Medical Mycology

92. Effect of sub-Minimal Inhibitory Concentrations (sub-MIC) of antifungal tioconazole on expression of esterase of Trichophyton rubrum.

Ana Lucia Fachin, Eucleia P. Betioli Contel and Nilce M. Martinez-Rossi. Dept of Genetics, FMRP-USP, Ribeiro Preto, SP, Brazil.

T. rubrum is a filamentous fungus that has the capacity to invade keratinized tissue (skin, hair and nails) of humans to produce infections. Tioconazole, one of the antifungals used to control T. rubrum, acts by inhibiting fungal ergosterol biosynthesis. In order to determine the relationship between resistance to tioconazole and esterases expression in this fungus, we isolated several tioconazole-resistant mutants by treating with UV light, an isolate from a patient from the University Hospital. The original isolate (MIC=0.5 mg/ml) and the resistant mutants (MIC=1.0 to 2.5mg/ml) were cultivated on Sabouraud liquid medium with and without tioconazole concentrations below the MIC (sub-MICs) of each strain. After the extration of intracellular total protein the esterase eletrophoretic pattern was developed with -naphthyl acetate. All strains cultivated on Sabouraud medium containing tioconazole (original isolate and resistant mutants) presented 5 bands, whereas those cultivated on Sabouraud medium alone presented only 2 clarly visible bands. Furthermore, the visualization of these 3 extra bands was found to depend on antifungal concentration. Although the physiological role of this esterase is not clear, its overexpression may be a response to the cellular stress caused by the presence of the antifungal, even in the resistant mutants, or may even play a role in cellular drug detoxification.

Financial support: FAPESP, CNPq and FINEP

93. Cloning and Characterization of a Putative Enolase from Pneumocystis carinii.

Deborah Fox and A. George Smulian, University of Cincinnati College of Medicine, VA Medical Center, Cincinnati OH.

The glycolytic enzyme enolase is one of the most highly abundant proteins expressed in fungi and has also been shown to be an immunodominant cell wall-associated antigen of the pathogenic fungus, Candida albicans. To investigate the expression and antigenicity of enolase in the opportunistic pathogen Pneumocystis carinii, the genomic and cDNA enolase were cloned and characterized. The genomic enolase clone was hybridized to restriction enzyme digested DNA, revealing the presence of a single P. carinii enolase gene. Hybridization of the enolase clone to electrophoretic karyotypes localized the gene to a 550 kb chromosome of the protoype form of P. carinii. The level of expression of P. carinii enolase mRNA was examined under a variety of growth conditions. Northern analysis identified a 1.4 kb transcript which was expressed at constant levels in either starvation or high glucose conditions. Sequence analysis of the enolase cDNA identified a 1350 bp continuous open reading frame. The predicted protein is 433 amino acid residues in length and shows extensive homology to other fungal enolase proteins, including C. albicans (87%), Aspergillus oryzae (88%) and Saccharomyces cerevisiae (85%). The active site and conformation metal ion-binding site residues are conserved in the predicted P.carinii enolase protein. Experiments are currently underway to purify the enolase fusion protein for immunoblot analysis with immune sera from both P. carinii and C. albicans. In addition, the enzymatic activity and subcellular localization of the enolase protein will be determined.

94. Disruption of Mitochondrial Ultrastructure and Function Induced by Exogenous Sphinganine in Neurospora crassa.

Jennifer A. Gerlach¹, Kenneth M. Bart² and Lawrence R. Aaronson¹. ¹Utica College and ²Hamilton College

Sphinganine, a sphingoid base found concentrated in mammalian epidermis, may serve as a natural antifungal barrier, preventing infection by pathogenic fungi. Neurospora crassa is being used as a model to study the effects of sphinganine in fungal pathogens. In Neurospora, exogenous sphinganine is metabolized into complex sphingolipids, such as ceramides and cerebrosides over a 1 hr period, suggesting that the lipid is being internalized. However, only sphinganine is associated with mitochondrial fractions during this time. Sphinganine appears to have a dramatic effect on mitochondrial structure and function. Transmission electron microscopy demonstrates that the disruption of ultrastructure is both a time- and concentration-dependent phenomenon. Exposure to 100 uM sphinganine for 1 hr completely disrupts the outer membranes of a majority of mitochondria. Analysis also shows that short-term exposure to subinhibitory concentrations of sphinganine causes swelling of mitochondria, while exposure to increasing concentrations of the lipid results in the complete disruption of outer membranes. The presence of cytoplasmic lamellar and mesosome-like structures near the plasma membrane, and enlarged vacuolar bodies, further indicate the deterioration of cellular vitality. The changes observed in mitochondrial ultrastructure appear to coincide with a loss of mitochondrial function. Using the fluorescent dye Rhodamine 123, it was observed that germinating cells treated with 100 uM sphinganine exhibit a severe decline in electrogenic mitochondrial activity. These results demonstrate that the loss of cell growth and viability observed in sphinganine-treated Neurospora may be due in part to the disruption of mitochondrial morphology and activity.

95. The Role of Multidrug Efflux Transporters in Antifungal Drug Susceptibility.

Yi Li and Chuck Staben. T.H. Morgan School of Biological Sciences, University of Kentucky.

