
proteins were fractionated using a 16-cm long, 0.75-mm thick, 12%
polyacrylamide gel containing 0.1% SDS (37). Separated proteins were
transferred to Immobilon-P membranes. The protein blots were
subsequently incubated with a previously characterized rabbit
anti-S. cerevisiae Arf1p sera (R40, a generous gift of R. Kahn, Emory University) diluted 1:50,000 in PBS containing 1% gelatin,
0.2% Tween 20, and 0.1% sodium azide. Antigen-antibody complexes were
detected using the Tropix Western-Light kit.
Synthesis of SC-61213--
Synthesis of this compound was
accomplished according to the scheme shown in Fig.
1 and involved reacting the amine
(1) with the 2-methylimidazole carboxylic acid
(2) in the presence of dicyclohexylcarbodiimide and
1-hydroxybenzotriazole (see Ref. 39 for a description of the seven-step
synthesis of compound 1). The resulting product was treated
with trifluoroacetic acid at ambient temperature, and the final product
(SC-61213) was isolated by reverse phase high pressure liquid
chromatography. The 1H NMR and mass spectral data of
SC-61213 were consistent with the structure presented in Fig. 1. A
stock solution of 40 mM SC-61213 was prepared in sterile
deionized water, and aliquots were stored at 20 °C prior to
use.
|
Kinetic Studies of the Inhibition of Orthologous Nmts by SC-61213-- The effects of SC-61213 on purified C. neoformans, human, and S. cerevisiae Nmts were defined using a previously described two-step enzyme assay (40). The amount of Nmt added to the 110-µl reaction depended upon the species: 50-100 ng of C. neoformans Nmt purified from E. coli (19); 30 ng of a homogeneous preparation of E. coli-derived human Nmt (25); or 3.5 ng of purified recombinant S. cerevisiae Nmt1p (41). The final concentration of [3H]myristoyl-CoA was 0.23 µM. An octapeptide representing the N-terminal sequence of a C. neoformans Arf (GLSVSKLL-NH2) was used as a substrate peptide (final concentration = 10 nM to 2 µM). [3H]Myristoyl-GLSVSKLL-NH2 was purified from the reaction mixture by reverse phase high pressure liquid chromatography using a C18 10-µm µBondapak column (dimensions = 3.9 × 300 mm; Waters Corp.) and a linear gradient from H2O, 0.1% trifluoroacetic acid, 0.05% triethylamine to 100% acetonitrile, 0.1% trifluoroacetic acid. The amount of labeled myristoylpeptide recovered was quantitated with an in-line scintillation counter (42). Peptide Km and Vmax values were determined for each Nmt from double-reciprocal plots. The Km value at each inhibitor concentration and the type of inhibition were determined using double-reciprocal plots (43). Ki values were calculated from Dixon plots (1/v versus [I]; see Ref. 43). All assays were performed at least twice, each time in triplicate.
Antifungal Susceptibility Assays-- The minimal inhibitory concentration of SC-61213 was defined by a broth microdilution anti-fungal test performed according to the protocol described by the National Committee for Clinical Laboratory Standards (NCCLS) tentative standard (44). Briefly, RPMI 1640, buffered to pH 7.0 with MOPS or to pH 5.4 with MES, was introduced together with varying amounts of SC-61213 into each well of a 96-well microtiter plate (Falcon). C. neoformans strains CPA8 and HMC1 were then added to the wells according to the NCCLS protocol. Microtiter plates were incubated at 35 °C without shaking. Forty-eight and 72 h after inoculation, the cells in each well were resuspended by pipetting, and the A550 of the resulting suspension was determined using a Molecular Devices Thermomax plate reader. SC-61213 was tested in a series of 2-fold dilutions over the concentration range 100 to 1.6 µM. Each dilution of the compound was tested on two separate occasions, each time in triplicate. Controls included (i) media without any inoculated C. neoformans to confirm that no contaminants were acquired during the course of the incubation; (ii) media with the isogenic C. neoformans strains but with no drug; and (iii) media containing the strains plus a known fungicidal agent (amphotericin B, 0.25-4 µg/ml).
