Isolation of a small DNA fragment carrying the gene for a dihydrofolate reductase from a trimethoprim resistance factor

Summary DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate reductase. The enzyme was found to be stable at 100°. Some aspects of the synthesis of dihydrofolate reductase are discussed.Dedicated to Professor Peter Karlson on the occasion of his 60th birthday     

A fluorescent derivative of methotrexate as an intracellular marker for dihydrofolate reductase in L1210 cells

Fluorescein isothiocyanate coupled via a diaminopentyl-linking group to methotrexate (G.R. Gapski, J. M. Whiteley, J. I. Rader, P. L. Cramer, G. B. Henderson, V. Neef, and F. M. Huennekens, 1975, J. Med. Chem.18, 526–528) produces a fluorescent compound which is a strong inhibitor of dihydrofolate reductase (K_i = 60 nM) purified from L1210 murine leukemia cells. The fluorescent methotrexate derivative is preferentially taken up by methotrexate-resistant rather than wild-type L1210 cells grown in culture and acts as a visual marker for dihydrofolate reductase (K_D = 50 nM) during both purification and polyacrylamide electrophoresis. Uptake, which is proportional to the level of dihydrofolate reductase (often an indicator of the degree of acquired cellular methotrexate resistance), occurs slowly and via a route that is distinct from the carrier-mediated system utilized by these cells to transport methotrexate.     

Isolation and characterization of aminopterin-resistant cell lines in maize

Aminopterin-resistant cell lines of maize were isolated by two different procedures of callus selection and by plating suspension cultures on drugcontaining medium after mutagen treatment. Efficiencies of different methods of variant selection were compared. Four aminopterin-resistant cell lines were shown to be 10–40 times more resistant than the parental cell line, and they were also resistant to another folate analog, methotrexate. The results suggest that alterations in at least three different cell properties could be responsible for resistance; 1) increased dihydrofolate reductase activity, 2) altered aminopterin sensitivity of dihydrofolate reductase, and 3) reduced drug uptake. One of the resistant cell lines showed more than one alteration, but its resistance proved to be unstable. The results suggest that stable changes which may or may not be of genetic origin and also unstable physiological changes or a combination of both could lead to aminopterin resistance in maize cell cultures.Abbreviations AMPT aminopterin - MTX methotrexate - DHFR dihydrofolate reductase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate Research supported by the College of Agriculture and Life Sciences and by the Graduate School, University of Wisconsin Madison, Wis, USA     

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 M, and for NADPNH 8.46±0.25 and 9.32±0.18 M, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 M, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 M, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

DNA sequence of a plasmid-encoded dihydrofolate reductase

Summary The sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R-388 was determined. The gene was subcloned and mapped by an in vitro mutagenesis method involving insertion of synthetic oligonucleotide decamers encoding the BamHI recognition site. Sites of insertion that destroyed the methotrexate resistance fell in two regions separated by 300 bp within a 1.2 kb fragment. One of these regions encodes a 78 amino acid polypeptide homologous to another drug-resistant DHFR. The second region essential for DHFR expression appears to be the promoter of the DHFR gene.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations bar/PAT bialaphos resistance gene/phosphinotricin acetyltransferase - 2,4-D 2,4-di-chlorophenoxyacetic acid - dhfr/DHPR dihydrofolate reductase gene/enzyme - gus/GUS -glucuronidase gene/enzyme - hpt/HPT hygromycin phosphotransferase gene/enzyme - Kn kinetin - PEG polyethylene glycol - RH relative humidity     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

Summary The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the a insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the a insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob · 1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.     

Inhibition of the differentiation of Candida albicans by the chelator 1,10-phenanthroline

Several chelators were examined for their ability to prevent the synchronous release of 24- to 48-hour stationary phase singlet cells of the dimorphic yeast Candida albicans into either the mycelial or the budding phenotypes (in a defined liquid medium at 37°C; at pH 6.5 or pH 4.5, respectively). The only chelator that was found to inhibit mycelium formation completely and to restrict bud formation to about 10% was 1,10-phenanthroline at minimal concentrations of 50 M and 230 M, respectively.The inhibition of both phenotypes could be reversed completely by the addition of 200 M of ZnSO₄. The synchrony of recovery from inhibition by the addition of zinc paralleled that of the controls for both phenotypes, and the final number of mycelia or buds as a percentage of the control was the same (100%).These findings support the hypothesis that the lag period between the release from stationary phase and the onset of development for Candida represents the time of acquisition of a minimum threshold amount of a cation, such as zinc. The involvement of zinc in phenotypic development is discussed, suggesting that while zinc is involved in the initiation of development, it may not determine the phenotype of Candida albicans.     