Fungi carefully control influx and efflux of small molecules, including antifungal drugs, toxins, and mating factors. Fungi apparently have 2 main classes of drug efflux transporters: ABC (ATP-binding cassette) proteins and MFS (major facilitator superfamily) transporters. These transporters endow some wild type fungi with inherent resistance to many inhibitory drugs. Alterations in activity or specificity of efflux appears to be a common method of acquiring drug resistance. Genetic characterization

of the Saccharomyces cerevisiae SGE1 gene indicates that its product mediates resistance to pentamidine and other structurally related inhibitory compounds, including G418, gentian violet, and ethidium bromide. Sge1p appears to be a typical transmembrane efflux protein of the MFS class that utilizes the proton gradient to transport its substrates. The activity of this exporter is modulated by environmental conditions, being much lower under respiratory conditions than during fermentative growth. S. cerevisiae and other fungi, including Candida albicans, have multiple members of the export protein families that have overlapping specificities. MFS efflux transporters also function in export of toxins associated with virulence in plant pathogens. Manipulating the transport capacity of fungi may be an effective means of modulating virulence or increasing drug susceptibility. Ability to manipulate the activities of these pumps relies upon a molecular understanding of pump function.

96. Isolation of a Cryptococcus neoformans gene that encodes a protein which elicits a delayed-type hypersensitivity response in mice.

M. Alejandra Mandel¹, Juneann W. Murphy², Kris I. Orsborn¹, John N. Galgiani¹ and Marc J. Orbach1. ¹University of

Arizona, Tucson, AZ. and ²Oklahoma HSC, Oklahoma City, OK.

Control of cryptococcal infection is critically dependent on T cell-mediated immune responses but little is known about the antigens which induce this activity. In mice immunized with cryptococcal extracts from a virulent encapsulated strain, delayed-type hypersensitivity (DTH), measured as footpad swelling, has been used as a marker for T cell response. A 20 kDa protein isolated from the culture filtrate of an acapsular mutant of C. neoformans (cap-67; from strain B-3501, serotype D, mating type) was found to stimulate DTH in this model.

We took a reverse genetic approach in order to clone the gene encoding this protein. Following gel purification of the protein, 20 amino acid residues were sequenced from the N-terminus and 20, 1 1 , and 6 residues were obtained from peptides produced by endoproteinase LysC cleavage. Degenerate oligonucleotides were designed for the N-terminal peptide and two of the three internal peptides for use in RT-PCR. Using oligonucleotides corresponding to the N-terminal sequence and one of the internal peptides, a 778 bp fragment was amplified. Translation of the sequence of this fragment revealed it contains sequences corresponding to amino acids adjacent to those in the peptides used for oligo design, as well as to one of the other internal peptides. Analysis of the genomic sequence and its expression will be presented.

Further work involvng the production of recombinant protein to assess the the vaccine potential of this protein is now possible.

97. Development ot sulfonamide resistance as a selectable marker for the transformation of Pneumocystis carinii (Pc).

A. George Smulian and Melanie T. Cushion. University of Cincinnati and VA Medical Center, Cincinnati OH.

An initial step critical in the development of a transformation system for the opportunistic pathogen, P. carinii, is the availability of a selectable marker allowing positive selection for transformed organisms. This is especially important given the inability to sustain long term in vitro culture of the organism and the reliance on immunosuppressed animals as a source of organisms. We have explored the use of many conventional markers in Pc. Tested in an in vitro cytotoxicity assay, Pc appeared resistant to G418, hygromycin and bialophos at levels up to 10 ug/ml. Pc is extremely sensitive to sulfonamides both in vivo and in vitro. The gene encoding dihydropteroate synthase (DHPS), the target of sulfa, has previously been cloned from Pc (i.e. Folic Acid Synthesis [FAS] gene). Sulfa resistance in Plasmodium falciparum and many bacteria has been associated with a few well characterized mutations at positions flanking the presumptive catalytic site of DHPS. We introduced two mutations (T-> F at residue 517 and G ->S at residue 687) associated with high level of sulfa resistance into the DHPS cDNA of Pc by site directed mutagenesis. The mutant FAS cDNA was cloned downstream of a Pc promoter and used to transform S. cerevisiae. The resultant yeast transformants had increased MICs to sulfadiazine compared to the parental yeast strain or yeast transformed with the identical plasmid construct containing the wild type FAS cDNA. Biochemical characterization of the recombinant enzyme is currently underway. The activity of sulfas against Pc in in vivo and in vitro model systems will allow development of a transformation system with selection for transformed organisms in short term in vitro culture and in the immunosuppressed rat model.

98. Nuclear Proteins Involved in Dimorphic Growth of Candida aibicans.

Phillip Stafford, Douglas Rhoads, University of Arkansas, Fayetteville, AR.

Candida albicans is the most prevalent of the human pathogenic yeasts. The Candidas are dimorphic in that they display one or more alternative growth forms. It is generally accepted that alternative growth forms are important for tissue invasion and immune-avoidance during infections. We have been characterizing growth-form-specific transcripts of C. albicans using ddRT-PCR to dissect this fundamental differentiation process. Differentially expressed mRNAs are characterized for sequence and time of expression during morphogenesis. We have identified two cDNAs for transcripts which are apparently unique to hyphal cells. Both cDNAs appear to encode proteins homologous to known nuclear proteins. One transcript (Caml) is expressed only transiently during early germ tube formation. This transcript is most similar to a Saccharomyces cerevisiae ORF related to the yeast nuclear transport protein NIP80. The other CDNA (Cam3) encodes a polypeptide most similiar to a uracil phosphoribosyl transferase. Portions of the gene align with a protein which interacts with Sin3, a general transcriptional regulator in S. cerevisiae. Further characterization of the genes for these mRNAs is under way. These genes will be analyzed for phase-specific upstream promoter sequences as well as the effects of gene disruption and constitutive expression on growth form.