Protein Synthesis Assays-- To determine the effects of SC-61213 on protein synthesis, cells were grown overnight at 30 °C in YNB, pH 7.0, to an A595 of 0.8. One-milliliter aliquots of the culture were treated with either 3.5 µM cycloheximide or 100 µM SC-61213 for 30 min at 30 °C. [35S]Methionine (100 µCi; 1000 Ci or 37 TBq/mmol) was added to each sample, and the cells were shaken for an additional 30 min at 30 °C. Labeled cells were then harvested by centrifugation, washed once in PBS, and resuspended in 250 µl of 4% SDS, 80 mM Tris, pH 6.8. Cells were disrupted by vortexing three times in the presence of 0.5 ml of zirconia/silica beads as described above. The resulting lysates were then boiled for 5 min and spun at 12,000 × g for 10 min at room temperature. Triplicate aliquots (10 µl each) of the supernatants were spotted onto Whatman Grade 3 filters. Filters were boiled in 10% trichloroacetic acid, washed, and counted in a scintillation counter. All assays were done in triplicate in two independent experiments.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of Isogenic Strains Containing Single Copies of NMT or nmt487D and Characterization of Their Growth Phenotypes-- A purified 9.7-kb HindIII fragment from pCN28-14, containing nmt487D and ADE2 as the selectable marker (Fig. 2A), was used to transform a serotype A strain of C. neoformans with an ade2 allele (MO49). Transformants were initially selected on plates lacking adenine and then screened for myristic acid auxotrophy at 24 and 37 °C. Five percent of the adenine prototrophs exhibited auxotrophy for the fatty acid at 37 °C. Southern blotting confirmed that all myristic acid auxotrophs had undergone homologous recombination at the NMT locus. Two-thirds of these auxotrophs had a single copy of nmt487D (e.g. see strain CMD19 in Fig. 2B). Replacement of the endogenous NMT allele was verified by an allele-specific PCR (Ref. 19; data not shown).
|
|
Levels of Arf N-Myristoylation Correlate with the Growth Phenotype
Produced by nmt487D--
Earlier [3H]myristate labeling
studies of a wild type strain of C. neoformans had shown
that during log phase growth, an Arf was the most prominently labeled
cellular N-myristoylprotein (45). An ARF gene was
subsequently recovered from C. neoformans and sequenced
(24). Co-expression of C. neoformans Nmt and this Arf in
E. coli, a bacterium with no endogenous Nmt activity,
confirmed that the Arf is a substrate for the acyltransferase (24).
N-Myristoylation of Arfs in E. coli changes their
electrophoretic mobility during SDS-polyacrylamide gel electrophoresis:
acylation produces more rapid migration compared with the corresponding
nonmyristoylated recombinant protein (24, 38). This mobility shift can
be used to assay levels of protein N-myristoylation in
eukaryotic cells. For example, in S. cerevisiae Arf1p and
Arf2p are necessary for growth and dependent upon their
covalently bound myristoyl moiety for function (46-48). Western blot
analysis of cellular lysates prepared from isogenic S. cerevisiae strains containing (i) NMT1 or
nmt1-451D (corresponding to C. neoformans
nmt487D) and (ii) wild type or null alleles of ARF1 or
ARF2 established that a 50% reduction in
Arf1p/Arf2p acylation is associated with a loss of viability
(38).
|
|
Complementation of nmt487D by Human Nmt--
An in vivo
test was designed to examine whether human and C. neoformans
Nmts have distinct substrate specificities for C. neoformans
proteins. The test consisted of expressing wild type fungal and human
Nmts in an nmt487D recipient strain and defining cellular
phenotypes under conditions where the activity of the "endogenous"
nmt487D enzyme was insufficient to support growth. CMD19 (one copy of
nmt487D) was chosen as the recipient strain for
transformation with orthologous NMTs because it has a
distinctive, easily scored phenotype (strict myristic acid auxotrophy
at 24 °C) that is stably maintained (reversion frequency
<108).
|
|
Use of Isogenic Strains Expressing Human or C. neoformans Nmt to Identify an Nmt-dependent Fungicidal Inhibitor of the Enzyme-- Isogenic strains of C. neoformans expressing wild type fungal or human Nmt can be used to establish whether an in vitro inhibitor of the purified acyltransferase with anti-fungal properties exerts its growth inhibitory effect through in vivo inhibition of the enzyme. Specifically, if an Nmt inhibitor is selective for the fungal compared to human enzyme in vitro, and if that compound also produces a greater degree of inhibition of growth of a fungal Nmt-producing, compared to human Nmt-producing strain, then an Nmt-dependent mechanism for its biological effect can be invoked.
In Vitro Characterization of a Fully Depeptidized Inhibitor of C. neoformans and Human Nmts-- Nmt has an ordered reaction mechanism: the apoenzyme first binds myristoyl-CoA, forming a binary Nmt:myristoyl-CoA complex; peptide then binds, forming a ternary complex; this is followed by catalysis and release of the CoA and myristoylpeptide products (41, 49-51). As noted in the Introduction, the acyl-CoA substrate specificities of orthologous Nmts are highly conserved (30, 52), whereas their peptide substrate specificities are somewhat different.