The border fresidues of the dihydrofolate reductase domain inEscherichia coli β-galactosidase correspond to the positions of introns 1 and 5 of dihydrofolate reductase of chicken

Summary In-galactosidase ofEscherichia coli residues 820–934 are similar to residues in dihydrofolate reductase ofE. coli. Dihydrofolate reductase ofE. coli and chicken are also similar and have identical tertiary structures. I used the similarity of the three-dimensional structure of prokaryotic and eukaryotic dihydrofolage reductases to align the chicken dihydrofolate reductase and the similar residues of-galactosidase. The positions of introns 1 and 5 of the chicken dihydrofolate reductase gene correspond exactly to the start and the end of the dihydrofolate reductase-like domain in the-galactosidase polypeptide chain. This equivalence of intron positions in a eukaryotic gene and domain structure in a prokaryotic protein was interpreted as evidence for a common origin of both genes.     

Transfer of MHC genes into hematopoietic stem cells by electroporation: A model for monitoring gene expression

Abstract

Previous attempts in using recombinant viral vectors to transfer genes into bone marrow cells have resulted in expression frequencies of 2‐15% (8,9,12,13,14,24). These vectors, however, require complex gene vector constructions and culture methods and have been difficult to utilize. We therefore have used electroporation to establish a less complicated and equally efficient technique for gene transfer. Conditions yielding high gene transfer into bone marrow precursor cells by electroporation were determined using a mutant dihydrofolate reductase gene and/or murine MHC class II genes. Successful transfer and expression of these genes were assessed by (1) fluorescence staining using monoclonal antibodies, (2) acquired resistance to the anti‐folate drug methotrexate and (3) Southern blot analysis. Stable integration of the newly acquired mutant dihydrofolate reductase gene was observed in > 25% of murine hematopoietic progenitor cells (CFU‐M). Electroporation of class II genes in conjunction with the mutant dihydrofolate reductase and subsequent selection with methotrexate resulted in expression of transfected la molecules in 12‐15% of bone marrow derived macrophages. Integration of the transferred major histocompatibility genes was verified by Southern blot analysis of high molecular weight cellular DNA following electroporation and methotrexate selection. The frequencies of gene transfer and expression achieved in this study suggest that electroporation is a viable option for monitoring gene expression in bone marrow derived cells.     

Fitness trade-offs in the evolution of dihydrofolate reductase and drug resistance in Plasmodium falciparum

Background

Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness.

Methodology/Principal Findings

We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine.

Conclusions/Significance

Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed.     

The antibacterial agent,moxifloxacin inhibits virulence factors of <Emphasis Type="Italic">Candida albicans</Emphasis> through multitargeting

Fluoroquinolines are broad spectrum fourth generation antibiotics. Some of the Fluoroquinolines exhibit antifungal activity. We are reporting the potential mechanism of action of a fluoroquinoline antibiotic, moxifloxacin on the growth, morphogenesis and biofilm formation of the human pathogen Candida albicans. Moxifloxacin was found to be Candidacidal in nature. Moxifloxacin seems to inhibit the yeast to Hyphal morphogenesis by affecting signaling pathways. It arrested the cell cycle of C. albicans at S phase. Docking of moxifloxacin with predicted structure of C. albicans DNA Topoisomerase II suggests that moxifloxacin may bind and inhibit the activity of DNA Topoisomerase II in C. albicans. Moxifloxacin could be used as a dual purpose antibiotic for treating mixed infections caused by bacteria as well as C. albicans. In addition chances of developing moxifloxacin resistance in C. albicans are less considering the fact that moxifloxacin may target multiple steps in yeast to hyphal transition in C. albicans.     

A new member of the adenylate kinase family in yeast: PAK3 is highly homologous to mammalian AK3 and is targeted to mitochondria

Summary Making use of the polymerase chain reaction primed by oligonucleotides corresponding to regions conserved between members of the nucleoside monophosphate kinase family, we have isolated the yeast gene PAK3. Pak3p belongs to the subgroup of long-form adenylate kinase isozymes (deduced molecular mass 25.3 kDa) and exhibits highest sequence similarity to bovine AK3 rather than to the yeast isozyme, Aky2p. The gene is shown to be non-essential because haploid disruption mutants are viable, both in the presence and absence of a functional AKY2 allele. It maps on chromosome V upstream of RAD3. Its expression level is low when cells are grown on glucose or other fermentable carbon sources and about threefold higher on glycerol, but can be significantly induced by ethanol. A PAK3/mouse dihydrofolate reductase fusion construct expressed in yeast is targeted to mitochondria. Transformation with PAK3 on a multicopy plasmid complements neither adenylate kinase deficiency in an aky2-disrupted yeast strain nor in Escherichia coli cells conditionally defective in adenylate kinase.Abbreviations Ap5A P¹,P⁵-di(adenosine-5)pentaphosphate - adenylate kinase ATP: AMP phosphotransferase (EC 2.7.4.3) - Pak3p (Aky2p) protein product of the PAK3 (AKY2) gene - DHFR mouse dihydrofolate reductase     

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.     

Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase

Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + α-ketoglutarate) of the novel α-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-α-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.     

Candida albicans Hyphal Formation and Virulence Assessed Using a Caenorhabditis elegans Infection Model

Candida albicans colonizes the human gastrointestinal tract and can cause life-threatening systemic infection in susceptible hosts. We study here C. albicans virulence determinants using the nematode Caenorhabditis elegans in a pathogenesis system that models candidiasis. The yeast form of C. albicans is ingested into the C. elegans digestive tract. In liquid media, the yeast cells then undergo morphological change to form hyphae, which results in aggressive tissue destruction and death of the nematode. Several lines of evidence demonstrate that hyphal formation is critical for C. albicans pathogenesis in C. elegans. First, two yeast species unable to form hyphae (Debaryomyces hansenii and Candida lusitaniae) were less virulent than C. albicans in the C. elegans assay. Second, three C. albicans mutant strains compromised in their ability to form hyphae (efg1Δ/efg1Δ, flo8Δ/flo8Δ, and cph1Δ/cph1Δ efg1Δ/efg1Δ) were dramatically attenuated for virulence. Third, the conditional tet-NRG1 strain, which enables the external manipulation of morphogenesis in vivo, was more virulent toward C. elegans when the assay was conducted under conditions that permit hyphal growth. Finally, we demonstrate the utility of the C. elegans assay in a screen for C. albicans virulence determinants, which identified several genes important for both hyphal formation in vivo and the killing of C. elegans, including the recently described CAS5 and ADA2 genes. These studies in a C. elegans-C. albicans infection model provide insights into the virulence mechanisms of an important human pathogen.Candida albicans is the most common human fungal pathogen; however, our knowledge of its virulence mechanisms is incomplete, and our best antifungal agents are often ineffective in treating severe candidiasis (3). Infections with Candida species account for 70 to 90% of all invasive mycoses (32) and can be associated with devastating consequences, particularly in intensive care units where mortality rates reach 40% (24, 34). The drug resistance of pathogenic fungi exacerbates this problem and often limits therapeutic options (35). The identification of virulence pathways that can be targeted with novel antifungal therapies is urgently needed (31, 38, 46).One approach to understand the genetic mechanisms of virulence is to use invertebrates, such as the nematode Caenorhabditis elegans, as model hosts (43). Studies of C. elegans infection with Pseudomonas aeruginosa and Cryptococcus neoformans, for example, have led to the identification of evolutionarily conserved mechanisms of host immunity and microbial virulence (1, 21, 50). However, efforts to design an accurate nonmammalian model of C. albicans pathogenesis have been stymied, in part because it has been difficult to capture the role of Candida dimorphism in these systems.Morphogenesis in C. albicans is intricately related to pathogenesis and thus has been intensively studied. C. albicans hyphae are important for tissue destruction and host invasion (3). As such, C. albicans mutants and non-albicans Candida species that are unable to form true hyphae are attenuated for virulence (3, 37). However, C. albicans yeast cells also have virulence attributes (4, 33) that are likely involved in dissemination of the fungus through the bloodstream, and the establishment of infection at distant sites. To date, genetic screens to identify the determinants of Candida morphology have been conducted in vitro. Determining the role of these genes in virulence has traditionally involved separate and often laborious studies in mammals. Therefore, an expedient system to study morphogenesis of C. albicans in vivo and accurately model pathogenesis would offer many important advantages.Here, we study C. albicans pathogenesis using the invertebrate host C. elegans. C. albicans yeast cells are ingested into the gastrointestinal tract. In liquid media, the yeast cells form hyphae, which results in an aggressive infection that ultimately kills the nematode. Fungal hyphae destroy worm tissues and pierce the collagenous cuticle of the animal, a phenotype that is easily visible using a dissecting microscope. By studying mutants and genetically engineered C. albicans strains, we show that hyphal formation is required for full virulence in this system. Finally, we illustrate the utility of the C. elegans-C. albicans infection assay in a screen for genes involved in Candida morphogenesis and virulence.