Peptide transport in Candida albicans

Candida albicans has been shown to possess a complex system for the utilization of environmental peptides. The presence of multiple peptide permeases was identified through the creation of a mutant resistant to the effects of nikkomycin, a peptide-nucleoside antibiotic used as an example of a toxic peptide. Peptides transported by the cell are hydrolysed by at least eight peptidases present in the soluble fraction of the cell.

Nikkomycin was a growth inhibitor of C. albicans (MIC on malt extract = 25yg/ml), The effect was observed as a profound effect upon the chitin network of the cell wall through inhibition of the enzyme chitin synthase (Ki= 0.16 yM). The alteration of the cell wall in the presence of the drug was followed by phase contrast and fluorescence microscopy in addition to transmission electron microscopy. Three major effects were observed: the inhibition of primary septum formation; a protoplasting effect in growing cells due to the weakening of the cell wall; finally the lysis of the cell wall at a point adjacent to the septum. The effects were analogous to those observed upon treatment of sensitive fungi with polyoxins.

The presence of peptone mixtures or certain defined peptides interfered with the uptake of the drug which shows that nikkomycin enters the cell via the peptide transport system of C. albicans. Two important observations were the reversal of nikkomycin activity by serum and the high frequency of spontaneous resistance to the drug. These will have considerable influence upon the development of anticandidal peptide drugs.

The fluorescamine technique was used to follow peptide uptake. Two peptide permeases have been defined for C. albicans by comparison of the wild type with a mutant resistant to the effects of nikkomycin through a transport defect. System I has high affinity for dipeptides and a lower affinity for oligopeptides; System II has high affinity for oligopeptides but only a low capacity for a few dipeptides. The specificities of each system are described. It is the loss of System I which confers resistance to nikkomycin in the mutant of C. albicans (NIK5). System II has been shown to be inducible when peptides provide the sole nitrogen source for the cell and an equivalent induction appeared to occur in System I.

A new technique for the visualization of peptidase isozymes has been developed based upon the electrophoretic transfer of native proteins from polyacrylamide gels to nitrocellulose sheets. Blotting is followed by an in situ stain for enzyme activity. By this method eight peptidases have been identified in the soluble fraction of C. albicans, six of which possessed overlapping dipeptidase activity. No differences were found between the peptidases of the wild type and the transport deficient mutant.

Following intracellular hydrolysis of [U¹⁴C]alanyl alanine, [U¹⁴C]-alanine undergoes rapid metabolism resulting in the release of ¹⁴C02 from a cell suspension. This loss of radiolabel has a considerable influence upon the apparent rate of peptide transport when monitoring the uptake of radiolabelled peptides.