5-Fluorouracil Altered Morphology and Inhibited Growth of Candida albicans

Fungal infections have recently emerged as a growing threat to human health. Fungal diseases due to Candida spp. are among the major causes of morbidity in seriously debilitated and immunocompromised hosts, such as human immunodeficiency virus and cancer patients (3, 5). Thus, research work in recent years has been mainly focused on screening of therapeutic agents for fungal pathogens and identifying drug targets, understanding the mechanisms at cellular levels, and designing new molecules or altering known agents to control pathogenicity. Recent studies have shown that C. albicans and other fungal pathogens possess a group I intron or self-splicing gene in the rRNA (4). The presence of these genes in the pathogens but not in the human genome and the intron-folded structure defining binding sites amenable to specific recognition by small molecules are distinct advantages, and they could be highly suitable as therapeutic targets. 5-Fluorouracil (5FU), an antimetabolite, widely used in treatment of various types of cancer, is effective and less toxic (2). It is well known that 5FU causes inhibition of the enzyme thymidylate synthase, thereby halting the DNA synthesis. Although DNA is the primary target site, a considerable amount of evidence suggests that 5FU also has other targets like RNA and could result in unique chemotherapeutic activity, which remains unaccounted. In our preliminary study (data not shown), we found that the self-splicing process of group I intron RNA from Tetrahymena thermophila was inhibited by 5FU in vitro in a concentration- and time-dependent manner. Since very limited work (1) has been done to document the effect of 5FU in controlling the growth of C. albicans and the molecular mechanisms remaining largely unknown, we aimed to study the effect of 5FU on growth and morphology of C. albicans.

Yeast isolates (clinical strains) were obtained from patients (Royapettah Government Hospital, Chennai, India). Subcultured isolates were maintained in Sabouraud dextrose agar-broth (BBL, Cockeysville, MD) at 1 × 10⁶ to 5 × 10⁶ CFU/10 ml media. A 2-mg/ml stock of 5FU (Pharmaceuticals, Chennai, India) prepared in sterile RNase- and DNase-free water was diluted to a 6-μg/ml culture for treatment purposes. Thirty microliters of cultured C. albicans (drug treated or nontreated) was spread over a clean glass slide and dried at 37°C for 5 min. Gram staining was performed using commercial kits as per instructions, and the slides were observed microscopically. Treatment with 5FU was found to markedly inhibit hyphae and ovoid growth (Fig. 1) of C. albicans, as revealed by Gram staining of drug-treated cells, in contrast to the controls that showed budding characteristics (Fig. 1). The hyphae of drug-treated cells showed breaks in their growth, suggesting possible uptake of 5FU. These changes were consistent with the inhibitory effects on the growth curve with respect to untreated cells (Fig. 2) and correlated with disk diffusion tests (data not shown).

FIG. 1.

Photographs showing morphology, as revealed by Gram staining, of C. albicans in the absence (A) and presence (B) of 5-fluorouracil (6 μg/ml). Magnification, ×57. Original magnification, ×100.

FIG. 2.

Growth curve of C. albicans in the presence or absence of 5-fluorouracil (6 μg/ml).

The inhibition of hyphal growth could be a direct effect of 5FU or due to eukaryotic cell toxin activated by 5FU, a base analogue that is converted into 5-FdUMP during DNA synthesis and incorporated into RNA transcription as a false base for uracil. It is possible that the nuclear-cytoplasmic factors such as thymidylate synthase, an enzyme that catalyzes dUMP, fail to recognize its target, bind to 5-FdUMP that mimics dUMP, and mediate the cytotoxic effects of the drug. Similarly, transcriptional-splicing molecules involved in RNA synthesis may have the same effect. The other possibilities include the fifth position of fluorine in 5FU, where a high electronegative charge could disrupt the folded nature of RNA or interact with receptors-integrins, which may have activated downstream genes through secondary responses in inhibiting Candida growth.

In view of outcomes of serious fungal infections and emerging drug resistance in high-risk patients, there is a need for novel therapies in clinical care. Our preliminary results showed that 5FU could inhibit both morphological forms of C. albicans, which may have therapeutic implications. Further studies to understand the precise molecular mechanisms and the drug targets of 5FU and investigations with other fungal pathogens may help in designing novel approaches for effective antifungal therapy.