Protoplasts from Yeast and Mycelial Forms of Candida albicans

Abstract

Protoplasts have been obtained in high yields from the yeast and mycelial forms of a variety of strains of Candida albicans by enzymic digestion of cells with commercially available lytic enzymes. The protoplast formation procedure was equally effective for exponential and stationary phase cells. Pretreatment with dithiothreitol and Pronase in the presence of EDTA and Tris was necessary. Other thiol reagents and conditions did not release protoplasts from all the strains of C. albicans tested. Treatment with digestive juice of the snail Helix pomatia required the addition of chitinase for the release of protoplasts from most strains tested. Conditions for maximizing the yield of protoplasts and the activities of P-glucuronidase and chitinase were determined. Electron microscopy of C. albicans showed that the pretreatment conditions removed the outer layers and the treatment itself completely removed the inner layers of the cell wall. More than 90 % of the protoplasts produced by this method were viable as assessed by vital staining with Janus Green B.

Characterization of the inverted duplication in the mitochondrial DNA of Candida albicans

“…The Candida albicans mitochondrial genome is a circle of about 40 kilobase pairs (15). The circular molecule contains a large inverted duplication analogous to that found in almost all chloroplast DNAs (14), in some other mitochondrial DNAs (mtDNAs) (7,16), and in other isolated instances.…”

“…We have previously reported the cloning of five of the six EcoRI restriction fragments by ligating a mixture of pBR322 DNA and whole mtDNA from C. albicans H317, both cleaved with EcoRI (15). This procedure did not yield a clone containing the largest fragment, El.…”

Current Topics in Medical Mycology

“…As mentioned above, physicochemical properties are different between growing and non-growing zones, and structural and chemical differences have also been noticed. In a series of papers, Wessels and co-workers (159,215,217,218,240), have also clearly established that newly-synthesized ~(1-3) glucans present in the apical region are watersoluble and that after some time they become insoluble in water and then insoluble in alkali. It has been observed that the apical region of S. commune does not contain chitin microfibrils and that the polysaccharide is extremely sensitive to chitinase degradation and dissolution with dilute Hel, whereas in the non-growing zone or in dead hyphae, microfibrils are readily observed and chitin becomes resistant to the above mentioned treatments (232).…”

“…As previously noted, efficient protoplast formation in C. albicans requires the use of S-S reducing agents, followed by proteases and l3-glucanases (78,101,240). Cell wall regeneration in C. albicans, in partial contrast to S. cerevisiae, occurs in simple liquid media.…”

“…An acid-soluble fraction and an insoluble fraction extracted from whole cells or isolated walls according to Duffus et al (74) received most of the focus. Less than 10% of total cell wall glucan is represented by the alkalisoluble species, in a 13 (1)(2)(3)(4)(5)(6) configuration (102,219 (78,101,240,262) is required for cell wall dissolution and protoplast formation. The insoluble glucan was a highly branched chain of 13(1-3)glucan with 13(1-6) extra-chain residues, at variance with glucan from S. cerevisiae, which has fewer 13 (1)(2)(3)(4)(5)(6) bonds and a minor degree of branching lO-Cell Wall of Candida albicans: Its Functions and Its Impact 261 (74,102).…”