THE MOLECULAR BASIS OF RESISTANCE TO 5-FLUOROCYTOSINE IN CANDIDA ALBICANS

Habib  M. ALLOUSH

Candida albicans is an opportunistic fungus that causes a variety of diseases ranging from superficial to systemic and life threatening infections. 5‑Fluorocytosine (5‑FC) is a relatively safe drug used in the treatment of patients with systemic candidosis; however, the effectiveness of this antifungal is severely hampered by the development of resistant strains during therapy. Approximately 40% of clinical isolates tested were found to be partially resistant to 5‑FC and are heterozygous giving rise to homozygous resistant variants responsible for therapy failure. Genetic and biochemical studies have shown that for the majority of strains tested resistance is associated with a lesion in the gene coding for uracil phosphoribosyltransferase (UPRTase,) a key enzyme of the pyrimidine salvage pathway. Resistance resulting from defects in other enzymes occurs only infrequently. The aim of this project is to study the enzyme  and  clone  the  FUR1  gene in order to provide an explanation for 5‑FC resistance in C. albicans.

UPRTase from C. albicans 72S, a strain with high enzyme activity, was purified 38 fold. Attempts at purifying the enzyme to homogeneity were faced with problems associated with the extreme liability of the enzyme and the rapid loss of activity at 4º C. The partially purified preparation was used to study the enzyme's general properties such as its stability, the optimal Mg++ and H+ concentrations for its activity and the ability of URPTase to utilize 5‑FU as a substrate. SDS‑PAGE suggested that UPRTase exists as a dimmer of two dissimilar subunits of M_r = 47 and 38 kDa, respectively. The K_m and V _max values of UPRTase for its substrates, uracil and PRPP,were determined at different concentrations of the second substrate. These values were higher than those determined in E. coli and baker's yeast but similar values were obtained in leukaemic cells. Graphic analyses of the data were consistent with the double displacement mechanism.

In order to gain insights into the genetic control of the biosynthesis of UPRTase, studies were carried out to clone the FUR1 gene. This gene was isolated by complementation of the furl mutation. The selection scheme used to screen for the expression of the FURI phenotype involved the construction of a strain blocked in both the de novo, and salvage pyrimidine synthesis. Such strain, when grown on cytosine or uracil, will depend on the presence of a functional UPRTase. Isolation of such a mutant involved the use of a double mutagenesis method to isolate furl mutants of C. albicans SGY-243 and the use of 5-fluoro-orotic acid (5-FOA) and N-phosphonoacetyl-L-aspartate (PALA) as inhibitors of the de novo pyrimidine synthesis in C. albicans 72R. Due to the failure to isolate a C- albicans strain suitable for  use  as  a  recipient   in   the  cloning  procedure , it  was  decided  to  isolate  a  mutant  of  the  yeast

S. cerevisiae DBY746.

A furl mutant was successfully obtained from the haploid S. cerevisiae and was used to screen for the expression of the cloned C. albicans FUR1 gene. A genomic DNA library of C. albicans MEN was prepared in the replicating plasmid pMK122 by transforming E. coli DHI to ampicillin resistance. The library was used to transform the furl strain to FUR1 phenotype by plating the cells on to plates containing cytosine as the exogenous pyrimidine source. Further molecular biology and biochemical experiments confirmed the cloning of the FUR1 gene in 3 out of the 7 FURl putative transformants obtained initially.