We have recently identified potent and selective peptidomimetic inhibitors of C. albicans Nmt (25-27, 29, 30, 52, 53). As with C. neoformans, Arfs are the most prominent C. albicans N-myristoylproteins labeled with [3H]myristate during log phase growth (45). Alanine scanning mutagenesis of an octapeptide derived from the N-terminal sequence of an Arf (GLYASKLS-NH2) revealed that Gly1, Ser5, and Lys6 play the most important role in recognition by the peptide binding site of C. albicans Nmt (28). Substitution of Ala for Gly1 yielded an inhibitor (ALYASKLS-NH2) that was competitive for peptide and noncompetitive for myristoyl-CoA. Replacement of the ALYA tetrapeptide with an 11-aminoundecanoyl group and replacement of the C-terminal Leu-Ser with N-cyclohexylethyl lysinamide moiety produced a dipeptide inhibitor that was more potent than the starting octapeptide inhibitor (28). Increased potency and selectivity for C. albicans versus human Nmt was obtained by substituting a 2-methylimidazole for the N-terminal glycyl amine and rigidifying the flexible undecanoyl chain with a 4-substituted phenylacetyl group. This compound (SC-58272) is a competitive inhibitor (Ki = 30 nM using GNAASARR-NH2 as the peptide substrate) and has 250-fold selectivity for the C. albicans enzyme (27). Unfortunately, it produces no growth inhibition when added to log phase cultures of C. albicans or C. neoformans, even at concentrations up to 100 µM (Ref. 38 plus data not shown). The Ser-Lys dipeptide that remains in SC-58272 is a potential site for cleavage by cellular proteases and may contribute to the lack of biological activity in the compound. Therefore, we synthesized a derivative (SC-61213) that lacks any peptide binds (see Fig. 1). The p-[(2-methyl-1-imidazol-1-yl)butyl]phenylacetyl moiety present in SC-58272 was retained in SC-61213 and used to represent the N-terminal ALYA tetrapeptide of the parental ALYASKLS-NH2 inhibitor. The C-terminal SKLS was represented in SC-61213 by a chiral tyrosinol scaffold. The imidazole group provided a mimic for the essential N-terminal amino recognition element in Ala1 of ALYASKLS-NH2. The critical Ser5 hydroxyl was retained in the form of an alcohol, and the important
|
Characterization of the Biological Effects of SC-61213-- CPA8 (NMT) and HMC1 (nmt487D plus 10 copies of human NMT) were chosen to test whether SC-61213 has any anti-fungal activity. These isogenic myristic acid prototrophs have similar growth kinetics at 35 °C in standard liquid medium (Fig. 8A) and, based on Western blot analysis, have similar steady state levels of Nmt (data not shown). At this temperature, a single copy of nmt487D will not support growth (see above), so growth and survival of HMC1 cells depends upon their human Nmt. SC-61213 is selective for the fungal compared with human enzyme in vitro. Therefore, a greater degree of inhibition of CPA8 compared with HMC1 cell growth would indicate an Nmt-dependent mechanism for its biological effects.
|
Prospectus-- Taken together, our results support the conclusion that the fungicidal effects of SC-61213 involve Nmt. A similar dose of SC-61213 has fungicidal activity against C. albicans Nmt (39), an observation that lends additional support for Nmt as a legitimate target for development of fungicidal drugs. Our isogenic C. neoformans strains, containing wild type C. neoformans or human Nmt, may prove useful for high throughput screens of large chemical or natural product libraries that seek to identify Nmt-based fungistatic or fungicidal compounds with relatively little activity against the human acyltransferase.
Finally, the reagents developed during the course of this study may also be generally useful for genetic manipulations of C. neoformans. NMT is a good selectable marker; the phenotype produced by nmt487D is distinctive (pronounced temperature-sensitive myristic acid auxotrophy) and stable (reversion frequency <10 ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dena Toffaletti, John Perfect, and Joe Heitman (all at Duke University) as well as Dwight Towler (Washington University) for their helpful suggestions and assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI38200 and Monsanto Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Edward A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO 63104.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Pharmacology, Box 8103, Washington University School of
Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7047; E-mail:
jgordon{at}pharmdec.wustl.edu.
1 The abbreviations used are: NMT, gene encoding myristoyl-CoA:protein N-myristoyltransferase; Arf, ADP ribosylation factor; kb, kilobase pair; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|